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. 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.     

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.     

Actinomycetes from solitary wasp mud nest and swallow bird mud nest: isolation and screening for their antibacterial activity

The aim of this study was to isolate and screen actinomycetes from solitary wasp and swallow bird mud nests for antimicrobial activity. The actinomycetes were isolated from soil of nests of solitary wasp and swallow bird, and identified on the basis of morphological characteristics and molecular biological methods. A total of 109 actinomycetal isolates were obtained from 12 soil samples (6 from each habitat) using two media. The highest number of actinomycetes were recovered on Humic acid vitamin agar media (65.13%, n = 71) as compared to actinomycetes isolation agar media (34.86%, n = 38). The antimicrobial activity of actinomycetes isolates was determined using the agar plug method. Among 109 isolates, 51 isolates (46.78%) showed antibacterial activity by agar plug assay. The morphological and molecular characteristics confirmed that the most of active isolates in both sample belonged to the genus Streptomyces, the other potential genera like Streptosporangium, Actinomadura, Saccharopolyspora, Thermoactinomycetes and Nocardia were also recovered, but in a low frequency. The isolates designated as 8(1)*, BN-6, MN 2(6), MN 2(7) and MN 9(V) showed most promising activity against various drug resistant bacterial pathogens. It seems that the promising isolates from these unusual/unexplored habitats may prove to be an important step in development of drug for treating multi-drug resistant bacterial pathogens.     

Lysine mannitol glycerol agar (LMG) and LMG with sulphamandelate for isolation of Salmonella spp. from clinical specimens

Lysine mannitol glycerol agar (LMG) was compared to xylose lysine deoxycholate agar (XLD), bismuth sulphite agar (BS), and Salmonella-Shigella agar (SS) for the ability to detect Salmonella spp. in clinical specimens, primarily faeces samples. During an 8-month period, 15 salmonellae were isolated from 940 faeces on LMG, while 14 strains were obtained on XLD, 11 on SS and only 3 strains on BS. Salmonella typhi was recovered from two blood cultures in 24 h on LMG, compared to 48 h on BS. LMG was augmented by addition of a sulphacetamide/mandelic acid (sulphamandelate) selective supplement (LMGS). During a 20-month period, 43 salmonellae were isolated from 2622 faeces on LMG and LMGS. The selectivity of LMGS was superior to that of LMG with no decrease in sensitivity of detection; all salmonellae isolated on LMG were isolated on LMGS. Both LMG and LMGS were suitable for routine use in the isolation of Salmonella spp. from clinical specimens.     

Biochemically aberrant Salmonella enteritidis ser. newington from human sources in Connecticut.

Three isolates of a lactose-fermenting, xylose-negative variety of Salmonella enteritidis ser. newington, identical in biochemical and serological reactions and in the antibiogram, were recovered from three patients in different areas of Connecticut in January 1974. Hydrogen sulfide production was not visible in Salmonella-Shigella agar, in triple sugar iron agar, and in Kligler iron agar but was noticed in lysine iron agar and on XLD agar, among others. The amount of fermentable carbohydrates present was found to correlate with failure to show hydrogen sulfide production (pH effect). In contrast to lactose-fermenting Salmonella strains reported by other authors, we could not elicit a direct transfer of the lac⁺ character at frequencies above 10⁻⁶. An epidemiological follow-up remained unsuccessful. Recommendations for the recognition of similar strains are presented.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Comparison of acetate differential agar with sellers citrate-mannitol-agar

A total of 245 isolates of Escherichia coli (paracolons) were employed in comparing the use of acetate differential agar (ADA) with the simple test described by Sellers to eliminate these organisms from consideration as enteric pathogens. At 18 hr after the media were inoculated, 83% of the isolates produced a positive citrate reaction on Sellers citrate-mannitol-agar (SCM) and 7% gave a positive reaction on ADA. At 24 hr, 92% of the cultures were positive on SCM versus 42% on ADA. At 48 hr, 244 (99.6%) of the isolates were positive on SCM, whereas a 72-hr incubation period was required for equal results with ADA. One isolate failed to grow on either medium.     

Isolation of Salmonellae and Shigellae from an Artificial Mixture of Fecal Bacteria

Numerous selective media, available commercially, act by suppressing "normal" bacterial inhabitants of the intestine while permitting the growth of so-called pathogenic representatives of the family Enterobacteriaceae. This investigation attempts to evaluate the action of Salmonella-Shigella (SS) agar, xylose lysine desoxycholate (XLD) agar, and hektoen enteric (HE) agar. Salmonellae and shigellae, isolated from clinical material, were mixed in various ratios with escherichiae, Klebsiella-Enterobacter-Serratia group bacteria, and members of the tribe Proteeae, also of clinical origin. Several of the mixtures were plated in multiple dilutions on the three media. Stools in preservative were also used for evaluation of the media after the addition of definite numbers of the pathogenic bacteria. Results indicate that SS agar suppresses the shigellae along with the autochthonous members of Enterobacteriaceae. XLD and HE agars readily permit the recovery of shigellae as well as salmonellae. This recovery is not obscured by the higher yield of other species obtained with these media.     

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.     

Isolation ofAcholeplasma spp. from coconut palms affected by lethal yellowing disease in Jamaica

A total of 35 isolates ofAcholeplasma spp. were recovered from phloem sap and rotting tissues of lethal yellowing-diseased coconut palms. The highest isolation rate, 33% of samples from two palms, was obtained in a conventional mycoplasma medium supplemented with Tween 80; no isolates were recovered from healthy palm tissues or from uninoculated media constituents. Metabolic and serological tests on uncloned isolates showed that about two-thirds were strains ofA. axanthum and the remainder were related toA. oculi. These results strongly suggest that acholeplasmas occur in or on plant tissues either as pathogens, epiphytes, or saprophytes.     

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%).     

High proportions of nonpathogenic Streptomyces are associated with common scab-resistant potato lines and less severe disease

Streptomyces isolates were obtained from potato tubers with common scab lesions from 2 fields over a 3 year period in Minnesota and a 5 year period in Maine. Isolates were obtained from different potato cultivars or breeding lines and types of scab lesions. A majority of isolates could be classified as putative pathogens based on the presence of genes for biosynthesis of the pathogenicity determinant, thaxtomin, but large numbers of streptomycetes lacking genes for thaxtomin biosynthesis (presumably nonpathogenic) were also recovered. Most Streptomyces isolates recovered from raised and pitted lesions were pathogens, whereas mostly nonpathogenic isolates were recovered from unblemished potato skin or nonscab lesions. Fewer pathogenic than nonpathogenic isolates were recovered from the most resistant potato lines. The proportion and diversity of nonpathogenic isolates recovered was higher in Maine than in Minnesota. The association between greater numbers of nonpathogenic Streptomyces and less severe common scab suggests that the interaction between plant genotype and Streptomyces microbial community is important in determining the severity of common scab on potato, and emphasizes the role of complex interactions between plants and microbial populations on and near plant roots in plant disease outcomes.     

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

AIMS: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. METHODS AND RESULTS: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Control of Shigella flexneri in Celebes black macaques (Macaca nigra)

Stool specimens collected systematically from a group of Celebes black macaques (Macaca nigra) with a high incidence of diarrhea were examined microbiologically. Numerous isolates of Shigella flexneri, Campylobacter jejuni and pathogenic Escherichia coli were recovered. Previous parasitology reports had revealed that the majority of the animals had Balantidium coli. Subsequently, the group was treated with trimethoprim-sulfamethoxazole, erythromycin and tetracycline. After treatment, Shigella flexneri was not detected in the stool of any animal for 1 year, and the clinical condition of the group was improved. Reduced recovery rates were obtained with other enteric pathogens.     

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.     

Pseudomonas putrefaciens Isolates from Clinical Specimens

A total of 109 cultures of Pseudomonas putrefaciens isolated from clinical specimens were studied. The cultures were separated into two groups. The majority of the group 1 isolates, comprising 31 cultures, were characterized by (i) growth in plain nutrient broth, but no growth in broth supplemented with NaCl at concentrations of 7% and above, (ii) no growth on Salmonella-Shigella (SS) agar, and (iii) production of acid from the carbohydrates, sucrose, maltose, arabinose, and dextrin. Most group 2 isolates, comprising 78 cultures, were (i) unable to grow in plain nutrient broth, but grew well in broth supplemented with NaCl at a concentration of 7 to 10%, (ii) able to grow on SS agar, and (iii) unable to produce detectable amounts of acid from any of the carbohydrates tested except for variable results with glucose and fructose.