MLAB 2434 – Microbiology Keri Brophy-Martinez Enterobacteriaceae MLAB 2434 – Microbiology Keri Brophy-Martinez

Overview- Enterics Family Enterobacteriaceae often referred to as “enterics” Four major features: All ferment glucose (dextrose) All reduce nitrates to nitrites All are oxidase negative, except Plesiomonas All except Klebsiella, Shigella and Yersinia are motile

Microscopic and Colony Morphology Gram negative bacilli or coccobacilli Non-spore forming Facultative anaerobe Colony morphology on BAP or CA of little value, as they look the same large, moist and grey on SBA Selective and differential media are used for initial colony evaluation ex. MacConkey, HE, XLD agars

Classification of Enterics Due to the very large number of organisms in the Family Enterobacteriaceae (see Table 19-1, page 429), species are grouped into Tribes, which have similar characteristics Within each Tribe, species are further subgrouped under genera

Virulence and Antigenic Factors of Enterics Ability to colonize, adhere, produce various toxins and invade tissues Some possess plasmids that may mediate resistance to antibiotics ESBL’s: Extended-Spectrum beta lactamases Inactivate extended spectrum antibiotics like penicillins, cephalosporins and aztreonam

Virulence and Antigenic Factors of Enterics Many enterics possess antigens that can be used to identify groups O antigen – somatic, heat-stable antigen located in the cell wall, made by all bacteria, stimulates EARLY antibody production H antigen – flagellar, made by bacteria with flagella, heat labile antigen,stimulates LATE antibody production K antigen – capsular, heat-labile antigen, made by some bacteria Vi- capsular antigen of S. typhii

Clinical Significance of Enterics Enterics are ubiquitous in nature Except for few, most are present in the intestinal tract of animals and humans as commensal flora; therefore, they are sometimes call “fecal coliforms” Some live in water, soil and sewage

Clinical Significance of Enterics (cont’d) Based on clinical infections produced, enterics are divided into two categories: Opportunistic pathogens – normally part of the usual intestinal flora that may produce infection outside the intestine Primary intestinal pathogens – not commensal flora, they produce infection from ingestion of contaminated food or water (Salmonella, Shigella, and Yersinia sp.)

Escherichia coli Most significant species in the genus Important potential pathogen in humans Common isolate from colon flora

Escherichia coli (cont’d) Infections Wide range including meningitis, gastrointestinal, urinary tract, wound, and bacteremia Urinary Tract Infections E. coli is most common cause of UTI and kidney infection in humans Usually originate in the large intestine Able to adhere to epithelial cells in the urinary tract

Escherichia coli (cont’d) Septicemia & Meningitis E. coli is one of the most common causes of septicemia and meningitis among neonates; acquired in the birth canal before or during delivery E. coli also causes bacteremia in adults, primarily from a genitourinary tract infection or a gastrointestinal source

Escherichia coli (cont’d) Gastrointestinal Infections Enteropathogenic (EPEC) – primarily in infants and children; outbreaks in hospital nurseries and day care centers; stool has mucous but not blood; identified by serotyping

Escherichia coli (cont’d) Enterotoxigenic (ETEC) – “traveler’s diarrhea”; watery diarrhea without blood; self-limiting; usually not identified, other than patient history and lactose-positive organisms cultured on differential media Enteroinvasive (EIEC) – produce dysentery with bowel penetration, invasion and destruction of intestinal mucosa; watery diarrhea with blood; do NOT ferment lactose; identified via DNA probes

Escherichia coli (cont’d) Enteroadherent E. coli Enteroaggregative (EAEC) Cause diarrhea by adhering to the mucosal surface of the intestine; watery diarrhea; symptoms may persist for over two weeks Diffusely adherent(DAEC) Associated with UTI’s and diarrheal disease, esp. in children and pregnant women

Escherichia coli (cont’d) Enterohemorrhagic (EHEC serotype 0157:H7) Origin Isolated in 1970 Undercooked hamburger, unpasteurized milk and apple cider have spread the infection Pathogenesis Circulating Shiga toxin binds to kidney endothelium causing an inflammatory response Macrophages and neutrophils damage the endothelium and glomerular basement membrane Associated with hemorrhagic colitis, TTP and hemolytic-uremic syndrome (HUS), which includes low platelet count, hemolytic anemia, and kidney failure; potentially fatal, especially in young children

Escherichia coli (cont’d) Enterohemorrhagic (EHEC serotype 0157:H7) Clinical Symptoms Starts with a watery diarrhea then progresses to bloody diarrhea. No WBC’s are found in stool Laboratory Diagnosis Does NOT ferment sorbitol Identified by serotyping, latex tests

Escherichia coli (cont’d) General Characteristics Dry, pink (lactose positive) colony with surrounding pink area on MacConkey

Other Escherichia species Escherichia hermannii – yellow pigmented; isolated from CSF, wounds and blood Escherichia vulneris - yellow pigmented; wounds

Escherichia coli (cont’d) Characteristics Ferments glucose, lactose, trehalose, & xylose Positive indole and methyl red tests Does NOT produce H2S or phenylalanine deaminase Simmons citrate negative Usually motile Voges-Proskauer test negative Fimbriae O, H, K antigens

Klebsiella, Enterobacter, Serratia & Hafnia sp. Usually found in intestinal tract Wide variety of infections, primarily pneumonia, wound, and UTI General characteristics: Some species are non-motile Simmons citrate positive H2S negative Phenylalanine deaminase negative Some weakly urease positive MR negative; VP positive

Klebsiella species Usually found in GI tract K. pneumoniae is mostly commonly isolated species Possesses a polysaccharide capsule, which protects against phagocytosis and antibiotics AND makes the colonies moist and mucoid Has a distinctive “yeasty” odor Frequent cause of nosocomial pneumonia

Klebsiella species (cont’d) Significant biochemical reactions Lactose positive Most are urease positive Non-motile

Enterobacter species Comprised of 12 species; E. cloacae and E. aerogenes are most common Isolated from wounds, urine, blood and CSF Major characteristics Motile Simmons citrate positive MR negative; VP positive

Enterobacter species (cont’d)

Serratia species Seven species, but S. marcescens is the only one clinically important Frequently found in nosocomial infections of urinary or respiratory tracts Implicated in bacteremia, septicemia, cardiac surgery, and burn units Fairly resistant to antibiotics

Serratia species (cont’d) Major characteristics Ferments lactose slowly Produce characteristic pink or red pigment, especially when cultures are left at room temperature S. marscens on nutrient agar →

Hafnia species Hafnia alvei is only species Has been isolated from many anatomical sites in humans and the environment Occasionally isolated from stools Delayed citrate reaction is major characteristic

Proteus, Morganella & Providencia species All are normal intestinal flora Opportunistic pathogens Deaminate phenylalanine All are lactose negative

Proteus species P. mirabilis and P. vulgaris are widely recognized human pathogens Isolated from urine, wounds, and ear and bacteremic infections Both produce swarming colonies on non-selective media and have a distinctive “burned chocolate” odor Both are strongly urease positive Both are phenylalanine deaminase positive

Proteus Lactose negative

Proteus species (cont’d) A: exhibits characteristic “swarming” B: shows urease positive on right

Morganella species Morganella morganii is only species Documented cause of UTI Isolated from other anatomical sites Urease positive Phenylalanine deaminase positive

Providencia species Providencia rettgeri is pathogen of urinary tract and has caused nosocomial outbreaks Providencia stuartii can cause nosocomial outbreaks in burn units and has been isolated from urine Both are phenylalanine deaminase positive

Citrobacter species Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses) Ferments lactose and hydrolyzes urea slowly Methyl red positive, Simmons citrate positive Resembles Salmonella sp.

Salmonella Produce significant infections in humans and certain animals Infections caused by ingestion of food or water contaminated with either human or animal feces

Salmonella (cont’d) Salmonella on MacConkey

Salmonella (cont’d) Lactose negative Negative for indole, VP, phenylalanine deaminase, and urease Most produce H2S Do not grow in potassium cyanide

Salmonella Virulence Factors Fimbriae- help in attachment Enterotoxin Anitgenic Structures O and H structures are used for serologic grouping Vi- helpful in ID of Salmonella typhi

Salmonella (cont’d) Clinical Infections Acute gastroenteritis or food poisoning Source = handling pets, insufficiently cooked eggs and chicken,milk, and contaminated cooking utensils Occurs 8 to 36 hours after ingestion Symptoms include vomiting, chills, watery diarrhea and abdominal pain Requires a high microbial load for infection Self-limiting in healthy individuals (antibiotics and antidiarrheal agents may prolong symptoms)

Salmonella (cont’d) Typhoid and Other Enteric Fevers Prolonged fever Bacteremia Involvement of the RE system, particularly liver, spleen, intestines, and mesentery Dissemination to multiple organs Occurs more often in tropical and subtropical countries

Salmonella (cont’d) Typhoid fever S. Typhii causative organism Invades intestinal mucosa causing constipation Gains entrance into lymphatic system and bloodstream then into the liver, spleen and bone marrow where they are phagocytized by PMN’s. They multiply in the PMN’s and are eventually released into the blood stream. Finally, they invade the gall bladder and other parts of the intestinal tract to initiate GI symptoms. Found in contaminated food originating from infected individuals Other causes, improper disposal of sewage, poor sanitation, lack of modern H2O systems Develops 9-14 days after ingestion with symptoms of fever, malaise, anorexia, myalgia, continuous dull headache, rash Severe

Salmonella (cont’d) Salmonella Bacteremia Carrier State Organisms shed in feces Gallbladder is the site of organisms (removal of gallbladder may be the only solution to carrier state)

Shigella species Closely related to the Escherichia Fragile organisms All species cause bacillary dysentery, not normal GI flora S. dysenteriae (Group A) S. flexneri (Group B) S. boydii (Group C) S. sonnei (Group D)

Shigella (cont’d) Characteristics Non-motile Do not produce gas from glucose Do not hydrolyze urea Do not produce H2S on TSI On differential, selective media appear as clear, NLF Lysine decarboxylase negative Citrate negative Possess O and some have K antigens lack the H antigen

Shigella (cont’d) Clinical Infections Cause dysentery (bloody stools, mucous, and numerous WBC) Endotoxin- LPS Exotoxin- Shiga toxin Confined to GI tract,septicemia rare Humans are only known reservoir Oral-fecal transmission, person-person Fewer than 200 bacilli are needed for infection in healthy individuals= takes a low infective dose

Shigella (cont’d)

Yersinia species Consists of 11 named species Yersinia pestis Causes plague, which is a disease primarily of rodents; transmitted by rat fleas Two forms of plague bubonic- flea bite, buboes Pneumonic- secondary to bubonic, organisms multiply in bloodstream and resp. tract

Characteristic bubo

Yersinia gram stain

Yersinia species Yersinia enterocolitica Most common form of Yersinia Found worldwide Found in pigs, cats and dogs Human also infected by ingestion of contaminated food or water Some infections result from eating contaminated market meat and vacuum-packed beef Is able to survive refrigerator temperatures (can use “cold enrichment” to isolate) Mainly causes acute gastroenteritis with fever, headaches Stool may be bloody Can mimic appendicitis

Laboratory Diagnosis of Enterics Collection and Handling If not processed quickly, should be collected and transported in Cary-Blair, Amies, or Stuart media Isolation and Identification Site of origin must be considered Enterics from sterile body sites are highly significant Routinely cultured from stool

Laboratory Diagnosis of Enterics (cont’d) Media for Isolation and Identification of Enterics Most labs use BAP, CA and a selective/differential medium such as MacConkey On BA, produce large, greyish, smooth colonies; can be β-hemolytic or nonhemolytic On MacConkey, lactose positive are pink; lactose negative are clear and colorless

Laboratory Diagnosis of Enterics (cont’d) For stools, highly selective media, such as Hektoen Enteric (HE), XLD, or SS is used along with MacConkey agar CIN- selects for Yersenia, hold at room temp. Identification Most labs use a miniaturized or automated commercial identification system, rather than multiple tubes inoculated manually

Laboratory Diagnosis of Enterics (cont’d) Identification (cont’d) All enterics are Oxidase negative Ferment glucose Reduce nitrates to nitrites

Laboratory Diagnosis of Enterics (cont’d) Common Biochemical Tests Lactose fermentation and utilization of carbohydrates Triple Sugar Iron (TSI) ONPG Glucose metabolism Methyl red Voges-Proskauer

Laboratory Diagnosis of Enterics (cont’d) Common Biochemical Tests (cont’d) Miscellaneous Reactions Indole Citrate utilization Urease production Motility Phenylalanine deaminase Decarboxylase tests

Identification Clues Lactose Fermenter with mucoid colony Think: E. coli, Klebsiella, Enterobacter, possibly Citrobacter Lactose Fermenter with mucoid colony Think: Klebsiella, Enterobacter H2S + Think: Proteus, Salmonella, Citrobacter Nonmotile Think: Klebsiella, Shigella Voges- Proskauer + Think: Klebsiella, Enterobacter, Serratia

Screening Stools for Pathogens Because stools have numerous microbial flora, efficient screening methods must be used to recover any pathogens Fecal WBC Fecal pathogens are generally lactose-negative Enteric pathogens include Salmonella, Shigella, Aeromonas, Campylobacter, Yersinia, Vibrio, and E. coli 0157:H7

References Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.