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Genus/species definition :

Genus/species definition The family Enterobacteriaceae . Gram-negative rods. Facultative anaerobes Reduce nitrate Ferment glucose, and other sugars, Without gas production Oxidase -negative Catalase -positive Motile at 22–30° C, but not at 37° C. Stain in a bipolar fashion - ‘safety pin ’ G + C -46–50 mol%.

Human pathogens:

Human pathogens Yersinia pestis Yersinia enterocolitica Yersinia pseudotuberculosis

The History:

The History Members of the genus Yersinia were formerly included in the genus Pasteurella and finally removed in the 1970s. The name Yersinia - the French bacteriologist Alexander Yersin , who first isolated the plague bacillus in Hong Kong in 1894 Y. pseudotuberculosis , first isolated by Malassez and Vignal in 1883

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Y. enterocolitica , Schleifstein and Coleman in 1939, Who first called it Bacterium entercoliticum Later Pasteurella pseudotuberculosis rodentium and Pasteurella X. It was given its current designation of Y. enterocolitica in 1964 by Frederiksen


Morphology Gram-negative coccobacilli Bipolar staining - Wayson ’s or Giemsa ’s stain Pleomorphic , particularly when colonies growing on nutrient agar medium are stained. No endospores -no envelope, Y. pestis , have an envelope in vivo and in vitro when grown at 37° C 0.5–0.8 μ m in diameter and 1 –3 μ m in length.

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Y. pestis is nonmotile , Y. enterocolitica and Y. pseudotuberculosis are motile , with peritrichous or paripolar flagella, when grown at 22° C , but not at 37° C When cultivated below 30° C, all species of Yersinia except Y. pestis produce flagella and are motile.

Cultural characteristics and growth requirements :

Cultural characteristics and growth requirements Grow at temperatures ranging from 4 to 43° C and over a pH range of 4–10 Aerobic and facultative anaerobic Colonies of Y. pestis can be as small as 0.1 –0.2 mm after 24 h of growth on nutrient agar at 37° C

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Blood agar- Non hemolytic opaque with a gray to yellow color in the center; they remain transparent and gray to white in color on the periphery ( ‘ Chinese hat ’ shape ) Hemin absoption

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Mac Conkey agar – Colourless The colony disappear after 2-3 days – autolysis Y.pestis

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Tellurite medium Y. pseudotuberculosis grow as small gray to black colonies

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Growth Characteristics in Brain Heart Infusion Broth After 24-48 hours of incubation, the cultures in broth can be described as suspended flocculent or crumbly clumps (" stalactites ")

Biochemical reaction :

Biochemical reaction Glucose - positive Maltose - positive Mannitol – positive Lactose – negative Sucrose – negative Rhamnose – negative Catalase – positive Oxidase – negative Esculin – negative Gelatin – not liquified Indole – negative MR – positive Vp – negative Citrate - negative

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Organism motility maltose Sucrose Salicin Indole Urease Oxidase Catalase Ornithine decarboxylase 22 0 c 37 0 c Y. Pestis _ _ + - + _ _ _ + _ Y. pseudotuberculosis + _ + _ + _ + + + _ Y. enterocolitica + _ + + _ _ + + + +

Resistance :

Resistance Easley destroyed by exposure to heat, sunlight , drying and chemical disinfectant 55 o C or by 0.5% phenol in 15 minutes Remains viable for long periods in cold,moist environment

virulence factors:

virulence factors Plasmid Chromosomal

Genetic mechanisms :

Genetic mechanisms Location Factor(s) Putative Function pPCP1 plasmid (9.5kb) Plasminogen activator ( Pla protease) Protease, : coagulase : promotes blockage of flea midgut pCD1 plasmid (~70-75kb) Yersinia outer protein ( Yop ) virulon Type III secretion system, inhibits phagocytosis and lymphocyte proliferation V antigen Facilitates intracellular survival; pMT1/ pFra plasmid (∼110€kb) Fraction 1 antigen Creates capsule that interferes with phagocytosis Yersinia murine toxin ( Ymt ) Phospholipase D activity colonization of flea midgut and blockage formation;

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Location Factor(s) Putative Function Chromosome (4.6Mb) Hemin storage system ( hms ) Proteins produced at lower temperatures; iron acquisition; colonization of flea proventriculus , biofilm production Yersiniabactin ( Ybt ) system Siderophore ; iron acquisition Yersinia Fe uptake system ATP-binding cassette transport system; iron acquisition Lipopolysaccharide Temperature-dependent remodeling of lipid A structure pH 6 fimbriae antigen ( psa ) Blocks phagocytosis ; pH dependent

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Y. enterocolitica and Y. pseudotuberculosis Yst - Invasion -toxin similar to the heat-stable toxin produced by E. coli Y. pseudotuberculosis YPM - Y. pseudotuberculosis -derived mitogen , acts as a superantigen ( exotoxin ) Invasin and Ail, two proteins under the control of chromosomal genes YadA - The plasmid-encoded protein. the 11 Yops control the antiphagocytic ability

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Virulence genes on Yersinia are controlled by two independent regulatory systems, temperature and cellular concentration of calcium

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Pathogenic strains of Yersinia can be divided into those with low virulence and those with high virulence based on their ability to obtain and store iron.

Cell-wall and envelope composition:

Cell-wall and envelope composition The cell wall Lipopolysaccharide (LPS) Lipid A Core region O-specific side chain

Antigenic structure:

Antigenic structure Y. pestis has more than 20 antigens The fraction 1 envelope antigen (F1) is a heat-labile water-soluble antigen stimulates immunity in both mice and humans and is produced when the organism is grown at 37° C Yersinia species V and W antigens. This complex is composed of two proteins The V protein is cell-bound The W protein is an excreted protein. Yops outer-membrane proteins- are associated with virulence

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Other antigens – Intracellular toxin - murine toxin, composed of toxin A and toxin B Lipopolysaccharide endotoxin

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Y. enterocolitica has more than 70 serotypes based on O and H antigens O:3 and O:9 are the most common cross-reaction Y. enterocolitica serotype O:9 and Brucella . There is some similarity between Y. enterocolitica and Vibrio cholerae Pathogenic strains also produce an enterotoxin similar in nature to E. coli ST.

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Y. pseudotuberculosis - six serotypes Serotype O:1 is associated with 90%of human disease

Classification :

Classification Y. aldovae Y. aleksiciae Y. bercovieri Y. enterocolitica Y. frederiksenii Y. intermedia Y. kristensenii Y. mollaretii Y. pestis Y. pseudotuberculosis Y. ruckeri 11 species


Plague 1320 BC Biblical plague The first pandemic, or Justinian Plague, during the sixth-century Byzantine Empire. Originated in central Africa and affected much of the Mediterranean basin(AD 542)

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“Black Death” - The second pandemic began in 1347 and spread rapidly throughout Europe, killing an estimated one fourth of the population.

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The third, or modern , pandemic began in China in the 1860s. By 1894, it had spread to Hong Kong , where Alexandre Yersin isolated the causative agent. In the 20th century -India was most severely affected by plague epidemics, with more than 20 million cases and 10 million deaths. In the 1960s and 1970s, war-torn Vietnam affected by plague

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In 1994, a total of 693 suspected bubonic or pneumonic plague cases were reported to WHO by Government of India 19 February 2002, reported a total of 16 cases of pneumonic plague including 4 deaths in Hat Koti village, Shimla district,

Habitat :

Habitat Y. pestis survives in nature in the stomach and proventriculus of the flea and in the soil of animal burrows Reservoir black or roof rat ( Rattus rattus ) brown sewer or Norway rat ( R.norvegicus ) Rattus rattus R.norvegicus

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Vector The oriental rat flea Xenopsylla cheopis X astia Ceratophylus fasciatus Human flea - Pulex irritans Oropsylla montana

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The sylvatic (forest) cycles Y. pestis is maintained in the environment by enzootic spread of the organism among rodents and low risk for transmission to humans The urban cycles The rodent is domestic Epizootic plague spreads rapidly among susceptible rodents, which die off rapidly An avid search by their fleas for new hosts, and an increased risk of spread of infection to humans.

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Y. pestis -3 biogroups Antigua Medievalis Orientalis based on their ability to reduce nitrate and to ferment glycerol and melibiose .

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variety Glycerol ferm Nitrate red Geographical distribution Y.Pestis var Orientalis _ + Primary foci india Myanmar , china 1894 pandemic Y.Pestis var Antigua + + Transbaikalia,mongolia , manchuria Justinian plague Y.Pestis var Medievalis + _ Southeast Russia

Pathogenesis and Pathology:

Pathogenesis and Pathology Two steps: Transmission via fleas The host response.

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Fleas become infected by feeding on a bacteremic host. Transmission factors are expressed that allow the bacillus to colonize the flea midgut , replicate, and create a blockage of the flea intestine. “ blocked ” fleas feed aggressively, regurgitating bacteria into the bite wound

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A few bacteria may be taken up by mononuclear cells unable to kill them Multiply intracellularly and begin to produce F1 envelope antigen Organisms are carried via lymphatics to the regional lymph nodes -bubo.

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Bubo. Microscopic examination Lymph nodes reveals invasion by polymorphonuclear leukocytes hemorrhagic necrosis with destruction of normal architecture and dense concentrations of extracellular bacilli.

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Bacteremia Sepsis Pneumonia Purulent, necrotic, and hemorrhagic lesions in various organs Excessive release of TNF-α and other kinins . SIRS -DIC

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Purpuric skin lesions are the most obvious manifestations of a bleeding diathesis These startling cutaneous signs may be the origin of the term “ Black Death .”

Clinical Manifestations:

Clinical Manifestations The incubation period is 2 to 8 days.


BUBONIC PLAGUE Bubonic plague has a usual incubation period of 2–6 days. chills; fever, myalgias ; arthralgias , headache, and a feeling of weakness. pain in one or more regional lymph nodes

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Inspection of the skin surrounding or distal to the bubo sometimes reveals the site of a flea bite marked by a papule, pustule, or ulcer. Without effective antimicrobial treatment, patients with typical bubonic plague manifest an increasingly toxic state of fever, tachycardiaconvulsions and delirium. Secondary plague sepsis may result in DIC, bleeding, shock, and organ failure.

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Axillary bubo with ulceration and small eschar at the site of an infective flea bite.

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Inguinal buboes in a young male showing marked edematous swelling, erythema , and overlying desquamation


SEPTICEMIC PLAGUE Bubo, are not present. Septic patients often present with gastrointestinal symptoms of nausea, vomiting, diarrhea, and abdominal pain Petechiae , ecchymoses , bleeding from puncture wounds and orifices, and gangrene of acral parts are manifestations of DIC Refractory hypotension, renal shutdown shock Adult respiratory distress syndrome (ARDS)

Pneumonic plague:

Pneumonic plague Secondary to bacteremia in bubonic or septicemic plague. primary pneumonic plague is acquired from inhalation of Y. pestis Primary pneumonic plague -short incubation period, ranging from a few hours to a few days. Sudden onset of dyspnea , high fever, pleuritic chest pain, and cough that may be accompanied by bloody sputum. Pneumonic plague is rapidly fatal unless an appropriate antimicrobial agent is begun within the first day of illness. The sputum may be clear, purulent, or hemorrhagic, and contains gram-negative rods .



Laboratory isolation and identification:

Laboratory isolation and identification A high index of suspicion must be maintained for the timely diagnosis of plague

Handling of specimens:

Handling of specimens Y. pestis requires Biological Safety Level II( BSL-2 ) facilities and practices. Specimens should only be handled in a BSL-2 laminar flow hood eyes protected closed-front laboratory coats with cuffed sleeves and gloves stretched over the cuffed sleeves should be worn. No activities that might create aerosols or droplet dispersal.

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Laboratory benches should be decontaminated after each use and contaminated supplies disposed of into proper receptacles. Laboratory works should avoid touching mucosal surfaces with their hands Should not eat or drink in the laboratory. Each time before leaving the laboratory, laboratory workers should take off and reverse their gloves before disposing them in a biohazard container, Wash their hands and remove their laboratory coat.

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The peripheral blood leukocyte count is typically elevated, in the range of 10,000 to 20,000 cells/mm 3 , with a predominance of neutrophils . Chest roentgenograms

Diagnostic specimens:

Diagnostic specimens Blood cultures Other materials Bubo aspirates Sputum or tracheo bronchial washes Swabs of skin lesions or pharyngeal mucosa Cerebrospinal fluid.

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Bubo aspirates obtained by Inserting a 20-gauge needle on a 10-mL syringe containing 2mL of sterile saline solution into the bubo and withdrawing the plunger several times until the saline becomes blood tinged

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Gram Stain Differential Stain ( Wayson,Wright / Giemsa ) bipolar safety-pin morphology

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Peripheral blood smear of septicemic plague patient showing large numbers of bipolar-staining bacilli.

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Serological diagnosis Detect antibodies to Y. pestis Micro hemagglutination , Complement fixation, ELISA

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Detect antigens Monoclonal antibody to detect F1 antigen present in large amounts in the blood and bubo fluid from patients infected with Y. pestis . Amplification of two plasmid-encoded genes, plasminogen activator antigen fraction

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Commercially available systems API 20E API Rapid32 IDE Micronaut E RapID onE Vitek

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Y. pestis susceptsble to ciprofloxacin, ofloxacin , ceftriaxone streptomycin, tetracycline and chloramphenicol


TREATMENT Appropriate and timely therapy can markedly improve patient outcomes and chemoprophylaxis can prevent spread. Drug of choice Streptomycin 30 mg /kg two divided doses daily IM for 7 days

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Other drugs Tetracycline (2 to 4 g/day in four divided doses orally) Doxycycline (100 mg PO or IV twice daily) Chloramphenicol (25 mg/kg IV as a loading dose followed by 60 mg/kg per day in four divided doses) Gentamicin (2.5 mg/kg IM every 12 hours) Trimethoprim-sulfamethoxazole (160/800 mg twice daily) All given for 7 days


Chemoprophylaxis Used for infective exposure within the previous 7 days Doxycycline is the prophylactic agent of first choice, Dose of 100mg bd for 7 days

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Other drugs Tetracycline Chloramphenicol Trimethoprim-sulfamethoxazole Ciprofloxacin

Isolation :

Isolation Patients suspected of having any form of plague should be placed on strict respiratory isolation until each of the following measures have been completed: Pneumonia has been ruled out At least 48 hours of effective antimicrobial therapy has been given Sputum cultures are negativ e

plague vaccines:

plague vaccines Formalin-killed vaccine , Plague Vaccine U.S.P. , Y. pestis in the laboratory Persons at high risk of exposure. Military personnel serving in Vietnam. The killed vaccine does not protect against respiratory exposures, requires multiple doses over time Its manufacture has been discontinued, There is now only one source worldwide (Commonwealth Serum Laboratories Ltd., Australia).

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Killed vaccine in India Haffkine institute , Mumbi Live attenuated vaccine Otten’s Tjiwidej strain

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At present Recombinant subunit vaccines that express F1 and V antigens of Y. pestis protect animals against infective aerosol exposures Other approaches under investigation include Passive immunization with aerosolized monoclonal antibodies Vaccines based on attenuated Y. pseudotuberculosis

Reservoir and Vector Control:

Reservoir and Vector Control Insecticides around rodent runs, nests, or burrows Removing rodent harborage Educating people on environmental sanitation, Early detection and treatment of suspected plague Avoidance of sick or dead rodents and cats. Flea control

Flea index :

Flea index Rat-flea Index = No. of rat flea collected from the rat examined Total no. of rat examined Mean number of flea per rat


Yersinosis Infections with yersiniae other than y.pestis

Y. Enterocolitica and Y. Pseudotuberculosis:

Y. Enterocolitica and Y. Pseudotuberculosis The enteropathogenic yersiniae hosts. The major clinical syndromes Enterocolitis, Mesenteric adenitis, Terminal ileitis, Septicemia, and Reactive arthritis.

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Genomic studies suggest that Y.pseudotuberculosis is the recent progenitor of Y. pestis

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Modes of transmission Fecal-oral, Ingestion of contaminated food or water, Rarely by transfusion of contaminated blood. The most common animal reservoir of Y. enterocolitica is pigs Can also be transmitted by rodents or other infected animal species, including wild birds

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Transmission routes of Yersinia enterocolitica


PATHOGENESIS An inoculum of 10 9 organisms may be required to cause infection. After an incubation period of 4 to 7 days,

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Mucosal ulcerations in the terminal ileum Necrotic lesions in Peyer’s patches, Enlargement of mesenteric lymph nodes. Septicemia may lead to focal abscesses in various organs e.g., lung, liver, meninges


CLINICAL MANIFESTATIONS Enterocolitis Fever, diarrhea Abdominal pain lasting 1 to 3 weeks. Nausea and vomiting Blood in stool Perforation of the ileum Rectal bleeding Mesenteric adenitis or terminal ileitis

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Reactive polyarthritis is due to molecular mimicry between HLA-B27 antigen and Yersinia antigens


DIAGNOSIS Isolated from Stool, mesenteric lymph nodes, pharyn geal exudates, peritoneal fluid, blood and from abscesses Isolation from feces is hampered by slow growth and by overgrowth of normal fecal flora.

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Culture of Yersinia Incubation at 25° C allows a selective advantage for the isolation of Yersinia from stool specimens Cold enrichment of the stool Y. pseudotuberculosis - BA- granular ,translucent with beaten copper surface , non heamolytic ,--

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Selective medium, Cefsulodin–Irgasan–novobiocin (CIN) medium. This medium inhibits the majority of other enteric bacteria Y. enterocolitica - red pigmented colonies surrounded by a clear zone Irgasan ticarcillin broth - from food

Serologic tests:

Serologic tests Agglutination assays ELISA Immunoblotting These assays can be used to detect IgG , IgA , and IgM class antibodies. Y. pseudotuberculosis cross-react with one another Brucella , Vibrio , Bartonella , Borrelia , and E.coli . Y. pseudotuberculosis types II and IV cross-react with Salmonella groups B and D.

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Intradermal test for Y. pseudotuberculosis Similar to tuberculin test

Susceptibility to antimicrobial agents:

Susceptibility to antimicrobial agents Y. enterocolitica susceptible to amoxycillin–clavulanic acid, cefoxitin , fosfomycin , gentamicin , kanamycin , neomycin, tetracycline, naladixic acid, norfloxacin , ciprofloxacin, and trimethoprim . Resistant to ampicillin

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Two chromosomally mediated β- lactamases Enzyme A -broad-spectrum enzyme Enzyme B is inducible and is a cephalosporinase Induction of β- lactamase production can be detected using an imipenem disk as the inducer and a cefotaxime disk as the substrate Streptogramin A acetyltransferase gene (sat)-resistance to streptogramins . Gram-negative organisms are naturally resistant to streptogramins due to the impermeability of their outer membrane

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Y. pseudotuberculosis , the only antibiotic found to be effective was ofloxacin (mouse model using a pathogenic strain)


Treatment Ciprofloxacin (500 mg twice daily) For the pediatric patient- trimethoprim-sulfamethoxazole (TMP 8 mg/kg per day and SMX 40 mg/kg per day in two divided doses). Alternative Doxycycline


PREVENTION Safe food processing and preparation and hand washing after exposure to pigs or pork products.

Typing methods :

Typing methods phenotypic markers biotyping , Serotyping , Bacteriophage typing, Antibiotic susceptibility patterns

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Genotypic REAP -restriction endonuclease digestion of plasmid REAC -restriction endonuclease digestion of chromosomal DNA Digestion of genomic DNA and separation by pulsed field gel electrophoresis (PFGE) Ribotyping and analysis of PCR-generated fragments. MLEE - Multilocus enzyme electrophoresis

Bacteriocins and bacteriophages:

Bacteriocins and bacteriophages Y. pestis produces two bacteriocins , Pesticin I - plasmid-mediated and active against Y. pseudotuberculosis and some strains of E. coli and Y. enterocolitica Pesticin II produced by both Y. pestis and Y. pseudotuberculosis - active only against select strains of Y. pestis

Plague as a Biological Weapons Agent:

Plague as a Biological Weapons Agent 1346 -plague victims into bio weapons with catapults World War II -Japanese developed plague into a biological weapons agent. The Japanese tested the weapon on Manchurian villages. Rats infested with plague-carrying fleas Plague-infested fleas

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1950s and 1960s, the United States and the Soviet Union biological weapons programs Aerosolized pneumonic plague remains one of the most deadly biological weapon

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