Isolation of Shigellae: VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with Stool Specimens

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.     

Isolation of Shigellae: V. Comparison of Enrichment Broths with Stools

Many enteric media are more efficient for the detection of salmonellae than of shigellae. Comparisons of three enrichment broths and three plating media were made during analysis of 1,405 stool specimens to choose a combination of media which would enhance detection of shigellae as well. Gram-Negative (GN), Selenite, and Silliker's Broths were streaked to E M B, Salmonella-Shigella (SS), and xylose lysine deoxycholate (XLD) Agars. The enrichment broths produced a twofold increase in isolations of both salmonellae and shigellae over direct streaking. All three broths performed equally well for Salmonella detection, but GN and Silliker's produced twice as many Shigella isolates as did Selenite. Comparison of the plating media showed that XLD was markedly more efficient than either E M B or SS Agar for the recovery of both genera. SS Agar was superior to E M B for isolation of salmonellae after enrichment, whereas E M B was better for isolation of shigellae by direct streaking. Both E M B and SS were more effective when used after GN and Silliker's than after Selenite. GN Broth and XLD Agar were the most efficient combination of media. During these analyses, 158 salmonellae and 49 shigellae isolates were obtained.     

Comparison of Brilliant Green Agar and Hektoen Enteric Agar Media in the Isolation of Salmonellae from Food Products

Brilliant Green (BG) agar and Hektoen enteric (HE) agar media were compared for their efficiency in isolating salmonellae from various food products. Of the 11,226 food specimens examined, 1,662 (or 14.9%) yielded salmonellae. Of this number, 1,475 (88.7%) were recovered from BG agar and 1,315 (79.1%) were recovered from HE agar media. The results indicate that BG agar is more effective in isolating salmonellae from food products. A smaller subsidiary study showed HE agar to be more selective than BG agar. Four hundred ten specimens yielded 92 nonlactose-fermenting isolants other than salmonellae on BG agar and only 11 such isolants on HE agar.     

Clinical Evaluation of Enteric Media in the Primary Isolation of Salmonella and Shigella

Over a 1-year period, media for the isolation of enteric pathogens were compared on 455 stool specimens. Fifty-three pathogens were isolated, of which 56% were Shigella sonnei and 13% were Sh. flexneri. Of these isolates, 90% were found on xylose-lysine-desoxycholate agar, 87% on Hekton enteric agar, and 80% on MacConkey without crystal violet with 2% agar and 0.007% neutral red, but only 28% were recovered on Salmonella-Shigella agar. Less than one-half of the shigellae were recovered after Selenite-F enrichment. On the other hand, enrichment was the most helpful method for isolating salmonellae. Studies on cultures from which mixed isolates were obtained indicated that numbers and chance distribution have an effect on the results obtained. The performance of Salmonella-Shigella agar in the isolation of enteric pathogens was inferior, and the effort involved to obtain those isolates was greater than for Hekton enteric and xylose-lysine-desoxycholate agars.     

Comparative Survival of Indicator Bacteria and Enteric Pathogens in Well Water

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.     

A New Plating Medium for the Isolation of Enteric Pathogens: I. Hektoen Enteric Agar

A new agar plating medium for the isolation of enteric pathogens is described. This medium contains greater quantities of peptone to offset the inhibitory effects of bile salts. Additional carbohydrates and larger quantities of those previously used have also been incorporated into the medium to differentiate pathogens from some of the slow lactose fermenters. The use of an indicator system never used before in a differential plating medium opens new possibilities for improvement over traditional and newer selective media. Known strains from the various genera of Enterobacteriaceae have been tested for colonial differentiation as well as for possible inhibitory effects. The results show the medium to be highly selective for the shigellae as well as for other enteric pathogens.     

Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport''s Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport's Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport's Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport''s Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

The structure of the population morbidity with specific intestinal infections in areas with different intensities in the development of the epidemic process and an evaluation of its manifestations]

The authors analyze the morbidity structure in five enteric infections (typhoid fever, dysentery caused by Shigella flexneri and Shigella sonnei, hepatitis A, and hepatitis E (non A, non B) with the fecal/oral mechanism of the agent transmission) in three towns of Turkmenia and in the town of Novomoskovsk, Tula Province. The incidence of S. sonnei dysentery was found higher in Novomoskovsk and that of the rest enteric infections under study in Turkmenia. The incidence of typhoid fever and hepatitis E was the highest among schoolchildren and adults, whereas preschool children suffered mostly from hepatitis A and S. sonnei dysentery. The authors discuss the specific features of the epidemic process manifestation in enteric infections.     

Comparison of reagent-impregnated paper strips and conventional tests for distinguishing Escherichia from Aerobacter: correlation with colonial morphology

The means for distinguishing Escherichia from Aerobacter (Enterobacter) differ in laboratories and range from complete dependence on colonial reactions on typical gram-negative media to reliance on one or more of the classical indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters. Three colonial types (one prejudged as Escherichia) of lactose-positive rods were catalogued on each of the most commonly used selective media, MacConkey Agar, Endo Agar, and E M B Agar. Each cultural type was presumptively diagnosed and then compared with the expected outcome of individual IMViC tests. The distribution of preliminary identifications was similar from growth patterns on MacConkey Agar and E M B Agar, but it differed markedly from Endo Agar. When organisms initially diagnosed by cultural methods were compared by single IMViC tests, it was found that for each colonial type one of the biochemical parameters was best suited. Thus, for those types initially considered Escherichia, the methyl red or Voges-Proskauer test results agreed most consistently; for other types, the citrate reaction was most satisfactory. In addition, when newly formulated reagent-impregnated paper strip methods for indole, Voges-Proskauer, and citrate were evaluated and compared to the standard methods, agreement was 97% for indole, 90% for Voges-Proskauer, and 95% for Simmons' citrate.     

The Evolution of Insertion Sequences within Enteric Bacteria

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.     

Comparative study of the prevalence and clinical profiles of diarrheas due to Aeromonas and other enteric pathogens

The prevalence of Aeromonas spp. and other enteric pathogens in stool specimens from diarrheic and non-diarrheic patients was studied over a 12 month period (January to December, 1986). Except for the absence of fever, all the clinical features in Aeromonas diarrhea were comparable to those associated with other diarrheagenic agents. These features included abdominal pain (30%), vomiting (24.5%), fever (31.5%), dehydration (9.5%) and hematochezia (19.5%). Aeromonas spp. were more frequently isolated from patients with gastroenteritis (2.5%) than from control patients (1.0%) (P less than 0.05). Isolates were recovered more often during the dry months (66.7%), than during the wet months (33.3%). Among the enteric pathogens isolated, Aeromonas spp. (2.5%) ranked next to Esch. coli (14.5%) and Shigella spp. (6.3%) in prevalence. Other bacterial isolates included Plesiomonas shigelloides (1.5%) Vibrio spp. (1.0%), Yersinia enterocolitica (1.0%) and Salmonella spp. (1.8%).     

Enteric pathogens and soil: a short review

It is known that soil is a recipient of solid wastes able to contain enteric pathogens in high concentrations. Although the role of soil as a reservoir of certain bacterial pathogens is not in question, recent findings show that soil may have a larger role in the transmission of enteric diseases than previously thought. Many of the diseases caused by agents from soil have been well characterized, although enteric diseases and their link to soil have not been so well studied. Gastrointestinal infections are the most common diseases caused by enteric bacteria. Some examples are salmonellosis (Salmonella sp.), cholera (Vibrio cholerae), dysentery (Shigella sp.) and other infections caused by Campylobacter jejuni, Yersinia sp. and Escherichia coli O157:H7 and many other strains. Viruses are the most hazardous and have some of the lowest infectious doses of any of the enteric pathogens. Hepatitis A, hepatitis E, enteric adenoviruses, poliovirus types 1 and 2, multiple strains of echoviruses and coxsackievirus are enteric viruses associated with human wastewater. Among the most commonly detected protozoa in sewage are Entamoeba histolytica, Giardia intestinalis and Cryptosporidium parvum. This article reviews the existing literature of more than two decades on waste disposal practices that favor the entry of enteric pathogens to soil and the possible consequent role of the soil as a vector and reservoir of enteric pathogens. Electronic Publication     

A prevalence survey for zoonotic enteric bacteria in a research monkey colony with specific emphasis on the occurrence of enteric Yersinia

Transmissible pathogenic and opportunistic zoonotic enteric bacteria comprise a recognized occupational health threat to exposed humans from non-human primates (NHPs). In an effort to evaluate the occurrence of selected enteric organisms with zoonotic and biohazard potential in a research colony setting, we performed a prevalence study examining 61 juvenile and young adult rhesus macaques participating in a transplant immunology project. Primary emphasis was directed specifically to detection of pathogenic enteric Yersinia, less well-documented and reported NHP pathogens possessing recognized significant human disease potential. NHPs were surveyed by rectal culture during routine health monitoring on three separate occasions, and samples incubated using appropriate media and specific selective culture methods. Enteric organisms potentially transmissible to humans were subcultured and identified to genus and species. Significant human pathogens of the Salmonella/Shigella, Campylobacter, and enteric Yersinia groups were not isolated throughout the survey, suggesting prevalence of these organisms may generally be quite low.     

Further Evaluation of Immunofluorescence Techniques for Detection of Shigellae in Fecal Specimens

The fluorescent antibody (FA) tests for group B and group D Shigella were reevaluated. Duplicate swab specimens from patients suspected of having shigellosis were cultured shortly after collection and after transport in a soft-agar holding medium. Smears for FA examination were made at the same time. Results obtained for the group D Shigella agree closely with those obtained in previous studies. The percentage of isolations of group B Shigella from transported specimens which were positive by FA was 59.6% as compared to 39.3 and 53.3% in previous studies. S. flexneri was isolated from 76.7% of the FA-positive specimens when they were examined shortly after collection. More isolates of Shigella were obtained when specimens were examined by both methods than when either method was used alone. The results indicated that FA tests for both group B and group D Shigella are practical and may be useful to laboratories engaged in Shigella isolation.     

A study of enteropathogenic organisms isolated in the public health laboratory, Lusaka.

A total of 328 specimens of stools were examined in the Public Health Laboratory during January and February 1973. Enteropathogens were isolated from 117 of these specimens. Besides these, 12 strains of Salmonellae were isolated from blood and 8 from urine. An occasional Salmonella was isolated from the pleural fluid (S. paratyphi A) pus from the knee (S. enteritidis) and from the C.S.F. of an infant (S. paratyphi C.). Salmonella typhi and Salmonella paratyphi A are the predominant Salmonella species. No Salmonella paratyphi B has been isolated. Shigella, was isolated with slightly less frequency than Salmonella, and Shigella flexneriis was the predominant species. E. coli 0112/K66 is the most common enteropathogenic E. coli. The majority of the Shigella and Salmonella species are sensitive to the common antibiotics used. The E. coli organisms show multiple resistance to a number of antibiotics.     

Reaction of anti-HLA-B monoclonal antibodies with envelope proteins of Shigella species. Evidence for molecular mimicry in the spondyloarthropathies

Immunologic cross-reactivity between enteric bacteria and the HLA-B27 protein may play a role in the etiology of Reiter's syndrome and reactive arthritis. The reactivity of two anti-B27 mAb (B27M1 and B27M2) with envelope proteins of Shigella flexneri isolated from Reiter's syndrome patients was studied by Western blot analysis. Proteins with an apparent Mr of approximately 36 and 23 kDa reacted with both mAb in ascites. mAb against related HLA class I Ag B7 and B40 did not react with the 23 kDa protein. Relatively high concentrations of antibody were required for reactivity, suggesting a low affinity interaction. Additional evidence for cross-reactive epitopes was obtained by ELISA against whole envelope and by using unsolubilized envelope to inhibit binding of M1 and M2 to B27-positive cell lines, as measured by quantitative flow microfluorimetry. The presence of cross-reactive proteins was not related to the presence of the intact virulence-associated plasmid or the invasive phenotype. Two Shigella sonnei isolates not implicated as causative agents of Reiter's syndrome or reactive arthritis showed a similar pattern of cross-reactivity. These results indicate that cross-reactive epitopes may be present on "arthritogenic" bacteria, but their presence is not a unique feature of such strains and is not the sole factor in induction of arthritis in B27-positive individuals.     

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.     

Variation in Plating Efficiency of Salmonellae on Eight Lots of Brilliant Green Agar

The plating efficiency of Salmonella anatum, S. cubana, S. dublin, S. tennessee, and S. typhimurium was determined for eight lots of Brilliant Green Agar made by two manufacturers. Washed cells were used as the inoculum and cultures were incubated at 41.5 C. All lots of Brilliant Green Agar were supplemented with 12 mg of sulfadiazine per 100 ml of medium. Of the eight lots of Brilliant Green Agar tested, average recovery of the test salmonellae in three did not differ from recoveries with Trypticase Soy Agar, which was used as a control to indicate the number of viable salmonellae in the test suspension capable of growth on a nonselective medium. Two lots of Brilliant Green Agar gave salmonellae recoveries with geometric means about 25% lower than, and significantly different from, those of the control agar. The remaining three lots of Brilliant Green Agar were generally unproductive.