University of Hawai‘i at Mānoa: Medical Technology Notes on Enterobacterales

Comparative Genomic Analyses

  • Comprehensive comparative genomic analyses performed by authors using:

    • 1548 core proteins

    • 53 ribosomal proteins

    • 4 multilocus sequence analysis proteins

  • Results supported existence of seven distinct monophyletic groups within the order ‘Enterobacteriales’.

    • Proposed new order: Enterobacterales ord. nov.

    • Consists of seven families:

    • Enterobacteriaceae

    • Erwiniaceae fam. nov.

    • Pectobacteriaceae fam. nov.

    • Yersiniaceae fam. nov.

    • Hafniaceae fam. nov.

    • Morganellaceae fam. nov.

    • Budviciaceae fam. nov.

  • Many genera previously associated with Enterobacteriaceae, such as Edwardsiella, Hafnia, Morganella, Proteus, Providencia, Serratia, and Yersinia, transferred into one of six new families.

  • Plesiomonas remains unassigned to a specific family.

Categorical Representation of Key Enterobacterial Genera

  • Enterobacteriaceae ≠ Enterics?

    • Enterobacteriaceae members referred to as enterics.

    • New taxonomy expands enterics to include additional families.

General Characteristics of Enterobacteriaceae

  • Normal flora of the GI tract.

  • Gram-negative bacilli (GNB) with:

    • Straight sides

    • Rounded ends

  • Facultative anaerobes.

  • Colony Morphology:

    • Most have large, gray, spreading colonies.

    • Klebsiella and Enterobacter exhibit mucoid colonies.

  • Biochemical characteristics:

    • Ferment glucose.

    • Reduce nitrates to nitrites.

    • Oxidase negative.

    • Most are motile (possessing peritrichous flagella).

Serologic Classification of Enterobacteriaceae

  • Cell-associated antigens:

    • O: Somatic (body) antigens

    • Heat stable.

    • Part of the cell wall (polysaccharide of outer membrane lipopolysaccharides).

    • Associated with endotoxin release.

    • K: Capsular antigens

    • Heat labile.

    • Capsular polysaccharide.

    • Strains with K are more pathogenic.

    • H: Flagellar antigens

    • Heat stable.

    • Composed of flagellar protein antigens, responsible for mobility.

Clinical Disease from Enterobacteriaceae

  • Classification based on clinical infections:

    • Opportunistic infections:

    • Generally normal flora causing infections outside habitat.

    • Primary intestinal infections:

    • Includes Salmonella, Shigella, Plesiomonas, Yersinia enterocolitica.

  • Opportunistic genera include:

    • Citrobacter, Edwardsiella, Enterobacter, Escherichia

    • Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia.

  • Most cause opportunistic and nosocomial infections:

    • Urinary Tract Infections (UTI).

    • Pneumonia.

    • Wound infections.

    • Catheter colonization.

  • Isolated from almost all body sites.

Laboratory Diagnosis of Enterobacteriaceae

  • Oxidase-negative with characteristic colony morphology and Gram Staining (GS).

  • Culture Techniques:

    • Use supportive and selective media for pathogen recovery.

    • Grows readily on routine lab media:

      • Blood Agar Plate (BAP).

      • Eosin-methylene blue (EMB) agar.

      • MacConkey (MAC) agar.

      • XLD.

      • Hektoen.

Case Study: Escherichia coli

  • 3-month-old male, prematurely born at 27 weeks gestation.

  • Transferred with recurrent Escherichia coli CNS infection.

  • Initially treated for culture-negative sepsis with ampicillin and gentamicin.

  • Developed intractable seizures and worsening hydrocephalus despite 6 weeks of treatment.

  • Endoscopic 3rd ventriculostomy revealed purulent fluid and significant pleocytosis.

  • Additional antibiotics led to sterility and resolution of pleocytosis after using ciprofloxacin.

  • Acridine Orange Staining:

    • Cell-permeant nucleic acid-binding dye.

    • Reveals green fluorescence when bound to dsDNA and red fluorescence with ssDNA or RNA.

Characteristics of Escherichia coli (E. coli)

  • Predominant facultative GNR in human intestinal tract.

  • Causes various infections:

    • UTI.

    • Bacteremia.

    • Neonatal meningitis.

    • Cholecystitis.

    • Cholangitis.

    • Pneumonia.

    • Diarrheal illnesses:

    • 1.7 billion cases and 525,000 deaths in children under five worldwide annually.

    • Second leading cause of infectious disease mortality in the US.

Pathotypes of Gastroenteritis E. coli

  • Six pathotypes:

    • Enterotoxigenic E. coli (ETEC).

    • Enteropathogenic E. coli (EPEC).

    • Enterohemorrhagic E. coli (EHEC).

    • Enteroaggregative E. coli (EAEC).

    • Enteroinvasive E. coli (EIEC).

    • Diffusely adhering E. coli (DAEC).

  • Diarrheagenic E. coli strains are antigenically distinct from commensal strains that colonize the intestine.

Enterotoxigenic E. coli (ETEC)

  • Common cause of bacterial diarrhea in developing countries (est. 840 million cases annually).

  • Causes acute watery diarrhea in infants and adults, characterized by:

    • Toxigenic, not invasive.

    • Produces heat-stable toxins (STa and STb) and heat-labile toxins (LT-I, LT-II).

  • Symptoms develop after 1-2 days and last 3-5 days:

    • Watery, non-bloody diarrhea,

    • Abdominal cramps.

    • Mild cases resemble cholera but are milder.

  • High mortality risk in malnourished individuals.

Enteropathogenic E. coli (EPEC)

  • Causes infantile diarrhea outbreaks and sporadic diarrhea in adults:

    • Non-toxigenic and non-invasive.

    • Uses intimin adhesin to bind intestinal cells.

    • Produces attaching and effacing lesions (A/E):

    • Disrupts brush border epithelium, increases secretion and leads to watery diarrhea.

    • Rapid disease onset within hours; most infections resolve within days.

Shiga Toxin-Producing E. coli (STEC/VTEC/EHEC)

  • Causes bloody diarrhea and dysentery:

    • Invasive and systemic illnesses that require antibiotic treatment.

    • Defined by the presence of Shiga toxin 1 or 2.

    • Major serotype: 0157:H7.

    • Disease occurrence can result from ingesting fewer than 100 bacteria; person-to-person transmission possible.

  • Severity of disease includes:

    • Mild uncomplicated diarrhea,

    • Hemorrhagic colitis with severe abdominal pain,

    • Bloody diarrhea.

    • Hemolytic uremic syndrome (HUS):

    • Characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia.

  • Resolution of uncomplicated disease typically occurs in 4-10 days.

  • Severe sequelae may occur in up to 30% of HUS patients.

Laboratory Diagnosis for E. coli

  • All E. coli strains:

    • Ferment glucose, lactose, and xylose.

    • Indole and Methyl Red positive.

    • No H2S or urease production.

    • Citrate negative.

    • Can be motile or non-motile.

  • For E. coli O157:H7:

    • Grown on MacConkey with sorbitol (SMAC).

    • Does not ferment sorbitol (colonies appear clear).

Shigellosis

  • Serotyping (O antigen):

    • A: Shigella dysenteriae (15 serotypes).

    • B: S. flexneri (6 serotypes and 16 subserotypes).

    • C: S. boydii (23 serotypes).

    • D: S. sonnei (1 serotype).

  • Epidemiology:

    • Transmitted human-to-human (fecal-oral route), very communicable.

    • Shigella sonnei: Most common in the US.

    • Shigella dysenteriae: Least common in the US but most severe.

  • Symptoms onset occurs within 24-48 hours:

    • Organisms may be cultivated from stools up to 30 days.

  • Clinical presentation may vary:

    • Watery diarrhea and abdominal pain,

    • Dysentery characterized by bloody, mucoid stools.

Pathogenesis of Shigellosis

  • Requires very few organisms to infect (10-200).

  • Shiga toxin causes:

    • Inflammation and ulcerative lesions,

    • Destruction of epithelial cells leading to bloody, mucus-laden stools.

Laboratory Diagnosis for Shigellosis

  • Features:

    • Lactose negative.

    • TSI: Alkaline/Acid, no gas, no H2S.

    • Urease negative.

  • Confirmatory serotyping protocols:

    • WHO protocol using polyvalent or monovalent antisera.

    • Reportable to health department in the US.

Salmonelleae Tribe Features

  • Single genus: Salmonella.

  • Two species:

    • S. enterica (six subspecies)

    • S. bongori.

  • Antigens include somatic (O) and flagellar (H), with S. typhi additionally expressing capsular (Vi) antigens.

  • Disease manifestations include:

    • Gastroenteritis:

    • Symptoms include nausea, vomiting, fever, abdominal pain, and diarrhea.

    • Usually self-limiting; antimicrobial therapy not recommended unless worsens.

    • Enteric fever (Typhoid):

    • Symptoms include fever, prostration, bacteremia, and organ failure.

    • Blood cultures typically positive in the first week; stool and urine culturing more positive in later weeks.

    • Other infections: bacteremia, septicemia, osteomyelitis, meningitis, endocarditis (increased risk in immunocompromised).

Identification of Salmonella

  • Identification revolves around biochemical results,

    • Requires serogrouping with antisera.

    • For unknown serogroups, capsular antigens may need inactivation by boiling for retesting.

    • Public Health laboratory confirmation recommended.

Epidemiology of Salmonella in the USA

  • Estimates by CDC:

    • 1.35 million infections annually,

    • 26,500 hospitalizations,

    • 420 deaths.

  • Key sources associated with outbreaks include:

    • Ground beef, raw cookie dough, flour, pet turtles, poultry.

  • 2022 incidents reported associated with Salmonella Uganda linked to pet bearded dragons.

Yersinia Species Overview

  • Clinically significant species include:

    • Yersinia pestis: Plague.

    • Y. enterocolitica:

    • Epidemiology:

      • Food and water-borne illnesses.

      • Major reservoir is swine; infections can occur through non-pasteurized milk.

      • CDC estimates 117,000 illnesses, 640 hospitalizations, and 35 deaths in the US annually.

    • Clinical manifestations:

      • Intestinal and extraintestinal diseases;

      • Septicemia in iron overload syndromes;

      • Gastroenteritis with mesenteric adenitis mimicking appendicitis.

Laboratory Diagnosis of Yersinia enterocolitica

  • Grows on routine media;

  • Optimal growth conditions at 25-30°C;

  • Unique culture characteristics:

    • Small, lactose-negative colonies on MacConkey agar;

    • CIN agar yielding pink colonies with red centers (“bull's eyes”).

    • TSI results show Acid/Acid, without gas.

Plesiomonas shigelloides Overview

  • Epidemiology:

    • Source of water-borne gastroenteritis, found in freshwater, soil, animals including shellfish, especially in tropic regions.

    • Clinical manifestations consist of mild watery diarrhea or extraintestinal infections (bacteremia, meningitis).

Laboratory Diagnosis for Plesiomonas shigelloides

  • Cultured under routine conditions:

    • Grows on BAP, MAC, HE agars,

    • Non-hemolytic on BAP, displaying white-pink colonies;

    • Biochemical tests positive for oxidase and indole.

Klebsiella spp Characteristics

  • K. pneumoniae: most clinically isolated species;

    • Exhibits distinct mucoid colonies on MAC and EMB agar;

    • Associated clinical feature includes “currant jelly-like appearance” in sputum samples.

  • Other significant species include K. oxytoca.

Enterobacter spp Characteristics

  • E. cloacae and K. aerogenes are most commonly isolated;

  • Environmental organisms;

  • Members of the ESKAPE group, noted for being leading causes of resistant nosocomial infections.

  • Intrinsic resistances observed to ampicillin, amoxicillin, first-generation cephalosporins, and cefoxitin due to constitutive production of AmpC β-lactamase.