Generally

Enterobacteriaceae are the largest group of medically important bacteria which comprise a family of Gram negative rods that mostly inhabit the intestinal tract. The port of entry into the human body is usually orally.

Many enterics are harmless gut commensals or opportunists. However, some are pathogens (e.g., E coli which can cause bloody diarrhea, Klebsiella pneumoniae which can cause various types of pneumonia, Proteus mirabilis which can cause urinary tract infections, Salmonella tryphi which can cause typhoid fever, Shigella dysenteriae which can cause bacillary dysentery and Yersinia pestis which can cause the plague.

Each year, infections from major foodborn pathogens are responsible for an estimated 9.4 million illnesses, 56,000 hospitalization and 1,350 deaths in the U.S. To evaluate progress toward prevention of enteric infections in the US, the Foodborn Diseases Active Surveillance Network (FoodNet) conducts surveillance for laboratory-diagnosed infections causes by eith pathogens transmitted commonly through food at 10 U.S. sites. Campylobacter and Salmonella are the leading causes of bacterial enteric infections transmitted commonly by food. During 2022, FoodNet identified higher incidences of Shiga toxin-producing Escherichia coli, Yersinia, Vibrio and Cyclospora infections compared with 2016-2018. See CDC

 Detection and Isolation

Example: Frm a carbapenemas proudcing enterobacterales (CPE) surveillance specimen, asthe stool specimen was inoculated on CHROMID CARBA SMART agar. The next day, scant gray colonies grew on the OXA side of the plate. The organism was identified as E. coli by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectromety (Bruker Daltonics). owever, becasue the colonies were gray, not pink on the agar, and du to a known limitation that MALDI_TOF cannot distinguish E. coli from Sigella, serotyping uisng Shigella antisera and biochemical reactions were set up. Kligler ion agar was alkaline over acid with no H2S and produced litttle gas The organisms was negative for lysine, ornithin, indole and citrate. Motolity was negative, and the isolate showed agglutination with S. Flexneri group B antisera, types 1-6, and no agglutination with antisera for other Sigella groups. The identification was confirmed by the BD Phoenix system, which gave a 99% S. Flexneri identificaiton of the isolate. The isolate was forwarded to the Provincial laboratory and was serotyped as S. fleneri type 2a. Stool culture ordered as a reflex test after detection of Shigella species was multiplex PCR identified the organism when it grew as non-lactose fermenting colonies on MacConkey agar. DNA was extracted from an overnight culture of the bacterial isolate using the easyMag system (bioMerieux). Pufified DNA was prepared for sequencing using the Rapid Barcoding Sequencing Kit SQK-LSK114 (Oxford Nanopore Technologies, UK). Sequencing occurred on a GridION system using a R10.4.1 flow cell FLO-MIN114, Oxford Nanopore Technologies) with High-accuracy model basecalling with data captured over 26 hours. Data was evaluted with the MinKNOW software 23.04.5. A consensus genome was constructed using Flye 2.9. The consensus genome for botht he bacterial isoalte and plasmid were anlzyed thorugh the Reistance Gene Identificaiton tool within the Comprehensive Antibiotic Resistance Database. Hits demosntrating a greater than 95% identity and 95% lenght of reference sequence were included in analysis. The database identified 5 reistance genes present within the plasmid. The plasmid consensus sequence was visualized and annotated using Geneious Prime v2023.0.1 (Biomaters, US). “Detection of OXA-181 carbapenemase in Shigella flexneri”

1. Selective Media

MacConkey Agar: See outline

Eosin Methylene Blue Agar: See outline

Hektoen Enteric Agar: See outline

Major characteritiscs are the following:

• Growth on MacConkey agar: See differential media right hand column
• Gram negative rods
• Oxidase negative but most are catalase-positive
• acid production from glucose (with or without gas)
• Most reduce NO3 to NO2

2. Serological Classification

The serologic classification of Enterobacteriaceae is based on 3 major groups of antigens: somatic O LPS, capsular K antigens, and the flagellar H proteins. For example. “E. coli O157:H7” refers to the O antigen and flagellar protein.

One protocol might be to conduct whole-genome sequencing by extracting DNA using easyMag (bioMerieux), sequencing on a GridION system with a R10.4.1 flow cell (oxform nanopore Technologies) and analyzaing the data with MinKNOW 23.04.5 (oxform Nanopore Technologies) to construct a consensus genome using multiplex assay.

SeeGene Allplex GI-EB gastrointestinal multiplex assay

3. Antibody Susceptibility Testing:

NG Biotech CARBA-5 Assay 

Comprehensive Antibiotic Resistance Database

Specific Types of Enterobacteriaceae

Some members of this family like E coli and K. pneumonia are part of the normal bowel flora that can cause opportunistic infections whereas others like Salmonella, Shigella and Yersinia are always associated with disease. Virulence factors include –endotoxins, –capsules, –antigenic phase variation and –antimicrobial resistance.

Escherichia coli

E. coli are part of the normal flora. E coli and other “coliforms” are commonly found only in the bowel. (coliforms are used as a public health indicator of fecal contamination of water). They are facultative anaerobe, gram-negative rods and lactose positive fermentators. Most infections are endogenous (come from the gut and get into other parts of the body that they should not be in). Enterohemorrhagic E. coli (EHEC) causes a Shiga-like toxin which causes bloody diarrhea. Enterotoxigenic E. coli (ETEC) causes traveler’s diarrhea and produces heat labile and heat stable enterotoxins similar to cholera which causes essential metabolites to flow out of the cell rather than into it. Together with group B streptococci, they are the most common cause of neonatal meningitis. They are also the most common gram negative organisms in septic patients. Virulence factors of pathogens include –adhesins, –exotoxins, –alpha hemolysins (which disrupt plasma membranes) and siderophores (which bind necessary ion). Escherichia coli: are gram-negative, facultative anaerobic, rod shaped bacteria which are often found in the lower intestinal tract of warm blooded animals as part of the natural flora. Some serotypes can cause serious food poisening in humans. The fecal-oral route is the major route through which pathogenic strains cause disease. It is one of the most widely studied prokaryotic organisms and has an important role in recombinant DNA.  E coli can be distinguished from other coliforms (rod-shaped gram-negative non-spore forming bacteria which can ferment lactose and commonly used to indicate sanitary quality of foods and water) by the ability to ferment lactose at 44C in the fecal coliform test.  See WHO

Enterobacter aerogenes:

Enterobacter aerogenes is a gram negative oxidase negative, catalase positive, citrate positive, indole negative, rod-shaped bacterium. They are found in the gastroinestinal tract and are opportunistic bacteria.

Klebsiella pneumoniae 

K. pneumoniae, a member of the Enterobacteriaceae family, has long been recognized as a formidable human pathogen associated with a wide range of infections, including urinary tract infections, pneumonia, bloodstream infections, and surgical site infections. The clinical significance of K. pneumoniae is underscored by its ability to exploit an array of virulence factors that facilitate adherence, colonization, and evasion of the host immune response. These virulence factors include capsule polysaccharides, lipopolysaccharides, fimbriae, and siderophores. Notably, the polysaccharide capsule confers resistance to phagocytosis and complements bacterial virulence by facilitating biofilm formation and mediating adherence to host tissues.

Klebsiella pneumoniae usually infects immunosuppressed individuals and cause a bloody sputum. They have an anti-phagocytic capsule. Carbapenem-resistant Enterobacteriaceae (CRE) are considered an urgent threat in the United States because they are associated with high morbidity and mortality, limited treatment options, and potential for rapid spread among patients. Carbapenemases, enzymes that confer resistance to the carbapenem class of antibiotics, are believed to contribute to increasing transmission and regional spread of CRE because the genes encoding these enzymes can reside on mobile plasmids and can be transferred among bacterial species. Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase seen in the United States, but isolates with the New Delhi metallo-β-lactamase (NDM) are emerging. Known risk factors for carbapenemase-producing CRE, including NDM, include health care exposures such as hospitalization outside the United States, recent overnight admissions to short-stay and long-term acute care hospitals, residence in long-term care facilities, surgical procedures, and having indwelling devices. Community-associated CRE lack these health care exposures and are rare in the United States. During 2014–2016, NDM-producing CRE were isolated from patients in Colorado without known health care risk factors. See CDC

Salmonella

Salmonella enterica are lactose negative (unlike E coli which are lactose +). A large infectious dose (106) is necessary. S. enterica is ubiquitous in animals. The source of most infections is from contaminated food products. Exposure to Salmonella bacteria can occur through food, drinking water, animal contact, environmental sources such as soil and water, and infected persons.

Some Salmonella serotypes are highly host-specific. For example, S. enterica serovar Dublin is primarily associated with infections in cattle and sheep, whereas serovar Gallinarum is almost exclusively associated with infections in poultry. In contrast, other serotypes, such as serovars Enteritidis and Typhimurium, are associated with infection of a wider range of human and animal hosts. See Attribution of Salmonella enterica to Food Sources by Using Whole-Genome Sequencing Data

Nontyphoidal Salmonella infections place a large burden on public health; an estimated 79 million cases of foodborne nontyphoidal Salmonella infection occurred in 2010.

Salmonella enterica serovar Infantis>  presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR).  Salmonella enterica subspecies enterica serovar Infantis is becoming an increasingly prevalent serovar globally. A 167% increase in human infections was observed in the United States during 2001–2016. nfantis is the predominant serovar isolated from broiler flocks and broiler meat, accounting for 56.7% of Salmonella isolates from broiler meat in 2018. Higher levels have been observed in Japan, at 72.2% of isolates from ground chicken, and levels of 84% were seen in broilers in Ecuador.

Salmonella typhi are an obligate pathogen of humans (do not spread from species to species) and cause enteric (typhoid) fever. They may form a carrier state (typhoid Mary) and invade macrophages. The infectious dose is small (10) compared to S. enterica.

Shigella

Shigella is primarily a pediatric disease although infections in male homosexuals are also observed. Transmission is by the fecal-oral route. Because as few as 200 bacilli can establish disease, Shigella bacteria are responsible for shigellosis. shigellosis spreads rapidly in communities where sanitary standards are low. A shiga like toxin is produced by S. dysenteriae causing a bloody diarrhea much as with EHEC. Shigella invades M cells and replicates in the cytoplasm. It goes from cell to cell using the actin machinery of the infected cell. In the US, most Shigella is already resistant to antibiotics such as ampicillin and trimethoprim/sulfamethoxazole. Woldwide, resistance to Cipro is also on the rise.

Limmatech Biologics is currently evaluating the sfety and immunogenicity of a Shigella vaccine candidate in a Phase I/II clinical trail in Kenyan children. The Sigella vaccine is tetravelnt in that it incorproates four anitgens. It covers up to 85 of diseases produced by Shigella.

Fhigella flexnerii: infection leads to shigellosis, an acute gastrointestina disease. Shigellosis affects socioeconomically disadvantaged and densely populated communiteis that have unsafe water, poor sanitation, and poor hygeine. (Dhabaan, “Detection of OXA-181 Carbapenemase in Sigella flexneri Emerging Infectious Diseases, 30(5), 2024).

Shigella sonnei: causes about 500k cases of diarhea in the US each year and is becoming increasingly resistant to antibiotics such as ciprofloxacin (Cipro). Cipro is often presecribed for travelers visiting other countries who develop diarhea while travelling. However, this may also be contributing to the bacterial resistance. Washing hands with soap and water, choosing hot foods and drinking only from sealed containers are protection measures.

Treatment:

The emergence of multidrug-resistant Shigella strains is a concerning trend. Multidrug-resistant strains resist multiple first line oral antimicrobials (i.e., ampicillin, trimethoprim/sulfamethoxazole, and ciprofloxacin). The situaiton is further complicated by enzyme-mediated beta-lactam resistance in Shigella bacteria, further impacting empiric therapy and making the isolates extensively drug resistant. Alhtough extesnively drug resistant isolates have remained susceptible to carbapenem therapy, carbapenem reistance in Shigells spp. thorugh imipenemase-type metallo-beta-lactamase, New Delhi metallo-beta-Lactamase, and Verona integron-encoded metallo-beta-lacamase has been reported. (Dhabaan, “Detection of OXA-181 Carbapenemase in Sigella flexneri Emerging Infectious Diseases, 30(5), 2024).

Yersinia 

Yersinia consists of several species. The Yersinia genus encompasses 2 enteropathogenic species, Y. enterocolitica and Y. pseudotuberculosis. Those bacteria are the cause of foodborne infections that range from mild enteritis, especially in children, to systemic infections in the elderly or patients with underlying disorders. See Genomic Characterization of Yersinia enterocolitica Isolates, Costa Rica

Y. pestis is the species that causes bubonic plague and was so devastating in the 14 century. This “black death” killed 25 million in Europe. It is transmitted via fleas and the reservoir is rodents, dogs and rabbits. It is a facultative intracellular bacterium and a zoonaotic

There are three clinical types of plague. 1) the first is bubonic (infected lymph nodes) which causes high fever and painful bubo (inflammatory swelling of lymph node). 2) The Second is pneumonic is the deadliest (almost 100% fatality rate) and transmissible by aerosol. 3) The third is septicemic (blood-born organisms).

Serratia: 

This genus comprises gram-negative rods c . 0.9–2 μm long and 0.5–0.8 μm in diameter, and is part of the family Enterobacteriaceae . They consists of the following recognized species: Serratia entomophila , S. ficaria , S. fonticola , S. grimesii , S. liquefaciens , S. marcescens , S. odorifera , S. plymuthica , S. proteamaculans , S. quinivorans , S. rubidaea and S. ureilytica. Most Serratia spp. are motile by peritrichous flagella and are facultative anaerobic, chemoorganotrophic bacteria with both a respiratory and a fermentative type of metabolism. Being ubiquitous inhabitants of soil, water and plant surfaces, Serratia spp. are commonly associated with raw food materials and cause spoilage of various foods. In addition, they are capable of colonizing a wide variety of surfaces in the digestive tracts of rodents, insects, fish and humans

S. marcescens is implicated in a wide range of serious infections including pneumoni. S. marcescens infection has been attributed to many different sources. Outbreaks of infection have been traced to medical equipment including nebulisers. See Antimicrobes

Specific Types found in Non-humans

Edwardsiellosis, caused by Edwardsiella tarda, has been reported worldwide in economically important fish species, including Japanese eel (Anguilla japonica), red sea bream (Pagrus major), yellowtail (Seriola quinqueradiata), channel catfish (Ictalurus punctatus), and turbot (Scophthalmus maximus).  Edwardsiella tarda is one of the serious fish pathogens, infecting both cultured and wild fish species. Research on edwardsiellosis has revealed that E. tarda has a broad host range and geographic distribution, and contains important virulence factors that enhance bacterial survival and pathogenesis in hosts. Although recent progress in edwardsiellosis research has enabled the development of numerous, highly effective vaccine candidates, these efforts have not been translated into a commercialized vaccine. see Park