enterobacteriaceae 3 (non lactose fermenters)

ENTEROBACTERIACEAE 3(NON LACTOSE FERMENTERS)

DR. AMADIN A. OLOTU

LECTURER/CONSULTANT MEDICAL MICROBIOLOGIST

BOWEN UNIVERSITY/BOWEN UNIVERSITY TEACHING HOSPITAL OGBOMOSO

• Salmonella

• Shigella

Shigella dysenteriae

Shigella flexneri

Shigella boydii

Shigella sonnei

• Serratia

Serratia marcesans

Serratia liquifaciens

• Yersinia

Yersinia enterocolitica

Yersinia pestis

Yersinia pseudotuberculosis

• Proteus

Proteus mirabilis

Proteus vulgaris

• Providencia

Providencia alcalifaciens

Providencia rettgeri

Providencia stuartii

• Morganella

Morganella morganii

SOME BIOCHEMICAL CHARACTERISTICS OF SOME NONLACTOSE FERMENTERS

Taxonomy

• Classification of salmonellae is complex

• the organisms are a continuum rather than a defined species.

• The members of the genus Salmonella were originally classified on the basis of epidemiology , host range, biochemical reactions, and structures of the O, H, and Vi (when present) antigens.

• The names (eg, Salmonella typhi, Salmonella typhimurium) were written as if they were genus and species;

• This form of the nomenclature is still widely used but is incorrect

Taxonomy

• By DNA -DNA hybridization studies there are currently in the genus Salmonella only two species each with multiple subspecies and serotypes.

• Salmonella enterica and Salmonella bongori

• Salmonella enterica contains five subspecies: Subspecies enterica(subspecies I); subspecies salamae (subspecies II); subspecies arizonae(subspecies IIIa); subspecies diarizonae (subspecies IIIb); subspecies houtenae (subspecies IV); and subspecies indica (subspecies VI).

• Most human illness is caused by the subspecies I strains, written as Salmonella enterica subspecies enterica.

• Rarely human infections may be caused by subspecies IIIa and IIIb or the other subspecies frequently found in cold-blooded animals.

• Currently the correct classification for what was called Salmonella typhimurium is

• Salmonella enterica subspecies enterica serotype Typhimurium, which can be shortened to Salmonella Typhimurium with the genus name in italics and the serotype name in roman type.

BRIEF SUMMARY OF CLINICAL DISEASES CAUSED BY SALMONELLAE

Enteric Fevers Septicemias Enterocolitis

Incubation period 7–20 days Variable 8–48 hours

Onset Insidious Abrupt Abrupt

Fever Gradual; then high plateau with“typhoidal” state

Rapid rise; then spiking “septic”temperature

Usually low

Duration of disease Several weeks Variable 2–5 days

Gastrointestinal symptoms Often early constipation; later,bloody diarrhea

Often none Nausea, vomiting, diarrhea atonset

Blood culture Positive from first week of the disease

Positive during high fever Negative

Stool culture Positive from second week on;negative earlier in disease

Infrequently positive Positive soon after onset

Urine Culture Positive from third week

Adapted from Jawetz, Melnick, & Adelberg’s Medical Microbiology Twenty-Eighth Edition

• Enteric fever – consists of : • Paratyphoid fever - Salmonella Paratyphi A, Salmonella Paratyphi B and

Salmonella Paratyphi C

• Typhoid fever - Salmonella Typhi

• Sepsis: Salmonella Choleraesuis and others

• Gastroenteritis: Salmonella Enteritidis and Salmonella Typhimurium

Epidemiology• Contaminated food is the major mode of transmission for

non-typhoidal salmonellae because

• Salmonellosis (non-typhoidal) is a zoonosis and has an enormous animal reservoir

• common animal reservoirs are • chickens, turkeys, pigs, and cows

• pet reptiles

• major mode of transmission of typhoid fever and other enteric fevers is fecooral, person-to-person spread

• humans are the only reservoir

• In typhoid fever and non-typhoidal salmonellosis, two other factors have epidemiologic significance:

• Asymptomatic human carrier state exists for both

• Use of antibiotics • in animal feeds and

• indiscriminate use of antibiotics in humans

• increase antibiotic resistance in salmonellae by promoting transfer of R factors

Gastroenteritis

• Incubation period for Salmonella gastroenteritis (food poisoning) depends on the dose of bacteria.

• Symptoms usually begin 6 to 48 hours after ingestion of contaminated food or water

• Usually take the form of nausea, vomiting, diarrhoea, and abdominal pain

• Myalgia and headache are common;

• However, the cardinal manifestation is diarrhoea.

• Fever (38oC to 39oC) and chills are also common.

• At least two-thirds of patients complain of abdominal cramps.

• The duration of fever and diarrhoea varies, but is usually 2 to 7 days.

Enteric fevers• Are severe systemic forms of salmonellosis which may be

fatal if antibiotics are not promptly administered.

• The best studied enteric fever is typhoid fever- S Typhi,

• Incubation period of 10 to 14 days.

• Infectious dose 105-8 organisms

• Enteric fevers may be preceded by gastroenteritis, which usually resolves before the onset of systemic disease

• The symptoms of enteric fevers are nonspecific and include fever, anorexia, headache, myalgias, and constipation.

• Enteric fever refers to typhoid fever and paratyphoid fever and is caused by:

• Typhoid fever - Salmonella Typhi

• Paratyphoid fever - Salmonella Paratyphi A, Salmonella Paratyphi B, Salmonella Paratyphi C

Typhoid feverEpidemiology• Typhoid fever is a bacterial disease, caused by Salmonella Typhi.

Transmission is via food or drinks contaminated with infected feces or urine.

• Typhoid fever is a major problem for people living in developing areas with poor sanitation and fecal contamination of food and water.

• It is estimated that there are at least 21 million new cases of typhoid fever each year and 216,000 deaths

• Incubation period 7-14 days

• Salmonella Typhi infects only humans.

Pathogenesis

S. Typhi organisms are ingested and survive exposure to gastric acid before gaining access to the small bowel, The organisms penetrate ileal mucosa and reach mesentric lymph nodes via lymphatics , they then multiply and invade the blood stream via thoracic duct. This is classically known as the first bacteremia.

From the blood stream they seed into the liver, gall bladder,, spleen, kidney, and bone marrow and after multiplication bacilli pass into blood causing the second phase of and a heavier bacteremia

• Involvement of the peyer’s patches, leads to inflammatory reaction, and infiltration with monocular cells, necrosis, sloughing and formation of chacteristic typhoid ulcers

Clinical features

Fever, headache, abdominal pain with constipation or diarrhea, relative bradycardia( Fagets sign), splenomegaly, and leukopenia abdominal symptoms and 'rose spots' which are pink rashes on the skin.

Fever is present in 75% to 100% of cases, is often initially of the remittent type, described as stepladder pattern of temperature but later becomes sustained,

Rose spots

• May present with rash, rose spots 2 -4 mm in diameter raised discrete irregular blanching pink maculae's found in front of chest

• Appear in crops of upto a dozen at a time

• Fade after 3 – 4 days

Complications

• Intestinal hemorrhage and intestinal perforation

• Typhoid psychosis

Carriers

• 1-3% of patients who recover become carriers. In carriers the bacteria remain hidden inside cells and the gall bladder, causing new infections as they are shed from an apparently healthy host.

History of a famous typhoid carrier• In the early 1900s Mary Mallon, an Irish immigrant to the US

worked as a cook with different families in New York City.

• There were outbreaks of typhoid fever among the families wherever she went. However this was not known until one family hired a sanitation engineer George Soper to investigate the cause of the outbreak in their family.

• Soper published the results on June 15, 1907, in the Journal of the American Medical Association. He believed Mallon was the source of the outbreak after discovering that she was the common link among many people who had become ill from typhoid fever

• The New York City Health Department finally sent Public healthphysician Sara Josephine Baker to talk to Mallon.

• She was called “Typhoid Mary” in a 1908 issue of the Journal of the American Medical Association

Diagnosis of Carriers

• Useful in public health purpose.

• Useful in screening food handlers, cooks, to detect carrier state

• Typhoid bacilli can be isolated from feces or from bile aspirates

• Detection of Vi agglutinins in the blood can be determinant of carrier state.

Investigations

• Blood cultures positive from 1st week to beyond 3rd week

• Stool cultures positive from 2nd week

• Urine cultures positive from 3rd week

• Bone marrow most sensitive but invasive

Widal test

• Serum agglutinins raise abruptly during the 2nd or 3rd week

• The widal test detects antibodies against O and H antigens

• Two serum specimens obtained at intervals of 7 – 10 days to read the raise of antibodies.

• Serial dilutions on unknown sera are tested against the antigens for respective Salmonella

• False positives and False negative limits the utility of the test

• The interpretative criteria when single serum specimens are tested vary

• Cross reactions limits the specificity

Limitations of Widal test

• Classically, a four-fold rise of antibody in paired sera Widal test isconsidered diagnostic of typhoid fever. However, paired sera are oftendifficult to obtain and specific chemotherapy has to be instituted onthe basis of a single Widal test.

• False positive reactions occur as a result of non-typhoid febrileconditions

Control

• Vaccines are available for typhoid fever

• Improved hygiene and sanitation measures

General salmonellosis treatment measures include;

• Replacing fluid loss by oral and intravenous routes, and Controlling pain, nausea, and vomiting.

• Specific therapy consists of antibiotic administration.

• Typhoid fever and sepsis should be treated with antibiotics. IV Ceftriaxone is effective

• Antibiotics are not recommended for uncomplicated Salmonella gastroenteritis because they do not shorten the illness and they significantly prolong the fecal excretion of the organisms and increase the number of antibiotic-resistant strains

Shigella

• Shigellosis has two basic clinical presentations:

(1) watery diarrhea associated with vomiting and mild to moderate dehydration, and

(2) Bacilliary dysentery characterized by a small volume of bloody, mucoid stools, abdominal pain/cramps and tenesmus

Infectious dose may be as low as 10 organisms

Flies may be important in the transmission of bacillary dysentery

• The genus Shigella is differentiated into four species:

• S dysenteriae (serogroup A)

• S flexneri (serogroup B)

• S. boydii (serogroup C)

• S sonnei (serogroup D)

Clinical Characteristics of Shigellosis

• Shigellae are facultative intracellular pathogens and may cause acute bloody dysentery with high fever and systemic manifestations of malaise, headache, and abdominal pain. The incubation period ranges from 6 hours to 9 days but is usually less than 72 hours.

• Shigella species are the most common cause of bloody diarrhea in children, and the syndrome may be particularly severe in poorly nourished children

• Diarrhea often precedes the dysentery.

• Dysentery is also characterized by the daily loss of 200-300 ml of serum protein into the feces.

• Depletion of immune factors also increases the risk of concurrent, unrelated infectious disease and contributes to substantial mortality .

Pathogenesis

• Shigella infection is superficial, and only rarely does the organism penetrate beyond the mucosa, so blood cultures in patients with shigellosis inspite of hyperpyrexia and toxemia are usually negative

• Shigella invade colonic and rectal cells, including M cells of the follicle-associated epithelium, macrophages, and epithelial cells; invasion is followed by intracellular multiplication, spread of infection to adjacent cells, severe inflammation, and destruction of colonic mucosa.

• Apoptotic destruction of macrophages in subepithelial tissue allows survival of the invading shigellae, and inflammation facilitates further bacterial entry.

• Once the organisms are intracellular, they multiply within the cytoplasm and move from cell to cell by an actin-dependent process.

• It produces toxins with enterotoxic, cytotoxic and neurotoxic properties

Diagnosis• Positive culture=> blood-tinged plugs of mucus in

freshly passed stool

• Initial streaking for isolation on differential/ selective media with aerobic incubation .

• Commonly used primary isolation media include MacConkey, Hektoen Enteric Agar, and Salmonella-Shigella (SS) Agar (contain bile salts to inhibit the growth of other Gram-negative bacteria).

• Following overnight incubation of primary isolation media at 37° C, colorless, non-lactose-fermenting colonies are streaked and stabbed into tubed slants of Kligler's Iron Agar or Triple Sugar Iron Agar.

• In these differential media, Shigella species produce an alkaline slant and an acid butt with no bubbles of gas in the agar.

• This reaction gives a presumptive identification, and slide agglutination tests with antisera for serogroupand serotype confirm the identification

Treatment• Fluid and electrolyte replacement to correct isotonic

dehydration, metabolic acidosis, and significant potassium loss

• Effective antibiotic treatment reduces the average duration of illness from approximately 5-7 days to approximately 3 days and also reduces the period of Shigella excretion after symptoms subside.

• Ciprofloxacin (500mg bd for 5 days) is effective

Proteus• P. mirabilis and P. vulgaris

• Swarming motility

• Non lactose fermenting

• Urease positive

• Hydrogen Sulphide positive

• P. vulgaris is indole positive

• Phenylalanine positive

• UTI and other infections

SWARMING OF PROTEUS ON BLOOD AGAR

Yersinia

• The genus Yersinia contains three species of medical importance:

• Y pestis, the agent of bubonic and pneumonic plague,

• Y pseudotuberculosis

• Y enterocolitica- both of which can result in severe gastroenteritis, with local abscess formation and death as a result of peritonitis.

• Y. pestis is a gram-negative coccobacillus that exhibits bipolar-staining with Giemsa, Wright’s, or Wayson staining.

History and Epidemiology• Primarily an infection of rodents and their fleas, but

plague has repeatedly spread to humans, causing at least three major pandemics

• The first pandemic, or Justinian Plague, originated in central Africa and affected much of the Mediterranean basin during the sixth-century Byzantine Empire.

• The second pandemic began in 1347 and spread rapidly throughout Europe, killing an estimated one fourth of the population

• The third, or modern, pandemic began in China in the 1860s and spread to Hong Kong, where Alexandre Yersin isolated the causative agent named after him. Y. pestis was spread by rats on steamships to port cities on all inhabited continents.

Clinical features

Antigenic structures of Yersinia pestis

Pathogenesis

• Fleas become infected by feeding on a bacteremic host the bacilli colonize the flea midgut, replicate, and create a blockage of the flea intestine.

• These starved fleas with blocked gut feed aggressively, regurgitating bacteria into the bite wound at each attempt

• The inoculated bacteria express very little F1 capsular antigen, and are phagocytized and killed by polymorphonuclear leukocytes a few bacteria may be taken up by mononuclear cells which are unable to kill them

• the bacteria multiply intracellularly and begin to produce F1 envelope antigen. If lysis of the mononuclear cells occurs, the released F1-producing bacilli are relatively resistant to further phagocytosis

• The invading organisms are carried via lymphatics to the regional lymph nodes, where they initiate an intense inflammatory reaction, creating a bubo

• Microscopic examination of the involved nodes reveals invasion by polymorphonuclear leukocytes, hemorrhagic necrosis with destruction of normal architecture, and dense concentrations of extracellular bacilli.

• Bacteremia is common and, in the absence of antibiotic therapy, can lead to sepsis, pneumonia, and purulent, necrotic, and hemorrhagic lesions in various organs

Clinical Feature

• Plague may present as bubonic plague, septicemic plague or pneumonic plague

• The usual incubation period is 2 to 7 days, but can be as short as 1 day in primary pneumonic exposure

• Bubonic plague is characterized by the sudden onset of fever, chills, weakness, and headache, accompanied by regional lymphadenitis, usually in the groin, axilla, or neck. The swollen lymph nodes, or buboes, are so tender that the patient may avoid any motion that would provoke discomfort

• In septicemic plague which occurs rarely bacteria may proliferate in the body without producing a bubo and patients may become ill with fever and die with sepsis but without detectable lymphadenitis.

• Pneumonic plague occurs in two forms, secondary and primary. Secondary pneumonic plague is the more common form and arises through hematogenous spread of bacteria from a bubo or other source especially due to delayed treatment of bubonic infections.

• Primary pneumonic plague results from direct inhalation of bacteria into the lungs as a result of contact with another patient with pneumonic plague, exposure to animals with respiratory or pharyngeal plague, laboratory exposures, or, potentially, as a result of intentional aerosol release for bioterrorism or biowarfare

• Patients with plague pneumonia experience rapidly advancing tachypnea, dyspnea, hypoxia, chest pain, cough, hemoptysis, and general signs of endotoxemia.

Diagnosis

• Yersinia infections must be diagnosed quickly due to the extraordinary virulence of these organisms

• Death from pneumonic plague can occur in as little as 24 hours after the first appearance of clinical symptoms.

• Sputum specimens- Gram-negative coccobacilli.

• Blood cultures –positive

• lymph node biopsy material shows a massive inflammatory cell infiltrate, together with numerous cell-free coccobacilli

• The organisms can be identified using a fluorescent antibody staining technique

• Epidemiology of the outbreak can be traced by bacteriophage typing.

• Yersinia pestis poses a serious infectious hazard for nursing and laboratory personnel.

• Cultivation and virulence testing of this organism should be attempted only in P-3 containment facilities by staff who have been immunized recently with live attenuated vaccine

Providencia

• P. alcalifaciens, P. heimbachae, P. rettgeri, P. rustigianii, and P. stuartii.

• Phenylalanine deaminase -- +ve.

• lysine and ornithine decarboxylase + arginine dihydrolase-- negative

• Produce acid from D-mannose

• Recovered from urine, throat, perineum, axilla, stool, blood, and wound specimens

• P. stuartii has long been recognized as a pathogen for nursing home patients with chronic indwelling urinary catheters

• There have been incidents of P. rettgeri causing health care associated infections

• Urine isolates are susceptible to the expanded-spectrum oral cephalosporins, including cefaclor, cefuroxime, cefpodoxime, ciprofloxacin, and amoxicillin-clavulanic acid

• Alternative choices for antimicrobial therapy would include ceftriaxone, mezlocillin, imipenem, and trimethoprim-sulfamethoxazole

Morganella

• The genus Morganella currently consists of one species, Morganella morganii, with two subspecies, morganii and sibonii.

• M. morganii is an opportunistic secondary invader that was originally thought to be the cause of summer diarrhea

• M. morganii has caused infections in neonates and the immunocompromised

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