• Francisella tularensis results tularaemia in man and certain small mammals, such as rabbits, hares, beavers and several rodent species.
• Tularaemia was originally described in Tulare county, California.
• It can be transmitted by direct contact, by biting flies, mosquitoes and ticks, by contaminated water or meat or aerosols.
• Francisella tularensis is also know as Pasteurella tularensis or Brucella tulerensis)

Morphology of Francisella tularensis:

• It is a very small, nonmotile, nonsporing, capsulate, gram-negative coccobacillus, about 0.3 to 0.7 μm × 0.2 μm in size.
• In culture it tends to be pleomorphic to and larger, even filamentous, forms are present.
• It stains poorly with methylene blue but dilute carbol fuchsin (10%) produces characteristic bipolar staining.

Cultural Characteristics of Francisella tularensis:

• F. tularensis is strictly aerobic.
• It will not grow on ordinary nutrient media but grows well on blood agar containing 2.5 percent glucose and 0.1 percent cysteine hydrochloride.
• Minute droplet-like colonies develop in 72 hours.

Biochemical characteristics of Francisella tularensis:

• Under suitable conditions acid is formed from glucose and maltose. Indole and urease tests are negative.
• Two biovars are recognized. Strains of F. tularen­sis have been subdivided into biotypes based on their virulence and epidemiological behaviour.
• Highly virulent strains are found only in N. America, while strains of low virulence are seen in Europe and Asia also.

Pathogenesis of Francisella tularensis:

• The infection, which is a typical zoonosis, is mainly spread by insects or ticks among lagomorphs and rodents.
• It is transmitted to man through handling of infected animals, e.g. rabbits or hares tick, mosquito or fly bites, inhalation of contaminated dust, ingestion of contaminated water or meat.
• Laboratory workers are at higher risk while handling infected laboratory animals or cultures of the organism.
• Man to man transmission of infection apparently does not occur.
• In human beings, tularemia may present as a local ulceration with lymphadenitis, a typhoid like fever with glandular enlargement or an influenza like respiratory infection.
• The severity of disease is much greater with type A strains and case fatality rates may exceed 5 percent.
• Disease caused by type B strains is much less severe, with very low mortality.

Laboratory Diagnosis of Francisella tularensis :

• F. tularensis is extremely dangerous to handle in the laboratory and Category 3 containment is required for all manipulations and animal work.
• Diagnosis may be made by culture or by inoculation into guinea pigs or mice. A PCR has been described, but is not widely available.
• Serology is most likely to be positive after 3 weeks.
• Rising titres of agglutinins to F. tularensis or individual titres of 160 are diagnostic.
• Serum from cases of F. tularensis may cross-react with brucellosis and vice versa, usually to relatively low titre.
• An intradermal delayed hypersensitivity test has been used in the past but the antigen is not readily available.

Treatment of Francisella tularensis:

• Streptomycin or gentamicin are the antibiotics of choice in tularaemia and are usually curative.

Prophylaxis:

• A vaccine based on the live-attenuated LVS strain confers some protection.
• It can be administered by scarification to persons who are subject to high risk of infection.
• F. tularensis has been developed as a biological warfare agent and has potential application in bioterrorism.

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• Plesiomonas is derived from the Greek word where it means “neighbor”.
• It indicates Plesiomonas has a close association with
• It is included in the family Enterobacteriaceae.
• P. shigelloides is the only species in the genus.

Classification of Plesiomonas shigelloides:

• Kingdom: Bacteria
• Phylum: Proteobacteria
• Class: Gammaproteobacteria
• Order: Enterobacteriales
• Family: Enterobacteriaceae
• Genus: Plesiomonas
• Species: shigelloides

Clinical Significance of Plesiomonas shigelloides:

• Plesiomonas shigelloides is present in surface waters and soil.
• Different cold-blooded animals like frogs, snakes, turtles, lizards are infected by
• By the ingestion of contaminated food, humans become infected with it.
• Despite its less frequent recovery from human feces as compared to Aeromonas, Plesiomonas-induced gastroenteritis has been reported in children and adults.
• In humans, Plesiomonas-related gastroenteritis is manifested as mild watery diarrhea.
• In this condition, stools are free of blood and mucin.
• In the immunosuppressed patients or persons having GI malignancies, severe colitis or a cholera-like illness may be seen.
• The prevalence of this infection is more during the warm summer months and in the subtropical and tropical regions of the world.
• It produces enteropathogenic enterotoxin.
• It has caused illness after uncooked shellfish consumption.
• It is also a cause of travelers’ diarrhea.
• Extraintestinal infections have also been reported such as:
  • Septicemia
  • neonatal meningitis
  • cellulitis
  • septic arthritis
  • acute cholecystitis
• Other reported cases are:
  • Postsplenectomy infection
  • Plesiomonas shigelloides-associated persistent dysentery
  • pseudomembranous colitis

 Laboratory diagnosis of Plesiomonas shigelloides:

• P. shigelloides is a straight-to-rounded, short, Gram-negative bacteria.
• Motile
• Have lophotrichous flagella
• grows well on sheep blood agar and most enteric media.
• nonhemolytic on sheep blood agar
• Grows at the incubation of 24 hours at 30°C- 35°C.
• optimum  growth temperature is 30°C
• Colony characteristics of Plesiomonas shigelloides:
  • average 1.5 mm in diameter
  • gray
  • shiny
  • smooth
  • opaque
  • slightly raised in the center.

• Culture media for shigelloides:
  • MacConkey Agar
  • Deoxycholate Agar
  • Hektoen agar
  • xylose lysine deoxycholate Agar
• Glucose fermentation takes place and appears as yellow in:
  • Kligler’s iron agar
  • triple sugar iron agar
• Non-lactose fermenter in MacConkey agar and make confusion with Shigella spp
• oxidase-positive
• indole positive.
• decarboxylates arginine, lysine, and ornithine
• does not produce DNase or extracellular proteases
• ferments inositol but not mannitol

Biochemical Characteristics of Plesiomonas shigelloides:

Antibiotic Susceptibility of Plesiomonas shigelloides :

• Resistant to penicillin, ampicillin, carbenicillin, and other β-lactamase susceptible penicillins.
• susceptible to the aminoglycosides, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole and the quinolones, ciprofloxacin, and norfloxacin.

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• Kingella belongs to the family Neisseriaceae in the γ-subgroup of the
• Species:
  • K. denitrificans
  • K.oralis
  • K. potus.
  • K. kingae was formerly a member of the genus Moraxella.
• K. denitrificans was formerly called CDC group “TM-1.”
• Kingella oralis was recovered from human dental plaque of a patient with adult periodontitis.
• Kingella potus was isolated from the zookeeper’s wound. Three days earlier, he had sustained the bite of a kinkajou (Potus potus).

Clinical Significance of Kingella Species:

• K. kingae is being recognized as an important human pathogen though it’s part of the normal microbiota of the upper respiratory and genitourinary tracts.
• In pediatric patients, it has emerged as a significant pathogen.
• It has caused primarily bacteremia and skeletal infections in them.
• In infants after 6 months of age, K. kingae colonizes the upper respiratory tract.
• In between 6 months and 2 years of age, the rate of colonization increases 10% to 12%
• Then the colonization begins to decline after this time.
• In a study it was found, during the first 2 years of life, invasive K. kingae disease occurred most frequently in previously healthy children.
• Transmission can occur from child to child by the respiratory route.
• Organisms may get entrance into the bloodstream through breaches in the oropharyngeal mucosa.
• In children, it causes the bone and joint infections.
• These infections present as bacteremia, septic arthritis, osteomyelitis, discitis, tenosynovitis, and dactylitis.
• Systemic infections in infants presents:
  • low-grade fevers
  • viral upper respiratory tract infections
  • frequently stomatitis are present
• In most cases, K. kingae septic arthritis is an acute infection.
• From these patients, blood cultures are usually negative.
• K. kingae septic arthritis is usually monoarthritic.
• It involves joints being the knee, hip, and ankle.
• K. kingae also causes osteomyelitis affecting the femur, other long bones, the tibia, ulna, radius, and calcaneus bones.
• K. kinage also causes spondylitis and intervertebral discitis.
• It also causes pediatric osteoarticular infections of the lower sternum and the junction between the manubrium and the xyphoid process.
• Invasive K. kingae causes the following infection in children:
  • acute lymphocytic leukemia
  • congenital heart disease.
• Rarely, bacteremia and endocarditis are caused by K. kingae.
• In the patients with heart disease such as rheumatic heart disease, cardiac malformations, or those with cardiac prostheses, K. kingae endocarditis occurs.
• In adults and school-age children, K. kingae endocarditis occurs.
• Complications of kingae endocarditis:
  • Pericarditis
  • paravalvular abscess
  • pericardial abscess
  • embolic phenomena
  • mycotic aneurysms
  • cerebral and pulmonary infarcts
  • septic shock
  • congestive heart failure.
• In women with systemic lupus erythematosis, K. kingae endocarditis with meningitis has been reported.
• In the immunocompetent adults, K. kingae bacteremia without endocarditis has been reported.
• It causes dental manipulations.
• Additional complications of bacteremia and bone infections include:
  • Meningitis
  • hematogenous orbital cellulitis
  • Endophthalmitis
  • soft tissue infection
  • corneal abscess.
• The clinical presentation of K. kingae bacteremia may mimic systemic neisserial infections (i.e., meningococcemia or disseminated gonococcal infection).
• K. denitrificans has also been reported as a cause of septicemia and native/prosthetic valve endocarditis.
• K. denitrificans has also been isolated from:
  • empyema fluid of a patient with bronchogenic carcinoma
  • the bone marrow of a patient with AIDS
  • the amniotic fluid of a patient with chorioamnionitis
  • a corneal ulcer

Cultural Characteristics of Kingella species:

• Kingella species are Gram-negative bacilli or coccobacilli.
• They occur in pairs or short chains.
• They are oxidase-positive and catalase-negative.
• All species grow on chocolate and blood agar
• Do not grow on MacConkey agar or other enteric media.
• All Kingella are negative for:
  • arginine dihydrolase
  • lysine and ornithine decarboxylases
  • urease
  • esculin hydrolysis
  • ONPG hydrolysis

Biochemical characteristics for the identification of Kingella species are:

• NA means not available
• HEM SBA: Hemolysis on sheep blood agar
• NO₃RED: reduction of nitrate to nitrite
• NO₂RED: reduction of nitrite to nitrogen gas
• ODC: ornithine decarboxylase
• ONPG: o-nitrophenyl-β-D-galactopyranoside
• DNase: Deoxyribonuclease

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• Gardnerella vaginalis is known by various names such as Haemophilus vaginalis and Corynebacterium vaginale.
• It was first described in 1953.
• It was placed formally in the new genus Gardnerella in 1980.
• Ultrastructural studies show the cell wall is seen as Gram-positive but it has got a thinner peptidoglycan layer.
• The layer is thinner than that of the Corynebacterium, Lactobacillus, or Staphylococcus
• Its peptidoglycan content constitutes about 20% of the total cell weight. It is similar to Enterobacteriaceae, such as Escherichia coli ( contain about 23%).
• It may be seen as Gram-positive, Gram-negative, or Gram-variable depending upon the various strains of vaginalis.
• The compounds which are present in the Gram-negative cell wall lipopolysaccharides are not present in the cell wall extracts of vaginalis.
• Example: meso-DAP, 2-keto-3-deoxy-D-manno-2-octonoic acid, hydroxy fatty acids.
• The cell wall of vaginalis is distinct from the cell wall of Gram-positive ( Corynebacterium type ) because of the absence of meso-DAP, arabinogalactans, and mycolic acids.
• G. vaginalis is found to be closely related to the bifidobacteria which is shown during its molecular studies.

Clinical Significance of Gardnerella vaginalis:

• G. vaginalis is a member of the normal vaginal microbiota.
• It was found to have an association with Bacterial vaginosis (BV)
• As this clinical syndrome is not caused by a single organism it is termed BV.
• In the Gram-stained smears of the vaginal discharge, inflammatory cells (seen with both Candida and Trichomonas vaginal infections) are not observed.
• Clinical characters of bacterial vaginosis caused by Gardnerella veginalis:
  • Malodorous vaginal discharge
  • Significant overgrowth in a number of G. vaginalis.
  • Growth in obligate anaerobes like Prevotella bivia, Prevotella disiens, Mycoplasma species, Peptostreptococci, and Mobiluncus species
  • Concomitant decrease in the numbers of normal vaginal lactobacilli.
  • BV is a risk factor for preterm birth and can cause an impact on adverse pregnancy outcomes.
  • BV is also a risk factor for obstetrical infections and pelvic inflammatory disease.
  • Initially, G. vaginalis was hypothesized as the etiological agent of this condition. Later, it was found that the other organisms were also involved in it.
  • G. vaginalis was present in the women with BV.
  • But, the presence of G. vaginalis was found in greater than 50% of women without BV.
  • In about 14% to 70 % of healthy women, vaginalis can be found in healthy women without BV.
  • Bacterial vaginosis is also indicated by the presence of a large number of G. vaginalis in the vagina.
  • It was found that the presence of these bacteria at concentrations of ≥2 × 107cfu/mL of vaginal fluid, vaginal pH greater than 4.5, had 95% sensitivity and 99% specificity.
  • In the male urethra also G. vaginalis was found.
  • For the diagnosis of BV, isolation of vaginalis by routine culture is not recommended because it is itself part of the vaginal microbiota.
  • Different studies had suggested as sexual transmission may not occur but from the intestinal tract, it may have colonized the vagina endogenously.
  • G. vaginalis was isolated from semen specimens of 50% of the men.
  • The organism was found to adhere to the cell membrane and it could penetrate the cytoplasm of both vaginal and male urethral epithelial cells.
  • Recolonization (reinfection) of the female vagina might occur due to a male partner. Bacteria might colonize the male lower genital tract asymptomatically.
  • G. vaginalis has also been associated with the complications of pregnancy.

Gardnerella vaginalis has also been isolated from the infants born from those mothers, particularly during and after delivery like:

• intrauterine infections
• intraamniotic infections
• Chorioamnionitis
• postabortal pelvic inflammatory disease
• postpartum endometritis after cesarean delivery.

Systemic and localized G. vaginalis may occur with the complications such as:

• Amnionitis
• episiotomy wound infection
• bacteremias
• Meningitis
• Cellulitis
• Conjunctivitis
• osteomyelitis

G. vaginalis is also isolated from the oropharyngeal cultures, gastric aspirates, and tracheal suction specimens of neonates.

• It is passed from the birth canal which is heavily colonized.
• It has also been isolated from:
• Bartholin’s gland abscesses
• postcesarean section
• postsurgical wound infections
• abdominal surgeries
• hysterectomies
• episiotomies

Rarely Gardnerella vaginalis has also caused infections in males.

• Occasionally, infections rather than the genitourinary tract are also caused by G. vaginalis.
• Bacteremia with G. vaginalis in men has been reported following transurethral prostatectomy, urogenital surgical procedures, and in association with renal calculi, and urinary retention secondary to obstruction.
• In both men and women, G. vaginalis also play a role in urinary tract infections.
• G. vaginalis has been isolated more from the urinary tract infection of females than the male because of its presence in females as the normal vaginal microbiota.
• From the symptomatic and asymptomatic patients, G. vaginals has been isolated from both the upper and lower urinary tracts.
• Upper urinary tracts include the ureters, renal pelvis, and calyx.
• The lower urinary tracts include the bladder.
• In a study, its isolation rate was found to be more in pregnant women than the non-pregnant women.
• Many of these patients are asymptomatic too.

Diagnosis of Bacterial Vaginosis: 

• Diagnosis of BV usually involves a patient with a malodorous vaginal discharge.
• After sexual contact, there may be minimal irritation.
• exposure to alkaline pH of vaginal secretions after sexual contact and during menstruation may cause fishy odors.
• The fishy odor is due to the volatilization of amines.
• Production of the fishy odor is the basis of the whiff test.
• In this test, for the production of a fishy odor, KOH is mixed with the discharge.
• Vaginal discharge of BV:
  • Homogeneous
  • White-gray
  • Maybe frothy
  • Vaginal pH is greater than 4.6
• The specificity of diagnosis is being even greater when the vaginal pH is ≥5
• Moderate to many vaginal epithelial cells can be seen by wet mounts.
• The large number of adherent bacteria having various morphologies can also be seen ( clue cells)
• The margins of the epithelial cells may be completely covered by adherent microorganisms.
• For the diagnosis of BV, its absence or possible presence, it is evaluated by various criteria.
• The relative amount of lactobacilli is evaluated.
• The morphotypes of Gardnerella and the Mobiluncus are also studied for it.
• These bacterial morphotypes cover the vaginal epithelial “clue” cells.
• The study of Gram reactions and morphologies also aids this diagnosis process.

Cultural Characteristics of  Gardnerella vaginalis

• The culture of vaginal specimens for the isolation of G. vaginalis in the case of diagnosis of BV should be discouraged.
• Because BV is not caused by a single species of bacteria and its isolation can be done even from more than 50% of the asymptomatic women.
• Wet preparation or the Gram-stained smears of vaginal discharge should be examined carefully and interpreted.
• After the prolonged incubation, on the Columbia NaladixicAcid Agar (CNA) medium, G. vaginalis can be recovered.
• Isolation of G. vaginalis can be done on routine SBA, CNA, and chocolate agar from the clinical specimens.
• Semi-selective media include HBT agar or V agar.
• After 48 hours of incubation on HBT agar, vaginalis forms small, clear zones of β-hemolytic colonies surrounding colonies with diffuse edges.
• For “routine media”, vaginalis grows better on Columbia agar-based media (i.e., CNA agar) than on blood agar made with a tryptic soy base.
• On CNA, vaginalis shows a subtle, “diffuse” hemolysis surrounding the colonies.
• This is noted initially in confluent areas of growth or after incubation for >72 hours.
• Growth is best at 35°C to 37°C in a 5% to 7% CO2 atmosphere.
• After 48 to 72 hours, most of the isolates are recovered.
• vaginalis can grow in most blood culture media but the anticoagulant additive sodium polyanethol sulfonate (SPS) is inhibitory to some G. vaginalis.
• By the additional testing, the suspected vaginalis should be confirmed from the systemic body sites such as blood, joint fluid, etc.

Presumptive identification of Gardnerella vaginalis is done by:

• typical cellular morphology on Gram-stained smears (small gram-positive, gram-negative, or gram-variable coccobacilli)
• characteristic growth on CNA agar with “diffuse” weak β-hemolysis
• negative oxidase and catalase tests.

Definitive identification of G. vaginalis is done by biochemical test:

Biochemical Characteristics for the Identification of Gardnerella vaginalis:

Other characteristics for the confirmation of   Gardnerella vaginalis are:

• presence of α-glucosidase
• absence of β-glucosidase
• positive starch and hippurate hydrolysis reactions.
• Carbohydrate utilization tests are performed in medium containing Proteose Peptone no. 3, phenol red indicator, and 1% filter-sterilized carbohydrate.
• Production of acid from glucose, maltose, sucrose, and starch, but not from mannitol, sorbitol, raffinose, rhamnose, or salicin
• Hydrolysis of hippurate
• Absence of lysine or ornithine decarboxylases or arginine dihydrolase
• does not reduce nitrate
• does not produce indole, urease, or acetoin
• zones of inhibition around disks containing metronidazole (50 μg) and trimethoprim (5 μg)

Antimicrobial Susceptibility of  Gardnerella vaginalis:

• Susceptible to penicillin, ampicillin, erythromycin, clindamycin, trimethoprim, and vancomycin.
• Ciprofloxacin and imipenem show variable activity.
• Some strains may be resistant to tetracycline and minocycline.
• Most strains show marked resistance to amikacin, aztreonam, and sulfamethoxazole.

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 Classification:

• Order: Rickettsiales
• Tribe: Rickettsiae
• Family: Rickettsiaceae
• Genera: Rickettsia, Orientia, Ehrlichia

Introduction:

• Rickettsiae are obligate, intracellular, small Gram-negative bacilli.
• It multiplies within the cytoplasm of eukaryotic cells.
• Size: 0.3×1-2 µm.
• Genome: 1-1.5 million base pairs
• Rickettsiae are primary pathogens of arthropods like:
  • Lice
  • Fleas
  • Ticks
  • Mites
• Transmitted to humans by these arthropod vectors.
• Rickettsiae were originally thought to be a virus because:
  • Have small size
  • Stain poorly with Gram stain
  • Grows only in the cytoplasm of eukaryotic cells
  • Obligate intracellular parasites
• Rickettsiae are bacteria because:
  • Have Gram-negative cell wall
  • Contain both DNA and RNA
  • Contain enzymes for Kreb cycle
  • Contain ribosomes for protein synthesis
  • Susceptible to antibiotics

Morphology of Rickettsiae:

• They are small Gram-negative coccobacilli.
• Size: 0.3-0.6 to 0.8-2 µm.
• Non-motile
• Non-capsulated
• Stains poorly with Gram stain
• Stains well with these stains:
• Deep red with Machiavello and Gimenez stain
• Bluish purple with Giemsa and Castaneda stain

Culture characteristics of Rickettsiae:

• Rickettsiae do not grow in cell-free media.
• Most Rickettsia grows in the cytoplasm inside the cell.
• Rickettsia causing spotted fever grows in the nucleus of the cell.
• Cell lines:
  • HeLa, Hep2, Detriot-6, mouse fibroblasts, and other continuous cell lines.
• Chick embryo:
  • Grows in the yolk sac of 5-6 days old chick embryo.

Human Infections Caused by Rickettsia:

Antigenic Structure of Rickettsia:

1. Group-specific antigen:

• It is the soluble antigen.
• It is present on the surface of the organisms.
• From the repeated washings and centrifugation, it can be extracted.

2. Species- or strain-specific antigen:

• It is present in the cell wall of the bacteria.

3. Alkali-stable polysaccharide antigen:

• It is a surface antigen.
• It is present in some species of Rickettsia and some strains of Proteus (Proteus OX19, OX2 and OXK).
• This sharing of antigen form the basis of the Weil-Felix test.

Pathogenesis of Rickettsia:

• Rickettsia has the capacity of multiplication inside the cell.
• The important virulent factor is adhesion.
• Adhesins are the outer membrane protein that facilitates the entry of the organism into the host cells.
• When they enter the cell, multiplication occurs and accumulates in large numbers.
• It then lyses the host cells.
• Rickettsia can cause rickettsemia when it multiplies after reaching the circulation.
• In the endothelial cells of small arterial capillary and venous vessels, Rickettsia is localized.
• Then the endothelial cellular hyperplasia occurs at those sites.
• It results in multiorgan vasculitis.
• It may lead to the thrombosis and development of small nodules.
• Gangrene may result in the extremities, ear lobes, nose, and genitalia. It is due to the thrombosis of supplying blood vessels.
• Vasculitis may lead to:
  • Increased vascular permeability with consequent edema
  • Loss of blood volume
  • Hypoalbuminemia
  • Reduced osmotic pressure
  • Hypotension

Typhus Fever Group:

• Epidemic or louse-borne typhus caused by Rickettsia prowazekii
• Relapsing louse-borne typhus or Brill-Zinsser disease caused by prowazekii.
• Endemic or flea-borne murine typhus caused by Rickettsia typhi.

1. Epidemic or Louse-borne Typhus:

• It is caused by Rickettsia prowazekii.
• It is transmitted by the human body louse Pediculus humanus corporis causing the acute febrile illness.
• It is named after the scientist Von Prowazek. He died of typhus fever while studying this disease.
• prowazekii is an invasive bacterium.
• It leads to vasculitis by multiplying in the endothelial cells of blood vessels.
• The average incubation period is 8 days whereas it may vary low as 2-3 days.
• Characteristics of epidemic typhus:
  • High fever
  • Severe headache
  • Chills
• On the 4^th or 5^th day, the petechial or macular rash appears
• The rashes first start to appear on the trunk.
• Without affecting the face, palms, and sole it then spreads to the extremities.
• In nearly, 40 % of patients rashes are seen.
• The patient may become stuporous and delirious if they are left untreated.
• In the disease process, the cloudy state of consciousness appears. The name typhus is derived from it. The meaning of tyhus is cloud or smoke.
• Complications:
  • Myocarditis
  • Central nervous system (CNS) dysfunction
  • Mortality rate is as high as 60% in old or immunocompromised persons.

2. Relapsing or Recrudescent Typhus:

• Example of a recrudescent case of typhus fever is Brill-Zinsser disease.
• This condition was seen in the patients who were cured of the disease or the patients who were treated with antibiotics.
• Even after the antibiotic treatment, the recurrence of typhus fever has re-emerged after many months, years and decades.
• It is because of the persistence of prowazekii in the body tissues which re-emerges later.
• Primary reservoir of epidemic typhus: Human
• If a person is suffering from typhus fever from brill-Zinsser disease, and when lice feed on it, it will be infected with prowazekii.
• Vector of epidemic typhus: Body louse ( humanus corporis )
• Pubic louse does not transmit it.
• Occasionally transmission may occur by head louse (humanus capitis )
• In the alimentary tract of louse prowazekii, lives and multiplies.
• Within the 3-5 days of infection, the bacteria gets excreted in feces.
• After the infection, lice die.
• During the blood meal when the rickettsia-harboring louse bites the human, infection is transmitted.
• During the feeding, lice defecate.
• The contaminated louse feces gets inoculated into the minute lesion of the bite wound when the host scratches on it.
• From there the bacteria reach the circulation, multiplies and cause rickettsemia.
• Infection may also be transmitted rarely through the conjunctiva or inhalation of aerosols of dry louse feces.

3. Endemic or Flea-borne Murine Typhus:

• It is caused by typhi.
• It has a short duration and the disease is milder than epidemic typhus.
• Incubation period: 7 to 14 days.
• Symptoms:
  • Fever
  • Headache
  • Malaise
  • Myalgia
• In about 50% of infected patients, rash develops on the 3^rd to 5^th day of infection.
• Rash appears on the chest and abdomen.
• It may spread to palms and soles.
• May last up to 3 weeks in the untreated course.
• Reservoirs: Rat ( Rattus rattus ), mice, and cat
• Humans are the accidental hosts.
• Vectors for transmission of disease: Rat flea (Xenopsylla cheopis ) or cat flea (Ctenocephalides felis).
• It is transmitted from rats to rat by the rat flea.
• It is transmitted to humans accidentally by the feces of infected fleas.
• When the fleas feed on the mice, cat, or natural host, it becomes infected.
• It then transmits the disease to humans by biting.
• At the site of the bite, inoculation occurs.
• Disease transmission also may occur by:
  • Cat flea felis
  • Inoculation or inhalation of aerosolized infectious specimens
  • Ingestion
  • Contaminated food with infected rat urine or flea feces

Spotted Fever Group:

1. Rocky Mountain spotted fever caused by Rickettsia rickettsiae.
2. Rickettsialpox caused by R. akari

• Boutonneuse fever caused by R. conori
• Kenyatick-bite fever,
• African tick typhus
• The Mediterranean spotted fever
• Indian tick typhus
• Marseilles fever

1. Rocky Mountain Spotted Fever:

• Incubation period: 7 days
• Symptoms:
  • Fever
  • Severe headache
  • Chills
  • Myalgia
  • Development of rash three or more days
  • At first, a rash develops on the wrist, ankles, palms, and soles.
  • It then spreads to the trunk.
  • In the early stage, the rash is maculopapular but in the later stage, it becomes petechial and hemorrhagic.
• Complications:
  • Respiratory failure
  • Encephalitis
  • Renal failure
  • Patient may die within 5 days of onset of symptoms.
• Ticks are the host, reservoir, and vector of rickettsiae.
• Vectors:
  • Woodtick (Dermacentor andersoni )
  • American dog tick (Dermacentor variabilis)
  • Lone star tick (Amblyomma Americana ).
  • When the tick bites the human, it gets transmitted by saliva.

2. Rickettsial Pox:

• It is caused by akari.
• It is a milder form of infection.
• Natural reservoir: Common home mouse ( Mus musculus )
• By the bite of mouse mite (Liponyssoides sanguineus ), R. akari Iis transmitted from mouse to mouse.
• Incubation period: 7 days
• Papule develops at the site of the bite which progresses to ulcer and leads to eschar formation.
• In 3-10 days fever, headache, malaise, and myalgia develop.
• After the emergence of fever, a popular vesicular rash appears un 3-4 days.
• Recovery starts after the illness of 10-14 days.
• Without treatment complete healing of rash takes 2-3 weeks.

What are the conditions/ characteristics that differentiate Rickettsial pox from other rickettsial infections?

• Presence of eschar at the site of the bite
• Presence of vesicular pustular eruption

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• Corynebacteria are classified as Actinobacteria and are gram positive in nature.
• They are rod shaped bacteria that survives in aerobic environment and are non-motile.
• Phylogenetically, Corynebacteria are related to mycobacteria and actinomycetes.
• A diverse community of bacteria, including animal and plant pathogens, as well as saprophytes, make up the genus Corynebacterium.
• Some of the corynebacteria are part of the normal human flora, particularly the skin and nares, that tends to find an appropriate niche in almost every anatomical location.
• Corynebacterium diphtheriae, the causal agent of the disease diphtheria, is the best known and most widely studied species.
• The diphtheria bacillus is also termed as Klebs-Loffler bacillus as it was first observed and explained by Klebs in 1883 and was first cultivated by Loffler in 1884.
• The pathogenecity of C. diphtheriae is due to the development of a potent exotoxin active in a number of tissues, including the muscles of the heart and peripheral nerves.

Morphology of the Corynebacterium diphtheriae:

• They are thin, slender gram-positive bacilli, but, especially in old cultures, they are easily decolorized.
• They measure about 3-6 μm × 0.6-0.8 μm.
• They have a remarkable ability to clubbing at one or both ends.
• They are largely pleomorphic.
• Cells often exhibit septa, and branching is seldom observed.
• They are nonmotile, nonspore forming, and nonacid fast.
• The bacilli are arranged in smears in a characteristic fashion.
• They are typically seen in pairs, palisades (resembling stakes of a fence) or small groups or as individual cells lying at sharp angles to another, resembling the letters V or L.
• This unique arrangement with C. diphtheriae has been termed the Chinese letter or cuneiform arrangement.
• This is because of the incomplete separation of the daughter cells after binary fission.
• This organism has granular and irregular staining.
• The granules in the cell are metachromatically reddish-purple when stained with methylene blue or toluidine blue.
• These granules are termed as metachromatic granules, volutin granules or Babes-Ernst granules.
• They are often located at the poles of the bacilli and are termed as polar bodies.
• To clearly show the granules, unique stains such as Albert’s Neisser’s and Ponder’s have been devised.
• The granules are stained bluish black and the protoplasm green with Albert’s stain.
• The granules indicate the accumulation of polymerized polyphosphates.
• The development of granules is best seen on the Loffler’s serum slope.
•  They seem to represent storage depots for materials required to form high-energy phosphate bonds.
• Three strains of Corynebacterium diphtheriae are known, gravis, intermedius and mitis.
• They are listed here as the severity of the disease they produce in humans in decreasing order.
• The same toxin is produced by all strains and is capable of colonizing the throat.
• The variations in virulence between the three strains can be understood by their diverse abilities to produce the toxin in rate and quantity, and by their varying growth rates.
• The gravis strain has 60 minutes of generation time (in vitro); the intermedius strain has about 100 minutes of generation time; and the mitis stain has about 180 minutes of generation time.
• Usually, the faster growing strains generate a larger colony on most growth media.
• A faster growth rate in the throat (in vivo) may enable the organism to deplete the local supply of iron more rapidly in the invaded tissues, allowing for earlier or greater development of diphtheria toxin.
• Also if bacterial growth kinetics are accompanied by the kinetics of toxin development, the faster growing variety will reach an efficient toxin level before the slow growing varieties.

Cultural Characteristics of Corynebacterium diphtheriae :

• C. diphtheriae is an aerobe and facultative anaerobe.
•  The optimum temperature for growth is 37°C and optimum pH is 7.2.
• For primary isolation and characterization, complex media are required.
• Growth can occur on ordinary nutrient agar, however, its growth is enhanced by the presence of animal proteins like blood or serum.
• Two media are essential for this purpose:
  • Löffler’s serum slope.
  • Blood agar having  fresh, lysed or heated blood.

Löffler’s Serum Slope :

• On Löffler’s serum slope, diphtheria bacilli develop very quickly and colonies can be seen in 6-8 hours, long before other bacteria grow.
• Colonies are tiny circular white opaque disks at first but on continuous incubation they expand and may develop a distinct yellow tint.
• The medium of Löffler is also helpful because it does not support the growth of streptococci and pneumococci that may be present in the clinical sample and limit the activity of most oral commensals and retards the growth of others, behaving as a selective agent but having little effect on diphtheria bacilli, such as Candida albicans and Staphylococcus aureus.

Tellurite Blood Agar

• The introduction of potassium tellurite(0.03-0.04%) helps to make the medium selective for corynebacteria by suppressing most other pathogenic and commensal bacteria.
• C. diphtheriae exhibit gray/black, shiny or dull black colonies.
• This is because the tellurite ion passes through the cell wall and membrane into the cytoplasm, and here, it is reduced to the metal tellurium and is precipitated inside the cells.
• The tellurite salts are reduced by any other corynebacterial, yeasts and staphylococci producing distinctive gray to black colonies.
• The addition of cystine to a medium containing tellurite (Tinsdale’s medium) has significantly aided to isolate diphtheria bacilli.
• The growth of diphtheria bacilli on the tellurite medium may be delayed and it may take two days for colonies to appear.

Biochemical test for Corynebacterium diphtheriae

• C. diphtheriae degrades glucose and maltose along with the production of acid (but no gas).
• However, it cannot ferment mannitol, lactose, trehalose or sucrose.
• C. diphtheriae is H₂S positive and reduces nitrate to nitrite.
• It does not liquefy gelatin nor hydrolyze urea.
•  It does not form phosphatase as well.

Pyrazinamidase (PYZ) Test:

• In pyrazinamidase (PYZ) test, the organisms that are capable of producing pyrazinamidase (PYZ) converts pyrazinamide into pyrazinoic acid.
• This test is esssential to differentiate C. diphtheriae (PYZ-negative) from other corynebacterium species (mostly PYZ-positive).
• Exception are C. ulcerans and C. pseudotuberculosis which are also PYZ-negative but they are urease test positive which differentiate them from C. diphtheriae (urease negative).

Diphtheria Toxin

• A very powerful exotoxin is produced by toxigenic strains of C. diphtheriae
• The toxicity found in diphtheria is specifically related to the toxin secreted by the bacteria at the site of infection.

Lysogeny and Toxin Production:

• Corynephages (tox⁺) acts as the genetic determinant that controls the toxin production.
• The toxigenicity of the diptheria bacillus relies on the presence of corynephages (tox⁺).
• By infection with the related bacteriophage, non-toxigenic strains can be transformed to tox+.
• This is called as lysogenic conversion or phage conversion.          
• The bacillus loses its toxigenicity when the bacillus is cured of its phage, as by developing it in the presence of antiphage serum.                                                                                                                                           

Iron for Toxin Production

• The production of toxins is also affected by the iron concentration in the medium.
• For toxin production, the optimal level of iron is 0.1 mg per liter, while a concentration of 0.5 mg per liter inhibits toxin formation.
• The toxin is released in massive quantities only when the obtainable iron in the culture medium is depleted.

Properties of Diphtheria Toxin:

• Diphtheria toxin is a crystalline, heat-labile protein that is iron-free.
• It is highly potent and is fatal for humans in amounts of 130 ng per kg of body weight.
• The lethal dose of diphtheria toxin is 0.1 μg/kg or less in highly susceptible animals (guinea pig, rabbit).
• The diphtheria toxin has a molecular weight of around 62,000 and is a protein.
• Fragment A seems to have all the enzymatic activity where-as fragment B is accountable for binding the toxin to the cells.
• This toxin is thermo-labile.
• It is highly potent.
• In eukaryotic cells, the toxicity of the toxin is due to its capacity to inhibit protein synthesis.
• Two fragments A (active) and B (binding) of MW 24,000 and 38,000, respectively, constitute the toxin.
• For the toxic impact, both fragments are required.

Mode of Action of Diphtheria toxin :

• The bacterial cell secretes the toxin and is non-toxic unless exposed to trypsin.
• Two polypeptide fragments, A and B, which are connected together by a disulfide bridge, result from trypsinization.
• Fragment A is responsible for cytotoxicity; fragment B binds to eukaryotic cell receptors and mediates the cytoplasm entry of fragment A.
• By resisting the binding of the toxin to the cells, the antibody to fragment B is defensive.
• By inhibiting protein synthesis, the diphtheria toxin works.
• Primarily, fragment A separates nicotinamide adenosine dinucleotide (NAD) to form nicotinamide and adenosine diphosphoribose (ADPR).  
• ADPR binds to and deactivate elongation factor 2 (EF-2), an enzyme that is vital for the elongation of ribosome polypeptide chains.
• Protein synthesis inhibition is possibly responsible for both the toxin’s necrotic and neurotoxic impact.
• We may summarize the reaction as follows:
• NAD+ + EF-2 (active) = ADPR-EF-2 (inactive) + nicotinamide + H+

Resistance in Corynebacterium diphtheriae:

• C. Diphtheriae is more resistant than most non-spore forming bacilli to light, desiccation, and freezing action.
• Organisms live for at least 14 weeks on dried fragments of pseudomembranes.
• Nevertheless, they are killed by a 1-minute exposure to 100 ° C or a 10-minute exposure to 58^oC.
• They are sensitive to most of the regularly used disinfectants.
• In 0.85 percent NaCl solution, it dies rapidly, but stays alive in dust and fomites for weeks when dry and shielded from sunlight.
• It is prone to penicillin, erythromycin and broad spectrum antibiotics.

Antigenic Structure of Corynebacterium diphtheriae :

• Antigenically, diphtheria bacilli are heterogeneous.
• They carry three distinct antigens:
• A deep-seated antigen found in all corynebacterial species as well as in Mycobacterium tuberculosis
• A heat-labile protein (K antigen).
• A heat-stable polysaccharide (O antigen).

Pathogenesis of Corynebacterium diphtheriae:

• Diphtheria bacilli creates an inflammatory exudate in the upper respiratory tract and causes necrosis of the faucial mucosal cells.
• By destroying epithelial cells or neutrophils, the diphtheria toxin may help colonization of the throat or skin.
• Diphtheria is a toxemia.
• Organisms do not penetrate deep into the mucosal tissue and there is generally no bacteremia.
• Exotoxin is locally produced and distributed to distant organs via the bloodstream, with a particular affinity for the heart muscle, the peripheral nervous system, and the adrenal glands.
• The pathogenesis process can be summarized in two main steps:
• Invasion:
  • Colonization and subsequent bacterial proliferation leads to the invasion of the local tissues.
  • On the adherence mechanisms of C. diphtheriae, little is understood, however, many forms of pili are produced by it.
  • The toxin of diphtheria, as well may be involved in throat colonization.
• Toxigenesis:
  • The development of a toxin by bacteria is termed as toxigenesis.
  • By inhibiting protein synthesis in the cells, the diphtheria toxin triggers the death of eucaryotic cells and tissues.
  • Although the toxin is responsible for the lethal symptoms of the disease, toxigenicity alone cannot be correlated with the virulence of C diphtheriae, because toxigenesis is apparently followed by a distinct invasive process.
  • Due to short-range effects at the colonization site, the diphtheria toxin plays an important role in the colonization process.

Diseases caused by Corynebacterium diphtheriae :

• The main disease caused by C. diphtheriae is diphtheria. The Greek meaning of diphtheria refers to the leathery skin referring to the pseudo-membrane that is formed on the pharynx initially.
• Occasionally, this microorganism is responsible for wound and chronic skin infections.
• Throughout the world, the non-toxicogenic strains are linked with the endocarditis, cerebral abscess, meningitis and osteoarthritis.

i. Diphtheria:

• As described by CDC, diphtheria is a disease of the upper respiratory tract marked by sore throat, low fever, and an adherent membrane (called a pseudo-membrane on the tonsils, pharynx, and/or nasal cavity).
• Myocarditis, polyneuritis, and other systemic toxic effects may be caused by diphtheria toxin developed by C. diphtheriae.
• A relatively milder form of diphtheria can be limited to the skin.
• Diphtheria is an infectious disease spread either by direct physical contact or inhaling the aerosolized secretions of infected individuals.
• Diphtheria which was once much prevalent has largely been eliminated in developed countries by the use of the DPT vaccine.
•  Diphtheria is a quickly evolving, acute febrile infection which includes both local and systemic pathology.
• In the upper respiratory tract, a local lesion occurs and involves necrotic epithelial cell damage.
• Blood plasma spills into the area as a result of this injury and forms a fibrin network that is interlaced with fast-growing C. diphtheriae cells.
• This membranous network, referred to as a pseudo-membrane, covers the site of the local lesion, causing respiratory distress, including suffocation.
• The diphtheria bacilli do not appear to conquer tissues below or away from the surface epithelial cells at the zone of the local lesion.
• Though at this site they generate the toxin that is absorbed and circulated through lymph channels and blood to the prone tissues of the body.
• The systemic pathology of the disease results from degenerative alterations in these tissues, which consists of the heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen.

ii. Systemic Effects:

• The toxin is also absorbed and may cause a number of systemic effects affecting the kidneys, heart, and nervous system, as all tissues have the receptor of toxin and will be affected.
• Intoxication takes the form of peripheral neuritis and myocarditis and can be related to thrombocytopenia.
• There is also visual disturbance, trouble swallowing and paralysis of the arms and legs, but it usually recovers spontaneously.
• Myocarditis can result in complete heart block.
• Death is mainly caused by congestive heart failure and cardiac arrhythmias.

iii. Complications:

• The prominent complications are:
• The pseudomembrane causes the mechanical obstruction of the respiratory passage that results in asphyxia for which an emergency tracheostomy might be essential.
• Acute circulatory failure, which can be either cardiac or peripheral.
• Postdiphtheritic paralysis, that usually occurs in the third or fourth week of the disease; palatine and ciliary but not pupillary paralysis is indicative, and spontaneous recovery is the rule.
• Septic conditions such as pneumonia and otitis media arises.

iv. Cutaneous Diphtheria:

• In case of the cutaneous diphtheria that is present in the tropics, the toxin is also absorbed systematically but systemic complications are less likely as compared to the upper respiratory infections with C. diphtheriae.

Laboratory Diagnosis of Corynebacterium diphtheriae:

• The clinical experience is supported by diagnostic laboratory tests and is of epidemiological importance, but not for the treatment of specific cases.
• Without waiting for laboratory testing, specific care should be instituted immediately on suspicion of diphtheria.
• Any postponement can be fatal.
• Laboratory diagnosis consists of diphtheria bacillus isolation and evidence of its toxicity.

1. Specimens

• Before antimicrobial drugs are given, swabs must be taken from the nose, throat or other suspected lesions.
• In suspected cases of facial or nasal diphtheria, swabs should be taken from both the throat and the nose and, usually, two swabs should be taken from the most affected area.
• Swabs should also be taken from skin lesions and wounds where there is a possible diphtheria infection, and swabs should be taken from suspected carriers from both the throat and nose.

2. Microscopy:

• As C. diphtheriae is morphologically similar to other coryneforms, direct microscopy of a smear is inaccurate.
• Smears stained with alkaline methylene blue or Gram stain exhibit beaded rods in typical arrangement.
• Smear examination alone is therefore not sufficient for the diagnosis of diphtheria, but it is essential for the identification of Vincent’s angina.
• For Vincent’s spirochetes and fusiform bacilli, a Gram or Leishman stained smear is studied.
• Toxigenic diphtheria bacilli may be detected in smears by immunofluorescence.

3. Culture

• On the Löffler’s serum slope, tellurite blood agar, and blood agar, the swab should be inoculated.
• At 37^oC, the cultures should be incubated aerobically.
• It should be kept moistened with sterile horse serum unless the swab can be inoculated promptly, so that the bacilli can remain viable.
• i. Löffler’s Serum Slope
  • A growth smear from all parts of the slope mixed in the condensation water is made and after incubation for 6 hours or overnight, it is stained with the Albert-Laybourn method and  the presence of slender green-stained bacilli containing the purple-black granules characteristic of C. diphtheriae is looked.
  • The Löffler slant can yield organisms with typical “diphtheria-like” morphology in 12 to 18 hours.
• ii. Tellurite Blood Agar
  • Since growth may sometimes be delayed, blood tellurite agar is examined after 24 hours and after 48 hours.
• iii. Blood Agar
  • It is used for distinguishing streptococcal or staphylococcal pharyngitis, that may simulate diphtheria.

4. Identification Tests:

• Identification is based on fermentation reactions of carbohydrates and enzymatic activities.
• diphtheriae ferments glucose and maltose, producing acid but not gas, and is catalase positive.
• Nitrate is reduced to nitrite and is non-motile.
• Reliable identification is provided by commercial kits such as the API Coryne strip.

5. Virulence Tests:

• Before the bacteriologic diagnosis of diphtheria is definite, any diphtheria-like organism cultured must be sub-mitted to a’ virulence’ test.
• These tests are actual toxigenicity tests for an isolated diphtheria-like organism.
• Diphtheria diagnosis depends on demonstrating that diphtheria toxin is produced by the isolate.
• In vivo or in vitro methods may be used for virulence testing.
• It is rare to perform in vivo testing because the in vitro methods are reliable, less costly and free of the need to use animals.
• A. In Vivo Tests
  • Subcutaneous test.
  • Intracutaneous test.
• B. In Vitro Test
  • Precipitation test.
  • Tissue culture test.
  • Enzyme-linked immunosorbent assays.
  • Polymerase chain reaction (PCR).

Epidemiology of Corynebacterium diphtheriae:

• Diphtheria is a disease that is widely distributed, particularly in poor urban areas where crowding occurs and vaccine-induced immunity has a low protective level.
• C. diphtheriae is preserved in the population as the asymptomatic carriage occurs in the oropharynx or on the skin of immune people.
• The infection is limited to humans and usually requires contact with a case of diphtheria or a carrier.
• Humans are the only recognized reservoir, with oropharynx or skin surface carriage.
• Diphtheria is predominantly a pediatric disorder, but in areas where there are successful immunization services for infants, the highest prevalence has moved towards older age groups.
• Acquired diphtheria immunity is mainly due to the toxin-neutralizing antibody (antitoxin).
• Passive immunity in utero is gained transplacentally and can last for 1 or 2 years after birth.
• Skin infection with toxigenic C. diphtheriae (cutaneous diphtheria) also occurs.

Prophylaxis for Diphtheria:

• Active, passive or mixed are the immunization methods available.
• Of these only active immunization will provide herd immunity and result to elimination of the disease.
• Passive and combined immunization can provide emergency protection only to susceptible risk-exposed individuals.

Active Immunization for Corynebacterium diphtheriae:

• For active immunization , following preparations are used:
  • Toxin-antitoxin mixture: It is not without hazards.
  • Single vaccines: It is less frequently used.
  • Combined preparations.
• Combined Preparations:
  • DPT (diphtheria­pertussis­tetanus) vaccine  
  • DT (diphtheria­tetanus, adult type)• DT (diphtheria­tetanus toxoid)
• DPT Vaccine
  • Diphtheria toxoid is typically administered as a tri-valent preparation containing tetanus toxoid in children and pertussis vaccine is also known as the DTP, DPT or triple vaccine.
  • In immunization systems, the WHO advises that only adjuvant DPT preparations be used.
  •  Primary immunization schedule: The primary immunization schedule for babies and children consists of DPT administered at 6 weeks, 10 weeks, 14 weeks and 16/24 months of age, accompanied by a booster dose of DT at 5-6 years of age.
  • Reactions: Fever and mild local reactions following DPT immunization are prevalent.
  • Neurological (encephalitis/encephalopathy, excessive convulsions, infantile spasms and Reye’s syndrome) are the most serious complications following DPT immunization.
• Passive Immunization
  • This is an emergency method to be used, such as when a case of diphtheria is admitted to general pediatric wards, where vulnerable (non-immunized) are exposed to infection.
  • It consists of 500-1000 units of antitoxin (antidiphtheritic serum,) subcutaneous administration.
  • Precautions against hypersensitivity should be observed, as this is a horse serum.
• Combined Immunization
  • This consists of the administration of the first dose of the adsorbed toxoid, while the ADS is administered to the other arm, such that the complete course of active immunization continues, provided that the protection granted by passive immunization is short-lived.
  • Ideally, combined immunization should be provided to all cases that receive ADS prophylactically.

Treatment for Corynebacterium diphtheriae:

• Specific diphtheria treatment consists of antitoxic therapy and antibiotic therapy.
• As soon as clinical diagnosis is made, antitoxin should be given immediately to neutralize the produced toxin, since antitoxin is ineffective if given after the toxin is bound to cell receptor sites.
• The dosage suggested is 20,000 units intramuscularly for moderate cases and 50,000 to 100,000 units for severe cases, half the dose being given intravenously.
• Diphtheriae is susceptible to most antibiotics, including penicillin and erythromycin, and is used in both carriers and patients.
• The circulating toxin is not neutralized by antibiotics.
• By killing diphtheria bacilli, they stop further toxin production.
• Erythromycin can be given to penicillin-sensitive individuals.
• In the treatment of carriers, Erythromycin is more active than penicillin.

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• Bordetella pertussis (Bordet-Gengou Bacillus; formally known as Hemophilus pertussis)

Morphology of Bordetella pertussis :

• The Bordetella spp are small, gram-negative coccobacilli with slight pleomorphism measuring 0.2-0.3 μm by 0.5 to 1.0 μm.
• They appear singly, in pairs, and in small clusters.
• On primary isolation, cells are uniform in size, but in subcultures they become quite pleomorphic and filamentous, and thick bacillary forms are common.
• It is nonmotile and non-sporing.
• Bipolar metachromatic staining may be demonstrated with toluidine blue.
• Capsules may be demonstrable in young, freshly isolated cultures only by special stains.
• In culture films, the bacilli tend to be arranged in loose clumps, with clear spaces in between giving a ‘thumb print’ appearance.
• Freshly isolated strains of B. pertussis have fimbriae.

Cultural Characteristics of Bordetella pertussis:

• It is an obligate aerobe.
• The optimum temperature for growth is 35-36°C.
• It does not require X and V factors for its growth.
• Complex media are necessary for primary isolation.
• The medium in common use is the Bordet-Gengou medium (potato-blood-glycerol agar).
• Primary isolation of B. pertussis requires the addition of charcoal, ion exchange resins, or 15-20 percent blood to neutralize growth-inhibiting effects.
• Potatoes impart a high starch content to the medium that neutralizes toxic materials.
• Glycerol acts as a stabilizing agent. Charcoal blood agar is a useful medium.
• The plates are incubated at 35-36°C in a moist environment (e.g., a sealed plastic bag).
• After incubation for 48-72 hours, colonies on Bordet-Gengou medium are small, dome shaped, smooth, opaque, viscid, greyish white, refractile and glistening, resembling ‘bisected pearls’ or ‘mercury drops’.
• Colonies are surrounded by a hazy zone of hemolysis. Confluent growth presents an ‘aluminium paint’ appearance.
• Subcultures of B. pertus­sis may be obtained on less exacting media, e.g. nutrient agar to which charcoal or starch has been added.

Biochemical Reactions of Bordetella pertussis:

• It is biochemically inactive.
• It does not ferment sugars, form indole, reduce nitrates, utilize citrate or split urea.
• It produces oxidase and usually catalase also.

Antigenic Constituents and Virulence Factors of Bordetella pertussis:

• B. pertussis is a delicate organism.
• It can be killed by heating at 55°C for 30 minutes, drying and disinfectants.
• Outside the body it can survive for five days on glass, three days on cloth and a few hours on paper.
• The organism is usually sensitive to ampicillin and erythromycin and these drugs have a reasonable therapeutic record.
• B. pertussis produces a number of factors that are involved in the pathogenesis of disease.
• 1. Agglutinogens
  • Bordetellae possess genus specific and species specific surface 14 agglutinogens associated with the capsular K antigens or fimbriae.
  • Factors 1 to 6 are found only in strains of B. pertussis, all of which carry Factor I and one or more of the other factors.
  • All three mammalian species of bordetellae has common Factor 7.
  • Factor 12 is specific’ for B. bronchiseptica and Factor 14 for B. parapertussis.
  • Agglutinogens promote virulence by help- ing bacteria to attach to respiratory epithelial cells.
  • They are useful in serotyping strains and in epidemiological studies.
  • On the basis of the agglutinogens Bordetellae carry they are classified into various types.
• 2. Pertussis toxin (PT)
  • PT, also known as lymphocytosis-promoting factor, pertussigen, histamine-sensitizing factor and islet-activating factor, has a wide spectrum of biologic activity.
  • Pertussis toxin promotes lymphocytosis, sensitization to histamine, and enhanced insulin secretion.
  • PT is expressed on the surface of the bacillus and secreted into the surrounding medium.
  • PT has a molecular weight of 117,000 and is a classic A-B toxin(dissociated into A and B subunits) made up of 6 polypeptide chains consisting of a A subunit, toxic subunit (SI) and five binding subunits (S2 to S5; two S4 subunits are present in each toxin molecule).
  • B unit consists of the remaining 5 polypeptide chains binds the toxin to the target cells and helps A unit to cross the membrane.
  • It can be toxoided. Pertussis toxoid is the major component of acellular pertussis vaccines.
  • It is apparently responsible for many of the clinical signs and symptoms of pertussis, as well as the relative and absolute lymphocytosis observed during the clinical illness.
  • Antibody against PT is also protective in animal models of pertussis. It can be toxoided.
  • The major component of acellular pertussis vaccines is PT toxoid.
  • Antibody to PT can protect mice against intranasal, intra- peritoneal or intracerebral challenge.
  • Outside the B. pertussis cells, the filamentous hemagglutinin and pertussis toxin are found which are secreted proteins .
• 3. Filamentous Hemagglutinin (FHA)
  • The filamentous hemagglutinin and pertussis toxin are secreted proteins and are found outside of the B. pertus­sis cells.
  • It is a protein that acquired its name through its ability to agglutinate erythrocytes.
  • It mediates adhesion to ciliated epithelial cells.
  • FHA is used in acellular pertussis vaccines along with PT toxoid.
  • Antibodies against filamentous hemagglutinin are protective.
  • FHA and PT hemagglutinins also promote secondary infection by coating other bacteria such as Haemo­philus influenzae and or Streptococcus pneumoniae and assisting their binding to respiratory epithelium besides facilitating adhesion of B. pertussis to respiratory epithelium.
  • This potential “piracy of adhesins” by other organisms may contribute to secondary bacterial invasion in pertussis .
• 4. Adenylate Cyclase (AC)
  • All mammalian Bordetellae but not B. avium produce adenylate cyclase .
  • At least two types of AC are known, only one of which has the ability to enter target cells and act as a toxin.
  • This is known as AC toxin (ACT).
  • It has the ability to enter target cells (leukocytes) and act as a toxin. It can be activated by eukaryotic calcium-dependent regulatory protein, calmodulin.
  • Inside the cell, after activation by calmodulin, this enzyme synthesizes cAMP(as pertussis toxin does) which is responsible for the biological effects such as interfering with leukocyte functions (inhibition of phagocytosis and chemotaxis).
• 5. Heat Labile Toxin (HLT) or Dermonecrotic Toxin
  • The heat–labile toxin (HLT) produced by all species of Bordetella appears to be a cytoplasmic protein it is a heat- labile toxin it is dermonecrotic and at high doses, this toxin causes fatal reactions in mice.
  • Role in disease is unknown.
• 6. Tracheal Cytotoxin (TCT)
  • Tracheal cytotoxin is a low-molecular-weight cell wall peptidoglycan monomer that has a specific affinity for ciliated epithelial cells.
  • It induces ciliary damage in hamster tracheal ring cultures and inhibition of DNA synthesis in the ciliated respiratory epithelial cells, resulting in accumulation in the lungs of mucus, bacteria and inflammatory debris leading to severe cough.
  • The disruption of ciliary function may also contribute to the secondary bacterial infections.
  • The toxin also stimulates the release of the cytokine interleukin-l, which leads to fever.
• 7. Lipopolysaccharide (Heat-Stable Toxin)
  • It is present in all bordetellae and exhibits features of gram-negative bacterial endotoxins.
  • Their role in the disease process is unknown.

Pathogenesis of Bordetella pertussis:

• Whooping cough is predominantly a pediatric disease.
• Whooping cough in 95 percent of cases is caused by B. pertussis.
• B. parapertussis causes about 5 percent of the cases and by B. bronchiseptica very infrequently (0.1%).

Stages of Whopping cough Disease:

• In human beings, after an incubation period of about 1-2 weeks, the disease takes a protracted course comprising three stages-the catarrhal, paroxysmal and convalescent -each lasting approximately two weeks.
• 1. Prodromal or Catarrhal Stage
  • The first stage, the catarrhal stage, resembles a com- mon cold, with serous rhinorrhea, sneezing, malaise, anorexia, and low grade fever.
  • Clinical diagnosis in the catarrhal stage is difficult.
  • During this stage, large numbers of organisms are sprayed in droplets, and the patient is highly infectious but not very ill.
  • This is unfortunate as this is the stage at which the disease can be arrested by antibiotic treatment.
• 2. Paroxysmal Stage
  • After 1 to 2 weeks, the paroxysmal stage begins.
  • As the catarrhal stage advances to the paroxysmal stage, the cough increases in intensity and comes on in distinctive bouts.
  • During the paroxysm, the patient is subjected to violent spasms of continuous coughing, followed by a long inrush of air into the almost empty lungs, with a characteristic whoop (hence the name).
  • The paroxysms of coughing may be so severe that cyanosis, vomiting and convulsions follow, completely exhausting the patient.
• 3. Convalescent Stage
  • After 2 to 4 weeks, the paroxysmal stage is followed by convalescence stage, during which the frequency and severity of coughing gradually decrease but secondary complications can occur.
  • The disease usually lasts 6-8 weeks though in some it may be very protracted.
• Complications:
  • Subconjunctival hemorrhage, subcutaneous emphysema, inguinal hernia or rectal prolapse due to pressure effects during the violent bouts of coughing.
  • Respiratory (bronchopneumonia, lung collapse)- Respiratory complications are self-limited, the atelectasis resolving spontaneously.
  • Neurological (convulsions, coma.)-neurological complications may result in permanent sequelae such as epilepsy, paralysis, retardation, blindness or deafness.

Epidemiology of Bordetella pertussis:

• Whooping cough is predominantly a pediatric disease, the incidence and mortality being highest in the first year of life.
• Maternal antibodies do not seem to give protection against the disease.
• Immunization should, therefore, be started early.
• The disease is commoner in the female than in the male at all ages.
• It is worldwide in distribution.
• It occurs in epidemic form periodically but the disease is never absent from any community.
• The source of infection is the patient in the early stage of the disease.
• Infection is transmitted by droplets and fomites contaminated with oropharyngeal secretions.
• Whooping cough is one of the most infectious of bacterial diseases and nonimmune contacts seldom escape the disease.
• The secondary attack rates are highest in close household contacts.
• The disease is often atypical.
• In adolescents and adults and may present as bronchitis.
• They may serve as a source of infection in infants and children.
• Natural infection confers protection though it may not be permanent, and second attacks have been reported.

Laboratory Diagnosis of whooping cough:

• Blood changes in the disease are distinctive and helpful in diagnosis.
• Pertussis typically causes an elevated white cell count, sometimes in excess of 50,000 cells/ μl (normal range=4500-11000 white blood cells/μl during the latter part of the catarrhal or early paroxysmal phase.
• A marked leukocytosis occurs, with relative lymphocytosis (total leukocytic counts 20,000-30,000 per/ μl with 60-80 percent lymphocytes).
• The erythrocyte sedimentation rate is not increased, except when secondary infection is present.

Three lab diagnosis methods are available:

1. Isolation of B. pertussis by culture from a pernasal swab;
2. Identification of the organism in a smear from a pernasal swab by immunofluorescence microscopy;
3. Serological demonstration of specific antibodies in the patient’s serum.

1. Microscopy:

• Microscopic diagnosis depends on demonstration of the bacilli in respiratory secretions by the fluorescent antibody technique.

2. Specimen Collection and Transport:

• Though the disease is mainly in the lower respiratory tract, the organism can be recovered readily from the nasopharynx.
• ‘Cough plates’ and postnasal swabs are unsatisfactory because of overgrowth by commensal bacteria.
• The optimal diagnostic specimen is a naso-pharyngeal aspirate.
• i. The Cough Plate Method
  • Here a culture plate is held about 10-15 cm in front of the patient’s mouth during about of spontaneous or induced coughing so that droplets of respiratory exudates impinge directly on the medium.
  • This has the advantage that specimen is directly inoculated at the bedside.
• ii. The Postnasal (Peroral) Swab
  • Secretions from the posterior pharyngeal wall are collected with a cotton swab on a bent wire passed through the mouth.
  • Salivary contamination should be avoided.
  • A West’s postnasal swab may be conveniently employed.
  • Cotton swabs should not be used because they contain fatty acids that are toxic to B. pertussis so it is preferable to use dacron or calcium alginate swabs for specimen collection.
• iii. The Pernasal Swab
  • A sterile swab on a flexible wire is passed gently along the floor of the nose until it meets resistance.
  • The swab, which will collect mucopus, is withdrawn and either plated immediately on charcoal blood agar, or placed in transport medium.
  • The use of transport medium reduces the isolation rate.
  • A single swab may yield a negative culture, but isolation rates of up to 80 percent may be achieved by taking specimens on several successive days.
  • The pernasal swab has generally replaced the cough plates or post- nasal swabs that were used in the past.

3. Culture:

• The swab is inoculated immediately on charcoalhorse blood agar and Bordet-Gengou medium both with and without methicillin or cephalexin and incubated for at least seven days before being discarded as negative.
• The specimen may be transported in Regan-Lowe semi-solid medium if delay in transport is unavoidable.
• Plates are incubated in high humidity at 35-36°C and colonies appear in 48-72 hours.
• Typical ‘bisected pearl’ colonies appearing after 3-5 days must be investigated further.

4. Identification:

• Identification is confirmed by microscopy and slide agglutination with specific antisera.
• Immunofluorescence is useful in identifying the bacillus in direct smears of clinical specimens and of cultures.

5. Detection of Bacterial Antigens:

• Bordetella antigens may be detected in serum and urine in tests with specific antiserum.
• Alternatively, bacteria in nasopharyngeal secretions are labelled with fluorescein-conjugated antiserum and examined by ultraviolet microscopy.
• This method has the theoretical advantage, compared with culture, of detecting dead bordetellae.

6. Polymerase Chain Reaction (PCR):

• Polymerase chain reaction (PCR) is used for the detection of bordetella DNA in nasopharyngeal specimens, by the use of various primers with a sensitivity of 80 percent to 100 percent.

7. Serology:

• Rise in antibody titer may be demonstrated in paired serum samples by ELISA, agglutination.
• Complement fixation, immunoblotting, indirect hemagglutination, and toxin neutralization.
• Demonstration of specific secretory IgA antibody in nasopharyngeal secretions by ELISA has been proposed as a diagnostic method in culture negative cases.

Treatment of whooping cough:

• B. pertussis is susceptible to several antibiotics (except penicillin).
• The drug of choice is erythromycin (or one of the newer macrolides such as clarithromycin), which may reduce the severity of the illness if given before the paroxysmal stage.
• Chloramphenicol and cotrimoxazole are also useful.
• Treatment with pertussis immunoglobulin has been tried, but with limited success.
• Prophylaxis:
  • Preventing the spread of infection by isolation of cases is seldom practicable as infectivity is highest in the earliest stage of the disease when clinical diagnosis is not easy.
• Treatment and Quarantine:
  • Antibiotics and immunoglobulins currently available are not very effective for the treatment of patients or the protection of contacts.
  • Control of the disease by quarantine is unrealistic.

Vaccination for whooping cough :

• Specific immunization with killed B. pertussis vaccine has been found very effective.
• It is of utmost importance to use a smooth phase I strain for vaccine production.
• The vaccines in general use are suspensions of whole bacterial cells, killed by heat or chemicals.
• Adsorption of the bacteria on to an adjuvant, such as aluminium hydroxide, enhances the immune response (particularly important with factor3) and also causes fewer adverse reactions.
• B. pertussis acts as an adjuvant for the toxoids (diphtheria and tetanus toxoid) producing better antibody response.
• Infants and young children should be kept away from cases.
• Those known to have been in contact with whooping cough may be given prophylactic antibiotic (erythromycin or ampicillin) treatment for 10 days to prevent the infecting bacteria to become established.
• The best protection that can be given to an infant is to administer a booster dose of DPT/DT to his siblings before he is born.
• Adverse Reactions:
  • Pertussis vaccination may induce reactions ranging from local soreness and fever to shock and neurological complications like convulsions and encephalopathy.
  • Provocation poliomyelitis is a rare complication.
• Contraindications:
  • If severe complications such as encephalopathy, seizures. ‘shock or hyperpyrexia develop following the vaccine, subsequent doses of the vaccine are contraindicated.
  • Routine pertussis vaccination is not advisable after the age of seven years as adverse reactions are likely and the risk of severe disease is low.
• Acellular Pertussis Vaccine:
  • Acellular vaccines containing the protective components of the pertussis bacillus (PT. FHA. agglutinogens 1, 2. 3) first developed in Japan, cause far fewer reactions, particularly in older children.
  • Both whole cell and acellular vaccines have a protection rate of about 90 percent.

The post Bordetella pertussis: characteristics, virulence factors, pathogenesis, symptoms, treatment and vaccine appeared first on Online Biology Notes.

• Neisseria gonorrhoeae; commonly termed as gonococcus (gonococci in plural).
• In 1879, the gonococcus was first described by Neisser in gonorrheal pus.
• Members of the genus Neisseria dominate the mucous membranes of human and other animals.
• Neisseria gonorrhoeae is responsible to cause the sexually transmitted disease gonorrhoeae.
• Gonococci are suited to grow on mucous membranes and hence are not able to tolerate drying.
• Their fragility restricts the transmission to direct contact between mucous membranes or the exchange of contaminated secretions.

Morphology of Neisseria gonorrhoeae:

• Under the microscope, it appears as a gram-negative coccus which is present in pairs (diplococci) with the flattening of the adjacent sides.
• The diameter ranges from 0.6-1 μm.
• The diplococci have kidney/coffee bean shape.
• It is a non-spore forming bacteria and is able to move using twitching motility.
• Gonococci exhibit pili on their surface.
• Pili enhances the binding of the cocci to the mucosal surfaces and promotes virulence by restricting phagocytosis.
• Human red blood cells are agglutinated by piliated gonococci, but not red cells from other mammals.

Cultural and biochemical characteristics for identification of Neisseria gonorrhoeae:

• Gonorrhoeae is a fragile organism with strict environmental and nutritional requirements.
• At pH 7.0-7.4 and at a temperature of 35-36 °C, growth occurs best.
• It is an aerobe meaning it needs oxygen to grow, however it can grow in anaerobic conditions as well.
• The supply of 5-10 percent CO₂ is needed.
• They grow rapidly on chocolate agar and Mueller-Hinton agar.
• The Thayer-Martin medium (chocolate agar containing antimicrobials such as vancomycin, colistin and nystatin) is a common selective medium that inhibits most contamination, including nonpathogenic Neisseria.
• Trimethoprim lactate may be introduced to the Thayer-Martin medium to suppress swarming Proteus species that are sometimes present in cervicovaginal and rectal specimens.
• Neisseria gonorrhoeae shows positive oxidase test (having cytochrome c oxidase) and is catalase positive as well, i.e. it can convert hydrogen peroxide to oxygen.
• The acid production takes place only from glucose and not from maltose, and also it does not ferment lactose or sucrose.
• It tests negative for nitrate reduction test.
• In enzyme substrate test, it tests positive for hydroxyprolylaminopeptidase.

Colonial morphology of Neisseria gonorrhoeae:

• In a humid aerobic environment enriched with 5-10% CO₂, colonies are thin, round, translucent, convex or slightly umbonate, with finely granular surface and lobate margins after incubation for 24 hours.
• The colonies of Neisseria gonorrhoeae are pinkish brown.
• They are easily emulsifiable and soft.
• The colonies are larger (1.5-2.5 mm) after 48 hours, often with a crenated margin and an opaque elevated center.
• The transparent, golden-brown pigmentation noticeable in 48 hr cultures after incubation results from cell autolysis.
• With gonococcal colonies, substantial variance in size exists and the colony out-line is irregular in most culture media, unlike the circular colonies of N. meningitidis.
• Growth is slower on the Thayer-Martin medium.
• Even if colonies are similar to those on the MNYC medium, they are typically smaller.
• Types of gonococci:
• On the basis of colonial appearance, auto-agglutinability and virulence, Kellogg classified gonococci into four types (T1-T4).
• T1 and T2 form small brown colonies and possess several numbers of fimbriae (pilated types P1 and P2). They are virulent and auto-agglutinable.
• Types T3 and T4 are non-piliated (P-), are avirulent type and form smooth suspensions.
• The fresh isolates obtained from acute cases of gonorrhea normally form T1 and T2 colonies.
• They transition to T3 or T4 colonial morphology in serial subculture.
• The forms T1 and T2 are often referred to as P+ and P++, while T3 and T4 are known as P-.

Virulence factors of Neisseria gonorrhoae:

• Being typical gram-negative bacteria, Neisseria gonorrhoeae has thin peptidoglycan layer that is sandwiched between the inner cytoplasmic and outer membrane.
• A true carbohydrate capsule does not enclose the outer surface.
• Gonococci are antigenically diverse and are able to modify their surface structures invitro.
• They likely do so in vivo as well in order to prevent host defense.
• The following are included in the surface structures:
• Pili:
  • Pili are hair-like appendages that reach out of the gonococcal surface up to several micrometers.
  • By facilitating attachment to host cells and inhibiting phagocytosis, they serve as virulence factors.
  • Pili is composed of repeating subunits of protein (pilins), whose expression is regulated by the pil gene complex.
  • At the amino terminal end, pilin proteins have a conserved region and at the exposed carboxyl terminus, a highly variable region is present.
  • Pili goes through antigenic and phase variations.
• Por protein (Protein I):
  • The Por protein is an essential outer membrane protein present on all gonococcal strains.
  • It forms pores or channels in the outer membrane.
  • Even if the protein I shows considerable heterogeneity among different strains, protein I of single strain is antigenically constant.
  • Two types of Por proteins have been described (PorA and PorB), each with a range of antigenic variations.
  • Only one type of Por is expressed by each strain of gonococcus, but the Por of various strains is antigenically distinct.
  • Any single strain carries either IA or IB only, but not both.
  • Utilizing monoclonal antibodies to protein I epitopes, gonococci can be categorized into multiple serovars, AI to 24 and BI to32.
• Opa proteins (Protein II):
  • Opa are the opacity proteins that are variably expressed on gonnacoccal strains that is responsible for phenotypes of various colony.
  • These proteins promote bacterial attachment to each other along with eukaryotic cells and also for the clumping of cocci seen in urethral exudate smears.
• Rmp (Protein III):
  • Highly conserved Rmp proteins (reduction-modifiable proteins, formerly protein III) are the third group of proteins in the outer membrane.
  • These proteins induce antibodies which inhibit the serum bactericidal activity against N. gonorrhoeae.
• Lipo-oligosaccharide (LOS):
  • This antigen has endotoxic activity and is composed of lipid A and a main oligo-saccharide identical to gram-negative lipopolysaccharide (LPS).
• Other proteins:
  • The other important gonococcal proteins are IgA protease which degrades secretory IgA and beta-lactamase which degrades penicillin.
  • Fbp (iron-binding protein), similar to Por in molecular weight, is expressed when the supply of iron available is restricted, for example, in human infection.

Resistance showed by gonococcus:

• Gonococcus is a very sensitive organism, readily destroyed by drying, soap and water, and many other washing agents or antiseptic agents when used and diluted correctly.
• Organisms can remain viable in pus-contaminating linen or other fabrics for a day or so.
• In cultures, the coccus dies in 3-4 days at room temperature.
• The most effective method for long-term gonococcus storage is freeze-drying, but storage at-70 ° C or in liquid nitrogen might be more convenient for intermediate storage.

Pathogenesis of Neisseria gonorrhoeae:

• Gonorrhea is a sexually transmitted disease.
• Gonorrhea is a condition that is essentially limited to humans’ mucus-secreting epithelial cells.
• The first stage in the infection is the adhesion of gonococci to the urethra or other mucosal surfaces.
• For the first attachment, the existence of pili is important.
• As the adhesion is quick and firm, the micturition after exposure offers no protection against infection.
• By the third day following infection, the cocci penetrate into the intercellular spaces and enter the subepithelial connective tissue.
• Gonococci presumably penetrate between columnar epithelial cells.
• Stratified squamous epithelium is comparatively resistant to infection.
• The period of incubation is 2-8 days.
• Gonococci invade the genitourinary tract, eye, rectum, and throat mucous membranes, causing acute suppuration that can lead to tissue invasion; which are followed chronic inflammation and fibrosis.

Gonococcal Infection in men:

• A few days after unprotected vaginal or anal sexual intercourse, acute urethritis in males is the most common clinical presentation.
• Discharge or dysuria normally occurs within 1 week of exposure, but there are never any signs or symptoms for as many as 5-10 percent of patients.
• The discharge is characteristically purulent, and in the gram stain of the exudate, gram-negative intracellular diplo-cocci can be easily seen.
• Inguinal lymphadenopathy is often present, and on the penile shaft and corona, occasionally frank lymphangitis may develop.
• There may be an asymptomatic condition in men up to several weeks after infection.
• Chlamydial urethritis or non-gonococcal urethritis (NGU) is the differential diagnosis of gonococcal urethritis due to non-chlamydial etiology such as Mycoplasma genitalium.
• With a history of receptive rectal intercourse, symptomatic anorectal gonococcus disease exists in men.
• Around 50 percent have symptoms, including pain in the rectum, discharge, constipation, and tenesmus.
• Some acute sexually transmitted rectal infections (STIs), such as herpes simplex, chlamydia proctitis and syphilitic proctitis, are included in the differential diagnosis.
• Women who have endocervical gonorrhea and who have not usually had receptive rectal intercourse might also have anorectal diseases.
• In these cases, contamination is believed to have occurred by the tracking of secretions throughout the perineum.
• Indeed, up to 30% of such women also have rectal infections that coexist, but they are usually asymptomatic.
• In either sex, Gonococcal pharyngitis can follow orogenital contact.
• Conjunctivitis will typically occur by finger auto-inoculation.

Gonococcal infection in women:

• The endocervix is the main site of infection in women and spreads to the urethra and vagina, causing mucopurulent discharge to occur.
• In adults, the vaginal mucosa is typically not affected because the stratified squamous epithelium is immune to cocci infection and also due to the acid pH of vaginal secretions, but in prepubertal girls, serious vulvovaginitis can occur.
• Asymptomatic carriage is prevalent in females, especially in the endocervical canal.
• Vaginal discharge, dysuria, and abdominal pain are widely experienced by symptomatic patients.
• Bartholin’s glands, endometrium, and fallopian tubes can be affected by the infection.
• Gonococci ascend into the fallopian tubes during menstruation or after instrumentation, particularly at the end of pregnancy, to give rise to acute salpingitis, which may be accompanied by pelvic inflammatory disorders and a high risk of sterility if handled inadequately.
• Occasionally, peritoneal spread occurs and can result in perihepatic inflammation (Fitz-Hugh-Curtis syndrome).
• Clinical disease is less severe in women, many of whom may bear cervical gonococcus without any clinical symptoms.
• Asymptomatic carriage of gonococci is rare in men.

Disseminated Gonococcal Infection:

• An unusual complication is disseminated gonococcal infection (DGI, gonococcal septicemia).
• Constitutional signs, fevers, chills, and a distinct set of syndromes consisting of oligoarticular septic arthritis, tenosynovitis, and rash characterize the condition.
• The rash consists of pustular lesions on the extensor surfaces of the upper and lower extremities that are normally sparse (<20).
• There are two typical clinical developments.
• Tenosynovitis and rash patients are more likely to have positive blood cultures.
• In patients with septic arthritis, in just 50 percent of cases, healthy blood and/or synovial fluid cultures are present.
• In concert with the outcomes of genital and mucosal site cultures, the diagnosis is made clinically.
• Any young sexually active patient presenting with fever, dermatitis and rheumatological symptoms should be suspected of having a DGI diagnosis.

Gonococcal Diseases in children:

• Ophthalmic Neonatorum:
  • Ophthalmia neonatorum in newborns is a nonvenereal infection.
  • Babies born to infected women may suffer from ophthalmia neonatorum, in which gonococcus coats the eyes as the baby passes through the birth canal.
  • Within a few days of birth, a severely purulent eye discharge with periorbital edema develops.
  • When untreated, ophthalmia easily leads to blindness.
  • The practice of instilling 1 percent silver nitrate solution into the eyes of all newborn babies (Crede’s method) has controlled this.
  • Alternatively, it is possible to use topical erythromycin; this has the benefit of being active and less toxic against Chlamydia.
• Vulvovaginitis:
  • Vulvovaginitis can be caused by gonococci in pre-pubertal children.
  • This happens either through bad hygiene conditions or by sexual abuse.
  • It should always be carefully invested and placed in communication with social workers and other practitioners capable of coping with this difficult situation.

Epidemiology of Neisseria gonorrhoeae:

• Gonorrhea exists in humans only and no other reservoir is yet known.
• Although a proportion of those infected, especially women, may remain asymptomatic, it is never discovered as a normal commensal.
• Gonococcal disease has historically been a key factor in areas where there has been limited access to healthcare services.
• This is reflected in the high rates of gonorrhea in the developing world, particularly in sub-Saharan Africa and Asia, as well as in the rates currently rising in the former Eastern Bloc/Soviet Union.
• Typically, acute gonorrhea is quickly detected and treated, and was well managed until the 1960s in most of the world.
• The occurrence of new cases worldwide was estimated at 16 million in1970, making it one of the most common infectious diseases.
• In the 1980s, with the AIDS scare, there was a significant decrease in gonorrhea incidence, although this has not been sustained.
• A higher frequency of gonorrhea was found in patients belonging to group B of the blood, the basis for this is yet not understood.

Laboratory diagnosis of Neisseria gonorrhoeae :

• In the acute stages, the diagnosis can be easily carried out. However, the chronic cases are difficult to be diagnosed.
• Specimens:
• Specimens in Men:
  • In acute gonorrhea, gonococci are present in large numbers in the urethral discharge.
  • The meatus is washed with a saline-soaked gauze and a discharge sample obtained with a platinum loop for culture or directly on a slide for smears.
  • Purulent discharge can be conveyed and collected with a swab in the anterior urethra.
  • There may be no urethral discharge in chronic infections.
  • The morning drop of secretion may be investigated or some exudate after prostatic massage may be collected.
  • In cases where no urethral discharge is available, it may also be possible to demonstrate gonococci in centrifuged urine deposits.
  • Anal canal – rectal culture in homosexual males.
• Specimens in Women:
  • Urethral, cervical and rectal specimens should always be examined in women.
  • A single well-taken endocervical swab can detect about 90% of women’s gonococcal infections.
  • A high vaginal swab is not appropriate.
  • Infection of the throat also happens and should be checked where possible.
• Specimens in both male and female:
  • Blood, swabs of skin lesions, or pus aspirated from a joint.
  • Conjunctival swab, particularly in the context of neonatal ophthalmia.
• Transport of specimens:
  • For culture, specimens should be inoculated, immediately upon collection, on pre-warmed plates.
  • If this is not feasible, specimens should be obtained with swabs impregnated with charcoal and transmitted to the laboratory in the Stuart’s transport medium.
• Direct microscopy:
  • Gram staining is performed with a few extracellular species that are typical of gonococcal infection.
  • The smear is confirmed as positive, showing characteristic kidney-shaped gram-negative diplococci lying inside polymorphonuclear leucocytes.
  • About 95 percent of males infected would develop a positive smear.
  • It must be stressed that the diagnosis of gonorrhea by smear analysis in women is inaccurate since some of the typical genital flora has a morphology that is essentially identical.
  • The use of fluorescent antibody techniques in the smear identification of gonococci is more sensitive and specific diagnosis by microscopy.
• Culture:
  • In acute gonorrhea, cultures can be acquired commonly on chocolate agar or Mueller-Hinton agar incubated at 35-36°C along with 5-10% CO₂.
  • However, it is best to use a selective medium such as the Thayer-Martin medium in chronic cases where mixed infection is normal and when examining lesions such as proctitis.
  • After 24 hours of incubation, the plates are examined and morphology and biochemical reactions identify the growth.
  • Incubation of primary isolation plates is continued for 48 hours and the above-mentioned procedures re-examine cultures until any specimen can be negatively reported.
  • In a humid aerobic environment enriched with 5-10 percent CO2, colonies are thin, round, translucent, convex or slightly umbonate, with fine granular surface and lobate margins after incubation for 24 hours.
  • They are easily emulsifiable and soft. The colonies are larger (1.5-2.5 mm) after 48 hours, often with a crenated margin and an opaque center elevated.
  • Smear is obtained from the colony and Gram staining is done.
  • Gonococci are pair-arranged gram-negative cocci (diplococci) with concave (pear or bean shaped) adjacent sides.
• Identification:
  • Gonorrhoeae are preliminarily defined on the basis of the isolation of oxidase-positive, gram-negative diplo-cocci that are selective for pathogenic Neisseria species and grow on chocolate blood agar.
  • N. gonorrhoeae is oxidase positive.
  • It only ferments glucose with acid.
• Genetic probes:
  • Gonorrhoeae were created in clinical specimens to directly detect bacteria.
  • Tests are sensitive, precise, and rapid (results are available in 2 to 4 hours) using these probes.
• Serological diagnosis:
  • It is not really possible to obtain gonococci in culture from some chronic cases as well as from patients with metastatic lesions like arthritis.
  • In such cases, serological tests are essential.
  • Complement fixation tests
  • Immunoblotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay) tests.
  • However, for routine diagnostic purposes, there was no serological test found to be useful.
  • Such tests are neither sensitive nor precise and are not recommended for use.

Treatment of Gonorrhea:

• Penicillin is no longer the antibiotic of preference for treatment of gonorrhea.
•  Since the development and wide-spread use of penicillin, gonococcal resistance to penicillin has gradually risen, owing to the selection of chromosomal mutants, so that many strains now need high concentrations of penicillin G for inhibition (MIC 2 μg/ml).
• Empirical therapy:
  • It is troublesome to select successful empirical therapy since the incidence of antibiotic resistance in gonococci is increasing.
  • The Centers for Disease Control and Prevention (CDC) is currently promoting the use of ceftriaxone, cefixime, ciprofloxacin, or ofloxacin as an initial treatment for uncomplicated cases of gonorrhoea.
  • For infections complicated by dual Chlamydia infections, doxycycline or azithromycin should be added.

Penicillinase Producing Gonococci (PPNG):

• Gonococci generating β-lactamase (penicillinase) emerged in1976, making treatment with penicillin ineffective.
• These penicillinase-producing gonococci (PPNG) were isolated in the United States and England from widely separated areas and have spread widely.
• Penicillinase development is plasmid-mediated in gonococci.

Chromosomally Mediated Resistance gonococci (CMRNG):

• Isolation of penicillin resistant N. gonorrhoeae that do not produce β-lactamase have been carried out.
• This chromosomal mediated resistance (CMRNG) is not limited to penicillin alone, but also applies to tetracyclines, erythromycin, and aminoglycosides, resulting from cell surface changes that prevent the antibiotic from entering the gonococcal cell.
• In Africa, Southeast Asia, Australia, and some US cities, resistance to fluroquinolones like ciprofloxacin has also become prevalent.

Control of gonorrhea:

• Gonorrhoea prevention consists of early cases identification, contact tracing, health education, and other general steps.
• The rate of transmission is significantly decreased by barrier methods of contraception, condoms in particular.
• Owing to the increase in gonococcus antibiotic resistance, chemoprophylaxis is of minimal benefit.
• Since even clinical disease does not grant any immunity, vaccination has no place in prophylaxis.

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• Legionellae are thin, non-capsulated bacilli, 25 µm x 0.3­-1 µm, coccobacillary in clinical material and assum­ing longer forms in culture.
• Most are motile with polar or subpolar flagella.
• They are gram-negative but stain poorly, particularly in smears from clinical specimens.
•  They stain better by silver impregnation but are best visualized by direct fluorescent antibody (DFA) stain­ing with monoclonal or polyclonal sera.

Cultural characteristics of Legionella pneumophila

• Legionellae are nutritionally fastidious.
• Their growth is enhanced with iron salts and depends on the supple­mentation of media the L­-cysteine.
• They have fastidious requirements and grow on complex media such as buff­ered charcoal, yeast extract (BCYE) agar, with L-­cysteine and antibiotic supplements, with 5 percent CO2, at pH 6.9, 35°C and 90 percent humidity.
• Growth is slow and colonies take 3 to 6 days to appear.

Biochemical characteristics of Legionella pneumophila:

• The organisms are non-fermentative and derive energy from the metabolism of amino acids.
• Most species are motile and catalase-­positive, liquefy gelatin and do not reduce nitrate or hydrolyze urea.

Pathogenesis of Legionella pneumophila:

• Respiratory tract disease caused by LegionelIa species develops in susceptible people who inhale infectious aerosols.
• Legionellae are facultative intracellular para­sites that can multiply in alveolar macrophages and monocytes following entry into the alveoli through aer­osols.
• Dissemination occurs by endobronchial, hematogenous, lymphatic and contiguous spread.
• Because of their intracellular location, humoral antibodies are inef­fective. Cellular immunity is responsible for recovery.

Diseases caused by Legionella pneumophila:

• Asymptomatic Legionella infections are relatively com­mon.
• Symptomatic infections generally affect the lungs and are present in one of two forms
  • Pontiac fever- An influenza-like illness.
  • Legionnaires’ disease -­ A severe form of pneumonia
• Pontiac Fever:
  • Pontiac fever is a milder, nonfatal ‘influenza like’ illness with fever, chills, myalgia malaise, and headache but no clinical evidence of pneumonia. Outbreaks with high attack rates may occur.
• Legionnaire’s Disease
  • The incubation time is 2 to 10 days.
  • The disease pre­sents with fever, non-productive cough and dyspnea, rapidly progressing, if untreated, to pneumonia.
  • The primary manifestation is pneumonia.
  • Multiorgan dis­ ease involving the gastrointestinal tract, central nervous system, liver and kidneys is common.
  • Case fatality may be 15 to 20 percent but can be much higher in patients with severely depressed cell­-medi­ated immunity, the cause of death being progressive respiratory failure and shock.
  • All age groups are suscep­tible, though more cases have occurred in the elderly.
  • Legionnaires’ disease (legionellosis) is characteristically more severe and causes considerable morbidity, leading to death unless therapy is initiated promptly.
  • Legion­naire’s disease may be either epidemic or sporadic.

Laboratory Diagnosis of Legionella pneumophila:

1. Microscopy:

• Legionellae stain poorly with gram stain. Nonspecific staining methods, such as those using Dieterle’s silver or Gimenez’s stain, can be used to visu­alize the organisms.
• The most sensitive way of detecting legionellae microscopically in clinical specimens is to use the direct fluorescent antibody (DFA) test, in which fluorescein-labelled monoclonal or polyclonal antibod­ies directed against Legionella species are used .
• The test is specific, with false-positive reactions observed only rarely if monoclonal antibody preparations are used.

2. Culture:

• Although legionellae were difficult to grow initially, commercially available media now make growth easy.
• Legionellae require L­-cysteine and their growth is enhanced with iron (supplied in hemoglobin or ferric pyrophosphate).
• The medium most commonly used for the isolation of legionellae is buffered charcoal­ yeast extract (BCYE) agar, although other supplemented media have also been used.
• Antibiotics can be added to suppress the growth of rapidly growing contaminating bacteria.
• Legionellae grow in air or 3 to 5 percent carbon dioxide at 35°C after 3 to 5 days. Their small (1­ to 3 mm) colonies have a ground-glass appearance

3. Antigen Detection:

• Enzyme­-linked immunoassays, radio-immunoassays, the agglutination of antibody ­coated latex particles, and nucleic acid analysis studies have all been used to detect legionellae in respiratory specimens and urine.
• If an antigen test is used, culture should always be per­formed.
• Nucleic acid analyses have been disappointing to date.

4. Serology:

• Detection of serum antibody is done by ELISA or indi­rect immunofluorescent assay.

Treatment of Legionella pneumophila :

• For treatment of disease caused by Legionella pneumophila, the newer macrolides, ciprofloxacin, and tetracyclines are effective. Rifampicin is employed in severe cases.
• Beta lactamase antibiotics and amino­ glycosides are ineffective in treatment for Legionella pneumophila.

Prevention of Legionellosis:

• Prevention of legionellosis can be carried out by the identification of the environmental source of the organism and decrease in the microbial burden.
• Hyperchlorination of the water supply and the continuation of elevated water temperatures have proved relatively successful.
• How­ever, complete elimination of Legionella organisms from a water supply is often difficult or impossible.
• Because the organism has a low potential for causing disease, reducing the number of organisms in the water supply is frequently an adequate control measure.
• Hospitals with patients at high risk for disease should monitor their water supply on a regular basis for the presence of Legionella and their hospital population for disease.
• Continuous copper­-silver ionization of the water supply may be necessary if hyperchlorination or superheating of the water does not eradicate disease (complete eradication of the organisms in the water supply is probably not feasible).

Epidemiology of Legionella pneumophila:

• Sporadic and epidemic legionellosis has a worldwide distribution. Legionellae are widely distributed in natu­ral water sources, such as stagnant waters, mud and hot springs, where the nutritional and growth requirements for these fastidious bacteria are provided by some types of algae.
• The bacteria are commonly present in natural bodies of water, such as lakes and streams, as well as in air conditioning cooling towers and condensers and in water systems (e.g. showers, hot tubs).
• Legionellae sur­vive and multiply inside free-­living amoebae and other protozoa.
• They also multiply in some artificial aquatic environments, which serve as amplifiers.
• Human infection is typically by inhalation of aero­ sols produced by cooling towers, air conditioners and shower heads which act as disseminators.
• Aerosolized legionellae can survive for long and can be carried over long distances. No animal reservoir exists and infection is limited to human beings.
• No carrier state is estab­lished. Man ­to­ man transmission does not occur.
• The outcome of inhalation of legionellae depends on the size of the infecting dose, virulence of the strain and resistance of the host.
• Known risk factors are smoking, alcohol, advanced age, intercurrent illness, hospitaliza­tion and immunodeficiency.
• Men are more often affect­ed than women.
• In the developed countries, legionel­losis accounts for 1 to 3 percent of community acquired, and 10 to 30 percent of hospital acquired pneumonias.
• Its prevalence in the developing countries is not ade­quately known.

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• Dysentery is a clinical condition of multiple etiology characterized by frequent passage of blood-stained mucopurulent stool.
• The causative agent of bacillary dysentery (Disease characterized by severe abdominal cramps and the frequent painful passage of low volume stools containing blood and pus) belong to the genus Shigella.
•  Shigella is named after ‘Shiga’ who in (1896) isolated the first member of this genus from epidemic dysentery in Japan.
• Other Bacilli such as entero-invasive E. coli, Vibrio parahemolyticus, Campylobacter can also cause the clinical feature of dysentery.
• Enterobacteria (fermentative, facultative anaerobes, oxidase -ve, gram -ve rods).
• Non lactose fermenting, non-motile, mostly anaerogenic urease -ve, non-citrate utilizing and KCN sensitive.
• Shigella are killed at 56^oC in 1 hour and by 1% phenol in 30 minutes.
• In faeces they die wthin a few hours due to acidity produced by faecal coliform.
• S. sonnei are more resistant than other Shigella they can tolerate low temperature if adequate moisture is present.
• They can survive over 6 months in water at room temperature.

Morphology of Shigella:

• Shigella are short gram -ve rods.
• (2-4) x 0.6 ml, non-sporing, non-motile
• Non-capsulated
• Fimbriae are present only in S. flexneri

Antigenic structure of Shigella:

• Shigella are differentiated by their ‘O’ antigens into serotypes.
• These are classified into 4 structures or subgroups based on a combination of biochemical and serological characteristics.
• Shigella dysenteriae (Subgroup A):
  • These are mannitol non-fermenting, consists of 10 serotypes.
  • Shigella dysenteriae type-1 forms a toxin.
  • 3 types of toxic activity have been demonstrated in Shigella culture filtrates.
    ( Neurotoxicity, enterotoxicity, and cytotoxicity)
• Shigella flexneri (Subgroup B):
  • Named after Flexner, who first time described first of the mannitol fermenting Shigella from Phillipines (1900).
  • Based on type specific and group specific antigen, they have been classified into six serotypes (1-6) and several subtypes.
• Shigella boydii (Subgroup C)
  • Consists of dysentery bacilli that resemble S. flexneri biochemically, but not antigenically.
  • After Boyd who first described this strain from India (1931).
  • S. boydii isolates least frequently.
  • 15 serotypes have been identified.
• Shigella sonnei (Subgroup D)
  • 1^st time isolated by Sonne (1915) in Germany.
  • Ferment lactose and sucrose late, indole negative.
  • Causes mildest form of bacillary dysentery.

Cultural characteristics of Shigella:

• Aerobic and facultative anaerobes.
• Optimum temperature 37^oC (Exception S. sonnei grow even at 10^oC and 45^oC).
• They grow on ordinary media however less readily than other Enterobacteria.
• Nutrient agar and Blood agar:
  • On Nutrient agar and Blood agar, Colony are smooth, circular convex greyish or colorless, translucent often 2-3 mm diameter.
  • Those of S. sonnei are slightly larger and opaque than others.
• MacConkey agar (MA):
  • On MA, colonies are pale and yellowish (non-lactose fermenting). Exception S. sonnei being late lactose fermenting become pink when incubation period is prolonged.
• Deoxycholate citrate agar (DCA):
  • On DCA, excellent selective medium for isolation of Shigella from faeces.
  • Colonies are pale and similar to though usually slightly smaller 1-1.5mm diameter and more translucent than those of Salmonella. They do not form black center.
• Xylose lysine deoxycholate agar (XLD):
  • On XLD, probably the best selective media for Shigella being less inhibitory to S. dysenteriae and S. flexneri than DCA.
  • Colonies are red and unlike those of most Salmonella without black centers.
• Peptone water and nutrient broth:
  • Good growth with uniform turbidity on incubation over night at 37^oC.
  • In some cases, especially fimbriated form a surface pellicle on longer incubation.
• Selenite F-broth:
  • Selenite F-broth enrich S. sonnei and S. flexneri but inhibitory to other Shigella.

Biochemical tests of Shigella:

• Carbohydrates utilization:
  • Most strains utilize sugar to produce acid but not gas though some strain S. flexneri and S. boydii form gas.
  • Glucose is fermented by almost all strains.
  • Lactose is not fermented within 24hrs.
  • However, S. sonnei and some strains of S. dysenteriae produce acid from lactose after prolonged incubation.
  • Mannitol fermentation is important characteristics. This differentiated Group A strain (which do not ferment mannitol) from group B, C and D, most strains of which ferment it.
  • Dulcitol is not fermented by most Shigella.
  • Sucrose is not fermented except S. sonnei andsomestrains of S. flexneri.
  • Adonitol and Inositol are also not fermented.
  • Xylose is not fermented except mannitol -ve biotype of Shigella flexneri.
• Methyl red test: +ve
• VP test: -ve
• Reduce nitrate to nitrite
• Catalase +ve
• Indole -ve,
• Citrate -ve
• H₂S -ve
• Urease -ve
• KCN growth (-ve).
• Gelatin not liquified.
• Decarboxylation test:
  • Group A, B and C fail to decarboxylate lysine and ornithine.
  • S. sonnei decarboxylate ornithine but not lysine

Pathogenesis of Bacillary dysentery caused by Shigella:

• Shigella causes bacillary dysentery.
• Infection occurs by ingestion.
• The minimum infective dose is low (10-100) bacilli.
• Being capable of initiating the disease probably because they survive gastric acidity better than other enterobacteria.
• Their pathogenic mechanism resembles those of Entero-invasive E. coli.
• The epithelial cell of the villi is the large intestine and multiply inside them spreading laterally to involve adjacent cell and penetrate into the lamina.
• Inflammatory reaction develops with capillary thrombosis resulting in necrosis of patches of epithelium which slough offs and leaves transverse superficial ulcers behind.
• Bacteremia may occur in sever infection, particularly in malnourished children and in AIDs patient.
• Has short incubation period (1-7 days usually 48 hrs).
• The onset and clinical course are variable and are largely determined by the virulence of infective strains.
• Shigellosis has high death rate especially in young children. Most death are caused by S. dysenteriae type 1.

Clinical manifestation of bacillary dysentery caused by Shigella:

• The clinical features of Shigella dysenteriae type 1 infection includes:
•  toximea, sometimes bacteremia and severe dysentery leading to marked dehydration and protein loss
•  Inflammation and ulceration of the large intestine
•  Hemorrhage, abdominal pain and high fever
• Death occur from circulatory collapse or kidney failure

Shigella: antigenic structure, cultural characteristics and biochemical tests

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