Enterobacteriaceae (Gram-negative bacilli) PDF

## Enterobacteriaceae (Gram-negative bacilli) ### **Escherichia** * **Escherichia coli (Colon bacillus)** * It is the most significant species in the genus Escherichia. * It may inhabit the female genital tract although it is a microbiota of the large intestine. * It is a primary indicato...

## Enterobacteriaceae (Gram-negative bacilli) ### **Escherichia** * **Escherichia coli (Colon bacillus)** * It is the most significant species in the genus Escherichia. * It may inhabit the female genital tract although it is a microbiota of the large intestine. * It is a primary indicator of fecal contamination in water purification. * It is the leading cause of nosocomial urinary tract infection (UTI). * It has both the adhesive fimbriae and sex pili. * It has several serotypes causing diarrhea and UTI. * **Mode of transmission:** person-to-person contact, fecal-oral route, and through consumption of contaminated food and water. * **Virulence factors:** Endotoxin, common pili, K1 antigen, and intimin, attachment factors. * **Antigenic determinants:** O, H, and K antigens. * **Culture:** * **MAC:** Colonies appear flat and dry, and they exhibit a pink color (lactose fermenter or LF); some strains may be non-lactose fermenters or NLF. * **BAP:** Most strains are non-hemolytic though some exhibit a β-hemolytic pattern. * **EMB:** Colonies appear as greenish metallic sheen. * **IMViC (Indole, methyl red, Voges-Proskauer, and citrate) reaction:** + +-- * **TSIA (triple sugar iron agar) reaction:** A/A (acidic slant/acidic butt), (+) gas, (-) hydrogen sulfide or H₂S. * **Related infection/disease:** Bacteremia, UTI, hemolytic uremic syndrome (HUS), and neonatal meningitis. * **Escherichia hermanii** * It is formerly called E. coli atypical or enteric group II. * It has been isolated from CSF, wound infection, and blood. * **Culture:** Colonies have yellow pigmentation. * **Other Species of the Genus Escherichia** * E. albertii, E. blattae, E. fergusonii, and E. vulneris. ### **Klebsiella** * The species of this genus are usually found in the GIT of humans and animals. * **Culture:** * **MAC:** Colonies exhibit a pink color (LF) with mucoid consistency. * **Growth on media with potassium cyanide (KCN):** Positive. * **IMViC reaction:** - - + + * **TSIA reaction:** A/A, (+) gas, (-) H₂S * **Species:** * K. pneumoniae subsp. pneumoniae, * K. oxytoca, * K. ozaenae, * K. rhinoscleromatis and * K. ornithinolytica (Raoultella ornithinolytica), * K. planticola (Raoultella planticola), and * K. terrigena (Raoultella terrigena). * **Klebsiella pneumoniae subsp. pneumoniae (Friedlander's bacillus)** * It is the most commonly isolated species of Klebsiella. * It is the frequent cause of lower respiratory tract infections among hospitalized patients (hospital-acquired pneumonia) and in immunocompromised hosts such as newborns, elderly patients, and patients on respirators. * It is an agent of ventilator-associated pneumonia due to carbapenemase-producing K. pneumoniae. * It produces a "currant jelly-like" sputum among infected individuals. * **Virulence factor:** Polysaccharide capsule. * **Differential Test:** (+) String test * **Neufeld-Quellung Test:** Positive * **Growth on media with potassium cyanide (KCN):** Positive * **IMVIC reaction:** - - + + * **TSIA reaction:** A/A, (+) gas, (-) H₂S. ### **Enterobacter** * The members of this genus resemble those of Klebsiella when grown on a MacConkey agar. * The species can cause bacteremia, nosocomial pneumonia, and urinary tract infection. * **Culture: MAC:** Colonies exhibit a pink color and are sometimes mucoid (LF). * **Growth on media with KCN:** Positive. * **Ornithine decarboxylase test:** Positive. * **Lysine decarboxylase (LDC) test:** Positive (all species except E. cloacae and E. gergoviae). * **Sorbitol fermentation:** Positive (E. aerogenes and E. cloacae). * **Urease and Malonate test:** Positive (E. cloacae). * **IMVIC reaction:** - - + + * **TSIA reaction:** A/A, (+) gas, (-) H₂S. * **Clinically significant species:** * E. aerogenes, * E. cloacae subsp. cloacae, * E. cancerogenus, and * E. hormaechei. * **Common isolates:** E. aerogenes and E. cloacae. * **Enterobacter cancerogenus (formerly E. taylorae)** has been isolated from individuals with osteomyelitis following traumatic wounds. ### **Cronobacter** * **Cronobacter sakazakii** * It is formerly known as Enterobacter sakazakii. * It is found as a contaminant of powdered infant formula. * It is isolated from individuals with brain abscesses and respiratory and wound infections. * **Culture:** * **MAC:** Colonies exhibit a pink color (LF). * **BHIA:** Colonies exhibit a yellow pigmentation with mucoid appearance. * **IMVIC reaction:** - - + + * **TSIA reaction:** A/A, (+) gas, (-) H₂S. ### **Pantoea** * **Pantoea agglomerans** * It is formerly known as Enterobacter agglomerans. * It causes nosocomial outbreaks of septicemia due to contaminated IV fluids. * It shows a triple decarboxylase negative reaction. * **Culture:** * **MAC:** Colonies are clear or colorless (NLF). * **BAP:** Colonies are large with yellow color. * **IMVIC reaction:** - v + v * **TSIA reaction:** K/A (Alkaline slant/Acidic butt), (-) gas, (-)H₂S. ### **Serratia** * The species in this group are opportunistic pathogens that are usually associated with nosocomial outbreaks such as pneumonia. * The species are resistant to a wide range of antibiotics. * **Culture: MAC:** Colonies are clear and colorless (NLF); some strains may exhibit late lactose fermentation. * **IMVIC reaction:** - - + + * **TSIA reaction:** K/A or A/A, (+) gas, (-) H₂S. * **Other biochemical test:** (+) DNAse, gelatinase, lipase, and ONPG. * **Species:** * S. marcescens subsp. marcescens, * S. rubidaea, * S. plymuthica, * S. ficaria, * S. entomophila, * S. fonticola and * S. liquefaciens complex. * **Biogroups:** S. odorifera biogroups 1 and 2. * **Serratia marcescens** * It is the most clinically significant species of the genus. * It causes bacteremic outbreaks in nurseries and cardiac surgery and burn units. * A few strains of this species are late lactose fermenters, pink in color after 48 hours. * **Biochemical test:** (+) Urease, gelatinase, and ONPG; (-) arabinose fermentation * **Notes to Remember:** * S. marcescens, S. rubidaea, S. liquifaciens, and S. plymuthica produce pink to red colonies (due to the production of prodigiosin pigment) after incubation at 25°C. * S. odorifera has a musty and pungent odor or a "rotten potato-like" odor. * S. liquefaciens ferments arabinose and exhibits growth in a culture medium with KCN. * DNAse test distinguishes Serratia spp. (positive) from Enterobacter spp. (negative). ### **Proteus** * The members are isolated from urine, wound, and ear infection. * The species can infect the proximal kidney tubules and can cause acute glomerulonephritis (AGN), particularly in patients with UTI or catheterization. * The members of this genus are rapid urease producers. Urease splits urea leading to increased urine pH and eventually promote renal stone formation. ("staghorn calculi"). * **Species:** * P. mirabilis, * P. vulgaris, * P. penneri, and * P. myxofaciens. * **Human pathogens:** P. mirabilis and P. vulgaris. * **Most common isolate:** P. mirabilis. * **Culture: MAC:** Colonies are clear and colorless (NLF) with "swarming phenomenon" and "burnt-chocolate" or "burnt-gunpowder" odor. * **Phenylalanine deaminase (PAD) test:** Positive. * **Lysine iron agar (LIA) reaction:** R/A (red slant acid butt). * **IMVIC reaction:** * **P. mirabilis:** - + v v * **P. vulgaris:** + + - v * **TSIA reaction:** * **P. mirabilis:** K/A, (+) gas, (+) H₂S. * **P. vulgaris:** K/A, (+/-) gas, (+) H₂S. ### **Citrobacter** * **Citrobacter koseri (formerly C. diversus)** * It causes outbreaks of neonatal meningitis and brain abscess in nursery units. * **Table 19-7. Differential Tests for Citrobacter Species:** | Biochemical Tests | C. freundii | C. koseri | |---|---|---| | Indole | - | + | | H₂S production | + | - | | Growth in KCN | + | - | | ONPG | + | + | | IMVIC | +-+ | +++ | | TSIA | A/A or K/A, (+) gas, (+) H₂S | K/A, (+) gas, (-) H₂S | ### **Salmonella** * The species of this genus are the most pathogenic enterobacteria that cause enteric fever (typhoid fever) and acute gastroenteritis (food poisoning) to humans. * They are not part of the human intestinal flora but commensals of the gastrointestinal tract of animals. * They may be transmitted by human carriers. * **Modes of acquisition:** Ingestion of contaminated animal food products or improperly cooked poultry, milk, eggs, and dairy; and direct human contact. * **Species:** * S. enterica (type species) and * S. bongori. * **Subspecies of S. enterica:** * S. enterica subsp. enterica (I), * S. enterica subsp. salamae (II), * S. enterica subsp. arizonae (IIIa), * S. enterica subsp. diarizonae (IIIb), * S. enterica subsp. houtenae (IV), and * S. enterica subsp. indica (VI). * **Most common human isolate:** S. enterica subsp. enterica (I). * **Virulence factor:** Fimbriae and enterotoxin (S. enterica). * **Culture:** * **MAC:** Colonies are clear and colorless (NLF). * **SSA:** Colonies are colorless with black centers. * **Antigenic structures:** Somatic O and flagellar H - for serologic grouping. * **Vi antigen (Salmonella serotype Typhi):** antiphagocytic; may interfere with intracellular killing. (part of the capsule of the Salmonella). * **Salmonella bongori** * It is named after the town of Bongor in Chad, Africa, where it was isolated from a host lizard in 1966. * It can also be isolated from other cold-blooded animals. * **Salmonella Serotypes and its Significance** * All former species of Salmonella have been designated as serotypes under S. enterica subsp. enterica (e.g., S. enterica subsp. enterica serotype Typhi). * The genus Salmonella has thousands of serotypes. (Source: https://www.cdc.gov/salmonella/reportspubs/salmonella-atlas/serotyping-importance.html). * **Examples of Salmonella serotypes:** Salmonella serotype Typhi, Salmonella serotype Paratyphi A, B, and C, and Salmonella serotype Choleraesuis, and Salmonella serotype Typhimurium * **Main causative agent of enteric fever:** Salmonella serotype Typhi * **Etiologic agents of paratyphoid fever:** Salmonella serotype Paratyphi A, B, and C, and Salmonella serotype Choleraesuis. * **Biochemical Characteristics of Salmonella Species** * All species are motile except Salmonella serotype Pullorum and Salmonella serotype Gallinarum. * All species produce gas except for Salmonella serotype Gallinarum and Salmonella serotype Typhi. * All species produce H₂S except for Salmonella serotype Paratyphi A. * **LDC:** Positive (except for Salmonella serotype Paratyphi A * **Urease:** Negative * **Growth on media with KCN:** Negative * **IMVIC reaction:** - + - - * - + - - (Salmonella serotype Typhi) * **TSIA reaction:** * K/A, (+) gas, (+) H₂S. * K/A, (-) gas, (+) H₂S (Salmonella serotype Typhi) * **General Categories of Salmonella Infection** * Salmonellae are found in various animals that serve as reservoirs and sources of infections, except Salmonella serotype Typhi and Salmonella serotype Paratyphi. * The carriers of Salmonella excrete the organisms along with their feces and thus cause "accidental" contamination of food if the person is unhygienic or if there are issues on water sanitation. * The carriers can be treated by antimicrobial therapy if the gallbladder infection is not evident. * Isolation of salmonellae from culture plates is significant and specific for diagnosis of typhoid fever. * Cholecystectomy can be recommended to enteric carriers in a chronic state. * **1. Acute Gastroenteritis** * It is one of the most common forms of food poisoning. * It is commonly caused by S. enterica subsp. enterica that comes from animals. * The Salmonella serotype Typhimurium has been involved in outbreak from contaminated food such as peanut butter crackers and cereals. * **Sources of infection**: Poultry products, milk, and handling of pets. * **Mode of dissemination**: Contaminated kitchen utensils. * **Infective dose**: 10⁶ bacteria. * **Symptoms**: Nausea, vomiting, fever and chills, watery diarrhea, and abdominal pain. * **2. Enteric fever (Typhoid fever)** * It is a febrile disease that develops from eating contaminated food prepared by infected individuals or carriers. * **Mode of transmission:** Through fomites is also possible. The site of long-term carriage is the gallbladder. * **Most common agent causing enteric fever:** Salmonella enterica subsp. enterica serotype Typhi * **Other agents of enteric fever (paratyphoid fever):** Salmonella serotype Paratyphi A, B, and C and Salmonella serotype Choleraesuis. * **Sources of infection:** Human carriers, food handlers, contaminated food, and water. * **Causes of outbreaks:** Improper sewage disposal, poor sanitation, and lack of clean water supply. * **Symptoms:** Malaise, anorexia, myalgia, and severe frontal headache. * **Complications:** Necrosis in the gallbladder (necrotizing cholecystitis) and Peyer's patches or lymphoid issues of the bowel. * **Hallmark of infection:** Appearance of "rose spots" during the second week of fever. * **3. Bacteremia** * It occurs with and without extraintestinal infection that is caused by non-typhoidal Salmonella species. * It is characterized by prolonged fever and intermittent bacteremia. * **Causative agents:** Salmonella serotype Typhimurium, Salmonella serotype Paratyphi, and Salmonella serotype Choleraesuis. * **Specimens for Salmonella Identification:** * **Blood:** first week of infection. * **Stool:** second to third week of infection. * **Urine:** third week of infection. * **Bone marrow:** The best specimens for the detection of the carrier state of Salmonella serotype Typhi is bone marrow. * **Table 19-8. Biochemical Differentiation of Salmonella Serotypes** | Biochemical Tests | S. serotype Typhi | S. serotype Paratyphi | S. serotype Choleraesuis | Other serotypes | |---|---|---|---|---| | Citrate utilization | + | - | + | + | | Gas production | + | + | + | + | | H₂S production | + | + | v | + | | Lysine decarboxylase (LDC) | + | + | + | + | | Ornithine decarboxylase | + | + | + | + | | Arabinose fermentation | + | + | + | + | * *v - variable (10% to 89% of strains are positive).* ### **Shigella** * The species of this genus are closely related to those of Escherichia. * The members are neither human microflora or animal commensals; they are non-motile, intracellular pathogens that multiply within the cells of the intestinal epithelium (enterocytes). * Most of the species can cause bacillary dysentery. * **Modes of transmission:** Four F's (flies, fingers, food, fecal); food and water from infected persons, and fecal-oral route. * **Reservoir:** Human carriers. * **Species:** * S. dysenteriae, * S. flexneri, * S. boydii, and * S. sonnei. * **Most virulent species:** S. dysenteriae (produces Shiga toxin and is considered a cause of gay bowel syndrome). * **Serogroups:** * A (S. dysenteriae), * B (S. flexneri), * C (S. boydii), and * D (S. sonnei). * **Antigenic structure:** Somatic O. * **Culture:** * **MAC:** Colonies are clear, fragile, and colorless (NLF). * **SSA:** Colonies are colorless without black centers. * **Urease test:** Negative * **LDC test:** Negative. * **IMVIC reaction:** v + -- * **TSIA reaction:** K/A, (-) gas, (-) H₂S. * **Biochemical Characteristics of Shigella Species** * All species do not produce gas from glucose except some strains of S. flexneri. * All species are mannitol fermenters except S. dysenteriae. * All species do not decarboxylate lysine. * All species do not decarboxylate ornithine except S. sonnei. * S. sonnei is a late lactose fermenter and has a positive reaction with the ONPG test. * **Shigella dysenteriae** * It is the most virulent of the species and causes bacillary dysentery. * **Virulence factor:** Shiga toxin. * **Shigella sonnei** * The infection from this organism is self-limiting, and it is usually characterized by fever and watery diarrhea (stool without blood). * It has one serotype as opposed to the other species. * **Shigella flexneri** * It is one of the agents of gay bowel syndrome. * It causes gastroenteritis among males who have sexual relationship with another male. * **Bacillary Dysentery** * It is an enteric infection that is most commonly caused by S. dysenteriae type 1. * It is characterized by acute inflammatory colitis and bloody diarrhea (blood, mucus, and WBCs in the stool) due to the attachment of the organisms to mucosal surfaces and formation of ulcers after epithelial penetration. * It is highly communicable because of the low infective dose that is required to produce the disease (< 200 bacilli). * In young children, rectal prolapse occurs due to the excessive straining. * **Source of infection:** Human carriers. * **Modes of transmission:** 4Fs, person-to-person contact, and contaminated water from infected persons. * **Symptoms:** Fever, chills, abdominal cramps, painful bowel movement, and tenesmus (urge to pass stool even though the bowel is empty). * **Complications:** Ileus (obstruction of the intestine), seizure, and hemolytic-uremic syndrome (HUS). (low motility of some part of the intestine). * **Notes to Remember:** * Poor personal hygiene is an important factor in the transmission of Shigella spp. * Shigella causes outbreak in areas where houses are in close proximity, in military camps, in daycare centers and among travelers. * Shigellae are sensitive to pH changes (susceptible to acidic stool) so stool specimens should be inoculated immediately after collection to increase the recovery of the organisms. * Aside from stool specimens, rectal swabs can be used for the isolation of Shigella. ### **Yersinia** * The species are predominantly isolated from the environment such as soil and water. * **Human pathogens:** * Y. pestis, * Y. enterocolitica subsp. enterocolitica, and * Y. pseudotuberculosis. * **Yersinia pestis (Plague bacillus)** * It is a class A bioterrorism agent. * It is a recognized zoonotic bacterium. * It is not part of the indigenous microbiota of human GIT and is a non-motile enterobacterium. * It is the only enterobacteria that is transmitted to humans through the bite of an infected flea. * It is the causative agent of the bubonic plague. (black death). * It can be isolated on a routine culture media, and it grows best at 25°C to 30°C. * **Vector:** Xenopsylla cheopis (Oriental rat flea). * **Reservoir:** Rats. * **Virulence factors:** Endotoxin, coagulase, and fibrinolysin * **Microscopy:** * **Gram staining:** Short, plump rod. * **Wayson stain or Methylene blue:** Appear as bipolar bodies exhibiting "closed safety pin" * **Culture:** * **MAC:** Colonies are clear and colorless (NLF). * **BAP:** Colonies are pinpoint at 24 hours. * **Broth:** Colonies have a "stalactite-shaped" pattern. * **IMVIC reaction:** - + - - * **TSIA reaction:** K/A, (-) gas, (-) H₂S. * **Urease test:** Negative. * **Yersinia enterocolitica subsp. enterocolitica** * It is the most commonly isolated species of Yersinia. * It is the causative agent of enterocolitis or waterborne gastroenteritis. * It is motile in SIM at 25°C by peritrichous flagella but not at 35°C * It has been isolated from contaminated blood (packed RBC) units, hence it is considered as a blood transfusion hazard. * It has the ability to survive in cold temperature ("cold enrichment"). * **Modes of acquisition:** Ingestion of undercooked food (pork and pork intestines, vacuum-packed meat and chicken) and dairy products (chocolate milk), and handling pets. * **Reservoir:** Swine, dogs, cats, rabbits, and cows. * **Related infections:** Appendicitis-like syndrome, arthritis, and erythema nodosum. * **Selective culture medium**: Cefsulodin-irgasan-novobiocin (CIN) agar. * **Microscopy:** Coccobacilli with bipolar bodies. * **Culture:** * **MAC:** Colonies are clear and colorless (NLF). * **CIN:** "Bull's-eye" appearance of colonies or burgundy centers with transparent borders after incubation at room temperature for 48 hours. * **IMVIC reaction:** v + - - * **TSIA reaction:** K/A, (-) gas, (-) H₂S. * **Urease test:** Positive. * **Yersinia pseudotuberculosis** * It is a pathogen of the rodents, particularly guinea pigs. * It has been isolated in avian animals such as turkey and pigeons. * In humans, it causes septicemia with lymphadenitis while pseudotubercles infection is observed in animals. * It has similar morphological characteristics with the plague bacillus. * It is motile in SIM at 18°C to 25°C but not at 35°C. * It may be isolated after a cold enrichment technique. * **Modes of acquisition:** Direct contact with infected animals or their feces, and ingestion of contaminated food and water. * **Reservoir:** Birds. * **Culture:** * **MAC:** Colonies are clear and colorless (NLF). * **CIN:** Small, "red pin-like" colonies without transparent borders. * **IMVIC reaction:** - + - - * **TSIA reaction:** K/A, (-) gas, (-) H₂S. * **Other biochemical test:** (+) Urease and rhamnose fermentation. * **Table 19-9. Differential Tests for Yersinia Species** | Biochemical Tests | Y. pestis | Y. enterocolitica | Y. pseudotuberculosis | |---|---|---|---| | Indole | - | - | - | | Methyl red | + | + | + | | Voges-Proskauer (25°C) | - | + | - | | Motility | - | + | + | | 25°C | + | + | + | | 37°C | - | - | - | | Urease | + | + | + | | Ornithine decarboxylase | - | + | - | | Sucrose fermentation | - | + | - | | Beta-galactosidase | + | + | + | ### **Plesiomonas** * **Plesiomonas shigelloides** * It is the only species in the genus Plesiomonas. * It is not part of the indigenous human microbiota and is considered a true pathogen. * It is found in fresh and estuarine water, and it has been isolated from warm- and cold-blooded animals. * It often cross-agglutinates with Shigella, hence the species name "shigelloides". * It is the only oxidase-positive member of the Enterobacteriaceae. * It is motile by monotrichous or lophotrichous flagella. * It has been isolated from HIV-positive individuals with inflammatory bowel disease. * It is considered as an occupational hazard among veterinarians, fish handlers, and those working with aquatic sports. * **Mode of acquisition:** Ingestion of undercooked seafood (oysters and shrimps) and contaminated water, they gain entry thru skin cuts * **Virulence factor:** hemolysins, cytotoxins, production of exoenzymes * **Microscopy:** Straight bacilli which can occur singly, in pairs, in short chains, or filamentous * **Vibriostatic Test O/129:** Sensitive * **Related infection:** Secretory diarrhea, neonatal meningitis, and septicemia * **Cultural Characteristics** * **BAP:** Colonies are shiny, opaque, and non-hemolytic. * **MAC:** Colonies are clear and colorless (NLF). Some strains will not grow on MAC. * **Inositol-brilliant green-bile salt agar:** Colonies exhibit white or green to pink color while Aeromonas species are colorless. This medium enhances the recovery of plesiomonads from specimens. * **HEA:** Colonies exhibit growth. * **TCBS:** Colonies do not exhibit growth. (ink-ended for vibrio spp., particularly V. cholerae). * **Media with NaCl:** Colonies do not exhibit growth. * **Biochemical and Serological Characteristics:** * **Carbohydrate fermentation test:** Glucose, maltose, trehalose. * **Oxidase test:** Positive. * **Decarboxylase test:** Positive trio decarboxylate test, positive in arginine, ornithine lysine. * **Inositol fermentation:** Positive. * **IMVIC reaction:** ++-- * **TSIA reaction:** K/A, (-) gas, (-) H₂S. * **Antigenic determinant:** Somatic O and flagellar H * **Laboratory Diagnosis of Enterobacteriaceae** * **Specimens:** stool, rectal swab, blood, urine, wound discharge, and CSF * The members of Enterobacteriaceae are commonly isolated from stool specimens hence, fecal samples should not be used for routine testing unless the diagnosis involves possible true intestinal pathogens. * Stool specimens submitted for routine testing should also include culture or toxin detection to identify EHEC.