The family Enterobacteriaceae is the largest, most heterogenous collection of medically important gram negative bacilli. Currently at least 27 genera with 7 enteric groups and more than 110 species have been described. Some of them (e.g. Escherichia coli, Klebsiella pneumoniae, Proteus) are the part of normal enteric microflora and can cause opportunistic infections, Salmonella, Shigella, Yersinia pestis are always pathogenic for humans. The enteric bacteria are gram negative rods, not forming spores, have a complex antigenic structure, are facultative anaerobes or aerobes, are motile by peritrichous flagella or nonmotile, have simple nutritional requirements, ferment various carbohydrates, reduce nitrates to nitrites, are oxidase negative.

Laboratory diagnosis

Microscopy: medium sized (0,3 – 1,0 x 1,0 – 6,0 nm) gram negative rods, some genera (Klebsiella) form capsules (negative staining).

Culture: liquid culture medium - selenite broth for multiplication especially of Salmonella. Most genera of the family Enterobacteriaceae form circular, convex, smooth colonies on solid culture media. Klebsiella can be differentiated because of very mucoid colonies (capsule). On blood agar (solid nonselective culture medium) all enteric bacteria grow in grey colonies, some strains of E. coli produce beta hemolysis. Differential culture media are MacConkey agar, Endo agar. Lactose-fermenting species (e.g. E.coli, Enterobacter) form pink colonies, lactose-non-fermenting species (e.g. Salmonella, Shigella, Yersinia, Proteus) form colorless colonies. Selective culture media – desoxycholate citrate agar, is also differential because of the content of lactose. Salmonella form colorless colonies with black center (H₂S production); highly selective medium for the genus Salmonella is Wilson Blair agar (black metal colonies surrounded with reduced bismuth).

Biochemical identification: Short routine biochemic tests – HISU identify enteric bacteria according to saccharides fermentation, gas and H₂S production (Hain medium), indol production and motility test (peptone medium), NH₄  utilization (Simmons citrate medium), urea fermentation (urea medium).Numeric identification of Enterobacteriaceae – Enterotest I, II.- the results are compared with the Enterotest register and evaluated in a special program.

Antigenic structure and serotypization of enterobacteria: Somatic heat stable O antigens (lipopolysaccharide), external K antigens (polysaccharides, e.g. Klebsiella, E.coli), Vi antigens are the capsular antigens of Salmonella typhi, flagellar heat labile H antigens (protein) present in motile enteric bacteria can be agglutinated with specific antisera in indirect agglutination test.

Phage and colicin typing: Epidemiologic studies can describe 25 phage types in Salmonella. Colicin typing can be used for the proper identification of Shigella sonei or E. coli izolates.

Serologic examination: In typhoid and paratyphoid fever the specific antibodies against O, H and Vi antigens of Salmonella typhi and paratyphi can be detected by direct agglutination (Widal reaction). Two serum samples obtained in the onset of disease and after 2 – 3 weeks are examined together, at least 4 fold increase of specific antibody titer in the second sample is significant for the illness.

Escherichia coli (E. coli), a member of normal GI flora, is lactose and mannitol fermenting, gas producing, indole and lysin decarboxylase positive. Enteropathogenic E. coli (EPEC) which adheres to mucosal cells of the small bowel (adherence factors are encoded by genes on plasmids) causes watery diarrhea in infants. Well known EPEC strains are identified by O antigen slide agglutination. Also Enterotoxigenic E. coli (ETEC) adheres to the epithelial cells of the small bowel. It is a common cause of „travellers diarrhea“ and diarrhea in infants when contaminates foods. Some strains produce heat-labile exotoxin (genes carried on plasmids) similar to enterotoxin of Vibrio cholerae (activates adenylyl cyclase and increase of cAMP). Some ETEC strains produce heat-stable enterotoxin (plasmid encoding). Strains of Enterohemorrhagic E. coli (EHEC) produce verotoxin (similar to shigatoxin of Shigella dysenteriae) and cause severe diarrhea, hemorrhagic colitis. Clinical syndrome of diarrhea similar to shigellosis is caused by Enteroinvasive E.coli (EIEC), which invades intestinal mucosal cells. Acute or chronic diarrhea can be induced by Enteroaggregative E. coli (EAEC).

Other than GI clinical syndromes caused by E.coli are frequent urinary tract infections (both E. coli and hemolytic E.coli), sepsis in immunocompromised patients and in newborns, meningitis especially in infants (E. coli strains with K1 antigen).

Klebsiella pneumoniae and Klebsiella oxytoca cause mainly respiratory and urinary tract infections. Klebsiella species are not motile, perform large polysaccharide capsules (mucoid colonies), utilize citrate, are lysin decarboxylase and urease positive. They are resistant to ampicillin. Similar Enterobacter aergenes has smaller capsules, is motile, has similar biochemic activity except urease and is isolated from urinary and respiratory tract infections. It is resistent to cefalotin. Common etiologic agent of nosocomial infections is Serratia marcescens.  It belongs to the most resistent species with the resistance to penicillins and aminoglycosides. Tests of DNase, lipase and gelatinase production are positive.

The species of  Proteus, Morganella and Providencia group are motile, swarming is especially characteristic for Proteus. All ferment lactose slowly or not at all, they are phenylalanine positive, Proteus and Morganella urease positive. Proteus mirabilis, Proteus vulgaris, Morganella morgani, Providencia rettgeri often cause urinary tract infections, contaminate wounds, belong to the agents causing nosocomial infections. Some strains may be resistant to various antibiotics.

Citrobacter, similar to Salmonella, slowly ferments lactose. Tests for motility, utilization of citrate are positive,lysine decarboxylase negative.It is found in GI and urinary tract infections.

Nonmotile Shigellae ferment glucose, do not produce gas, except Shigella sonnei do not ferment lactose. They are closely related to E. coli. They are obligatory pathogens of humans where they cause widely spread bacillary dysentery with the invasion into mucosal epithelial cells of the large intestine and terminal ileum.  Shigella dysenteriae produces heat-labile exotoxin. This species causes most severe infections.  Most of  Shigella isolates are now of Shigella sonnei which is often transmitted by contaminated food. Shigella flexneri and Shigella boydii are isolated less often. Shigellae are susceptible to common antibiotics.

Salmonella-Arizona group contains the genus Salmonella and Arizona with hundreds of subspecies and serotypes which can be identified by O and H (and Vi) agglutination. Salmonella typhi, S. Paratyphi A and  B, S. choleraesuis are primarily pathogenic for humans. The majority of Salmonellae have an animal reservoir, e.g. S. enteritidis, S. typhimurium. The most important species is S. typhi, a causative agent of typhoid fever. Paratyphoid fever is caused by S. paratyphi, bacteremia by S. choleraesuis and other serotypes. S. enteritidis is the most common cause of enterocolitis caused by Salmonellae. Complex diagnostics of Salmonella infections include cultivation of the specimens chosen according to the type of infection, antibiotics susceptibility testig,  biochemical testing, serotypization, serologic examination (Widal agglutination test) in typhoid fever. The global prevention of the transmission of Salmonellae by the contaminated food is carried worldwide. The S. typhi carriers are screened, the prevention by vaccination is possible.

Laboratory excercise.

1.  Demonstration of growth of enterobacteria on various diagnostic culture media.
2. Determination of biochemical properties of enterobacteria. Identification by routine testing, demonstration of Enterotest I.
3. O antigen grouping of Salmonella by means of slide indirect agglutination test.

Procedure.
1. Put 3 drops of physiological solution on the slide.
2. Suspend the typed bacterial colony in each drop with the bacteriological loop. The suspension looks milky opalescent.
3. Put one loop (one drop) of anti-O diagnostic serum into one suspension and mix properly. Repeat the procedure with other two diagnostic sera.
4. The specific agglutination occurs within 2 minutes, it is visuable by a naked eye. Agglutinated bacteria look like the grains in a clear solution.
The proper grouping of Salmonella must be completed with H – antigen typing by specific anti- H antisera, also by indirect slide agglutination. The complete list of Salmonella based on the antigenic characteristics is called Kauffmann-White scheme.
5. Write the protocol of today experiment with the result of Salmonella typing.

Questions.

1. Which genera of Enterobacteriaceae family do you know?
2. Which are the main characteristics of Enterobacteriae?
3. What do you know about the pathogenicity of Enterobacteriae?
4. What are the main diagnostic procedures of Enterobacteriae infections?
5. Which diseases are caused by Salmonella and Shigella?
6. How could you diagnose Salmonella typhi infection?
7. What do you know about the serologic diagnosis of typhoid fever?