Comparison of methods for isolating Campylobacter jejuni from raw milk.

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Comparison of three procedures for isolating DNA from bacteria.

Three methods employing chloroform-isoamyl alcohol (CI), phenol, or enzymes, were evaluated for isolating DNA from Escherichia coli, Bacillus subtilis, and Arthrobacter globiformis. For the amounts of reagents employed at optimum conditions in the CI and phenol procedures, 0.4-0.9 mg of DNA/g wet weight of cells was isolated. Using the enzymatic procedure, approximately twice as much DNA was isolated. DNA isolated by the CI procedure contained 0.03-0.09% protein and 0.08-0.12% RNA. DNA isolated by the phenol procedure contained 0.02-0.05% protein and 2.2-2.6% RNA. DNA isolated by an enzymatic procedure, which is described in detail, contained 32.2-45.7% protein and 0.3-0.6% RNA. DNA isolated by all three procedures are double-stranded and at least 10(6) in molecular weight, as suggested by data from thermal transition analyses and transformations. These data emphasize that the desired characteristics of DNA for experimental purposes must be considered in selecting an isolation procedure.     

Effects of enrichment media and incubation conditions on isolating salmonellae from ground-meat filtrate.

Forty-eight combinations of enrichment media, secondary enrichment, incubation times and temperatures, and atmospheres were examined for their efficacy in recovering different serovars of Salmonella that had been inoculated into ground-meat extract. Variations included three selective-enrichment media, two (37 and 43 degrees C) incubation temperatures, two (24 and 48 h) incubation times, two (aerobic and anaerobic) incubation atmospheres, and secondary enrichment to two of the selective-enrichment media. The ratio of Salmonella to other microorganisms was 10: greater than 1,000,000. One-hundred and twenty-four tests were conducted for each enrichment under each condition of incubation. None of the methods recovered Salmonella in more than 60% of the trials. Salmonella typhimurium was recovered most frequently of the serovars tested; S. abortusovis was recovered least frequently. There was considerable variation in the results obtained by the different methods, but there was a statistically significant advantage in the 43 degrees C incubation temperature. Secondary enrichment in tetrathionate broth showed a statistically significant advantage over secondary enrichment in selenite broth. Secondary enrichment into a different medium from the primary enrichment also was advantageous.     

Comparison of four agar plating media with and without added novobiocin for isolation of salmonellae from beef and deboned poultry meat

Four plating media, Hektoen enteric (HE), xylose-lysine deoxycholate (XLD), tryptic soy-xylose-lysine (TSXL), and tryptic soy-brillant green (TSBG) agars with and without 10 mg of added novobiocin per ml, were evaluated for recovery of Salmonella from roast beef and deboned turkey. Colonies producing a reaction typical of H(2)S-positive salmonellae (alkaline with black centers) were picked. On the media without novobiocin, from 109 determinations on 75 samples, number of salmonellae found and false-positives were, respectively: HE-13, 58; XLD-17, 18; TSXL-23, 0; TSBG-22, 7. When novobiocin was present the corresponding results were: HE-17, 24; XLD-21, 2; TSXL-23, 3; TSBG-20, 7. A total of 25 determinations were positive on one or more agars. False-positives on HE and XLD without novobiocin were predominantly Proteus, which were almost totally eliminated by addition of 10 mg of novobiocin per liter. If alkaline H(2)S-negative colonies had been considered, many more false-positives would have been found on HE and XLD but not on TSBG or TSXL. Addition of novobiocin markedly improved isolations of salmonellae from XLD and HE and reduced the number of false-positives. Addition of novobiocin did not improve performance of TSXL and slightly impaired differentiation of salmonellae from Citrobacter on TSBG. XLD with novobiocin and TSXL are highly specific for H(2)S-positive salmonellae, and the appearance of Salmonella-like colonies on these media can be considered a presumptive test for H(2)S-positive salmonellae.     

Comparison of methods for isolating Campylobacter jejuni from raw milk

The method of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) was compared with that of Lovett et al. (Appl. Environ. Microbiol. 46:459-462, 1983) for the ability to recover Campylobacter jejuni strains inoculated into raw milk at a concentration of less than 1 cell per g. The method of Lovett et al. gave significantly greater recovery proportions.     

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.     

Comparison of enrichment methods and atmosphere modification procedures for isolating Campylobacter jejuni from foods.

A comparison was made of enrichment broths for recovery of Campylobacter jejuni from food by the methods of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353) and of Park et al. (Can. J. Microbiol. 27:841-842). No significant differences were found between the results obtained with the two broths. Recovery was greater, however, with a constant gas flow into the broths than with an evacuation-replacement method.     

Antimicrobial resistance ofEnterococcus spp. isolates from raw beef and meat products

E. faecalis (67%) and E. faecium (13.7%) were most frequently isolated among enterococci that contaminate cooled and frozen processed meat, follow-up heat-treated meat products and unheated fermented dry salami. Most isolates of both species were resistant to cephalothin (95 and 83 %) and clindamycin (77 and 67%, respectively). Furthermore, E. faecalis and E. faecium isolates were resistant to erythromycin (44 and 72%), tetracycline (34.5 and 17.4%), and streptomycin (13.3 and 4.3%, respectively). Only a few of the isolates were resistant to ampicillin, ampicillin-sulbactam, chloramphenicol, and vancomycin while all isolates were susceptible to gentamicin, penicillin, and teicoplanin. During the production of heat-treated meat products, numbers of resistant isolates increased in spite of the decreasing enterococcal contamination of the samples. An opposite situation was found in the production of fermented dry salami.     

Virulence determinants invA and spvC in salmonellae isolated from poultry products, wastewater, and human sources.

The presence of two virulence foci, invA and spvC, in Salmonella isolates obtained from poultry, wastewater, and human sources was determined. All isolates (n = 245) were positive for the invA gene sequence. Differences in degree of invasiveness were apparent with the Madin Darby canine kidney cell line, as only 79 of 159 randomly selected isolates (49.7%) tested were invasive at > 0.1% of the inoculum. 25% were invasive between 0.1 and 1.0% of the inoculum, and 24.5% were invasive at > 1.0% of the inoculum. There was a significant correlation between degree of invasion and source from which the isolate was recovered but no correlation between geographic origin of poultry isolates and degree of invasion. Only 37 of 245 isolates (15.1%) hybridized with the spvC DNA probe. All isolates that were recovered from a commercial egg production environment and chicken eggs and whose sequences exhibited homology with the spvC gene sequence were determined to be either Salmonella enteritidis PT 23 or PT 13. The sequences of few isolates from ceca and none from wastewater or humans demonstrated homology with the spvC gene.     

Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

Comparison of enrichment methods and atmosphere modification procedures for isolating Campylobacter jejuni from foods

A comparison was made of enrichment broths for recovery of Campylobacter jejuni from food by the methods of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353) and of Park et al. (Can. J. Microbiol. 27:841-842). No significant differences were found between the results obtained with the two broths. Recovery was greater, however, with a constant gas flow into the broths than with an evacuation-replacement method.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

Penetration of poultry meat byPseudomonas andLactobacillus spp.

The penetration of poultry muscle strips byPseudomonas putida, P. fragi, Lactobacillus plantarum andL. casei was studied at 37°C. The first three bacteria penetrated along the muscular septa whileLactobacillus casei did not invade the poultry muscle strips at all. The penetration by both pseudomonads occurred throughout the 3 cm muscle strip butL. plantarum showed minor penetration up to 2 cm only.     

Isolation and characterization of methicillin-resistantStaphylococcus aureus from livestock and poultry meat

Meat samples from sheep, bovine, camel and poultry were collected from Amman area and were processed and tested for the presence of methicillin (oxacillin) resistantStaphylococcus aureus (MRSA). Identity ofS. aureus was ensured by Gram-staining and a battery of biochemical tests. From 1260 meat samples, 157S. aureus positive isolates were identified. Of the 157 isolates, 30 were resistant to methicillin levels greater than 2 μg/ml and only 15 weremecA-positive MRSA originating mainly from sheep and chicken. Subjecting themecA-positive MRSA to antibiotic susceptibility testing revealed that all isolates were resistant to β-lactam antibiotics (ampicillin, penicillin, and oxacillin) and were sensitive to vancomycin, trimethoprim, chloramphenicol and cephalothin. Randomamplified polymorphic DNA (RAPD) analysis ofmecA-positive animal isolates generated six different patterns. Comparing these results with results of isolates of human origin of our laboratory there is some molecular epidemiological relatedness between both and could be a possible source of infections through consuming contaminated meat products, direct contact or meat processing.     

Isolation of Escherichia coli 0157 from raw meat products

This study has evaluated an enrichment and four subculture procedures for detection of Escherichia coli 0157 in raw meat products. The combination of enrichment in modified tryptone broth incubated at 42C for 6 h, followed by immunomagnetic separation and subculture on to cefixime, tellurite sorbitol MacConkey agar was the most sensitive and selective procedure. Traditional subculture using 10 μl and 100 μl inocula and culture of centrifuged deposits were less satisfactory. A most probable number method was used to enumerate E. coli 0157 in naturally contaminated samples associated with human cases. The results indicated that the samples contained <0.3 to 2300 cfu g^-1 of E. coli 0157 which confirms that the infective dose for this organism is low.     

Virulence ofEscherichia coli isolated from raw meat,food handlers and equipment of meat shops

Of 101Escherichia coli isolates from raw meat, food handlers and equipment 24.8% showed enterotoxigenicity, 22.7% surface hydrophobicity and there was a high incidence of resistance towards metal ions such as mercury, cadmium and silver. Only 6% of the strains were colicin producers. Significant statistical correlation was observed between enterotoxin production and cadmium and mercury ion resistance.

---

Résumé Center une souches d'Escherichia coli ont été, isolées de viandes crues, de manutantionneurs de nourriture et d'equipement de boucherie. Elles exhibent une enterotoxigénécité à concurrence de 24.8%, une hydrophobicité de surface à concurrence de 22.7%. Il y a une grande fréquence de résistance vis-à-vis d'ions métalliques comme le mercure, le cadmium et l'argent. A peine 6% des souches sont productrices de colicine. Il y a une corrélation significative sur le plan statistique entre la production d'enterotoxines et la résistance aux ions cadmium et mercure.

     

Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay.

Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerase chain reaction assay (MIPA). A direct polymerase chain reaction assay performed with chicken meat spiked with Salmonella typhimurium resulted in poor sensitivity (approximately 10(7) CFU/g of meat). The use of immunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, but enrichment was required for the detection of low levels of contamination. Enrichment for 6 h in either buffered peptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initial inoculum of 100 CFU per g of meat. Enrichment of the salmonellae present on 25 g of spiked chicken meat for 24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detection limit of approximately 0.1 CFU/g of meat. A detection limit of approximately 1 CFU/g of meat was obtained when the spiked meat was stored at -20 degrees C before enrichment for 24 h and analysis with the MIPA. Although the MIPA was developed for S. typhimurium, a MIPA in which a panel of six monoclonal antibodies specific for Salmonella serogroups A through E was used detected the presence of 0.1 CFU of Salmonella enteritidis per g of chicken meat. These data indicate that the method is applicable to other commonly isolated serotypes.     

Microtechnique for isolating fecal coliforms from soil.

A most-probable-number microtitration technique for isolating fecal coliforms from soil was developed. A correlation coefficient of 0.86, with a 95% confidence interval of 0.76 less than zeta less than 0.92, was obtained when this technique was compared with the standard elevated-temperature fecal coliform most-probable-number procedure.