Detection and enumeration of E. coli O157:H7 in water samples by culture and molecular methods.

The performances of three chromogenic agars were evaluated for the recovery of Escherichia coli O157:H7 from spiked dechlorinated tap, ground and surface water, and treated drinking water samples. The chromogenic agars: ChromAgar O157 (CHROM), Rainbow Agar O157 (RB) and HiCrome EC O157 (HC) were compared to cefixime-tellurite Sorbitol MacConkey (CT-SMAC), commonly used for the isolation of E. coli O157:H7. Confirmation of suspect E. coli O157:H7 colonies were performed by colony real-time PCR (C-RTi-PCR) based on the presence of Shiga-toxin genes (stx1 and stx2). Recovery of inoculated E. coli O157:H7 from dechlorinated tap water indicated that RB and CHROM agars demonstrated improved recovery when compared to HC or CT-SMAC. There was a significant drop in recovery on all agars tested after 120h (day 5). Twenty dechlorinated tap and/or treated drinking water samples were inoculated with a pure culture of E. coli O157:H7 (ATCC 43894), and a mixed culture of E. coli O157:H7 (ATCC 43894), E. coli strain K-12, and Enterococcus faecalis (ATCC 063589). After a 48-hour holding time, the recovery using CHROM (99%) and HC (12%) from samples contaminated with the pure culture were found to be significantly different (p<0.05). Recovery results using CHROM (39%) and CT-SMAC (32%) from samples contaminated with the mixed culture after a 48-hour holding time were not significantly different (p>0.05). Analysis by C-RTi-PCR of forty five environmental water samples (surface, sewage, and final effluents) which were negative for E. coli O157:H7 showed an incidence of false suspect positive colonies of 38% (CHROM), 53% (RB), 58% (HC), and 91% (CT-SMAC). Further analysis of eight of the environmental samples inoculated with E. coli (ATCC 43894) showed 100% recovery when utilizing CHROM, 50% when using RB and 40% when using HC. In addition, the C-RTi-PCR positive confirmation rate was 100% for CHROM and HC and 65% for RB. CHROM demonstrated improved recovery of E. coli O157:H7 over RB, HC, and CT-SMAC in terms of sensitivity and specificity.

Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples.

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p<0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p>0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p<0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P<0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.

Assessment of the application of an automated electronic milk analyzer for the enumeration of total bacteria in raw goat milk.

Automated electronic milk analyzers for rapid enumeration of total bacteria counts (TBC) are widely used for raw milk testing by many analytical laboratories worldwide. In Ontario, Canada, Bactoscan flow cytometry (BsnFC; Foss Electric, Hillerød, Denmark) is the official anchor method for TBC in raw cow milk. Penalties are levied at the BsnFC equivalent level of 50,000 cfu/mL, the standard plate count (SPC) regulatory limit. This study was conducted to assess the BsnFC for TBC in raw goat milk, to determine the mathematical relationship between the SPC and BsnFC methods, and to identify probable reasons for the difference in the SPC:BsnFC equivalents for goat and cow milks. Test procedures were conducted according to International Dairy Federation Bulletin guidelines. Approximately 115 farm bulk tank milk samples per month were tested for inhibitor residues, SPC, BsnFC, psychrotrophic bacteria count, composition (fat, protein, lactose, lactose and other solids, and freezing point), and somatic cell count from March 2009 to February 2010. Data analysis of the results for the samples tested indicated that the BsnFC method would be a good alternative to the SPC method, providing accurate and more precise results with a faster turnaround time. Although a linear regression model showed good correlation and prediction, tests for linearity indicated that the relationship was linear only beyond log 4.1 SPC. The logistic growth curve best modeled the relationship between the SPC and BsnFC for the entire sample population. The BsnFC equivalent to the SPC 50,000 cfu/mL regulatory limit was estimated to be 321,000 individual bacteria count (ibc)/mL. This estimate differs considerably from the BsnFC equivalent for cow milk (121,000 ibc/mL). Because of the low frequency of bulk tank milk pickups at goat farms, 78.5% of the samples had their oldest milking in the tank to be 6.5 to 9.0 d old when tested, compared with the cow milk samples, which had their oldest milking at 4 d old when tested. This may be one of the major factors contributing to the larger goat milk BsnFC equivalence. Correlations and interactions between various test results were also discussed to further understand differences between the 2 methods for goat and cow milks.

Quantitative effect of refrigerated storage time on the enumeration of Campylobacter, Listeria, and Salmonella on artificially inoculated raw chicken meat.

Active monitoring of pathogens on retail foods has been recommended and implemented in a number of developed countries. Because only a portion of retail food is contaminated with pathogens, a cost-effective and informative surveillance program at the retail level often involves a two-stage approach of initial presence-absence analysis and subsequent pathogen enumeration in any positive samples. Most-probable-number (MPN) methods are more resource intensive and therefore used only for samples considered positive by presence-absence methods. Interpretation of the results assumes that the initial bacterial count remains relatively stable between the initiation of the presence-absence analysis and the enumeration analysis. The objective of this study was to quantify the influence of 4 degrees C storage for 5 and 8 days on pathogen counts on raw chicken. The three pathogens examined were Salmonella Typhimurium, Campylobacter jejuni, and Listeria monocytogenes. No significant differences were found between treatments for Salmonella and Campylobacter. However, significant differences were observed for Listeria; counts at day 0 were lower than counts after 5 or 8 days of refrigerated storage (the maximum mean difference was less than 0.6 log units). These findings suggest that a two-stage approach could overestimate the number of Listeria cells on chicken at the time of purchase. By using an MPN analysis on the presumptive positive samples after 5 days of refrigerated storage, this difference will be reduced. These findings support the decision to reduce surveillance costs by performing a two-stage analysis for Salmonella and Campylobacter on retail chicken. This study provides direction for future sampling or surveillance programs that include enumeration of Listeria on retail food.

Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture.

The effects of various hydrocarbon substrates, and a chemical surfactant capable of enhancing crude-oil biodegradation, on the community structure of a mixed-bacterial inoculum were examined in batch culture. Of 1000 TSA-culturable isolates, 68.6% were identified at the genus level or better by phospholipid fatty acid analysis over 7-day time course experiments. Cultures were exposed to 20 g/L Bow River crude oil with and without 0.625 g/L Igepal CO-630 (a nonylphenol ethoxylate surfactant), 5 g/L saturates, 5 g/L aromatics, or 125 g/L refinery sludge. A group of six genera dominated the cultures: Acinetobacter, Alcaligenes, Ochrobactrum, Pseudomonas/Flavimonas, Stenotrophomonas, and Yersinia. Species from four of the genera were shown to be capable of hydrocarbon degradation, and counts of hydrocarbon degrading and total heterotrophic bacteria over time were nearly identical. Pseudomonas/Flavimonas and Stenotrophomonas normally dominated during the early portions of cultures, although the lag phase of Stenotrophomonas appears to have been increased by surfactant addition. Acinetobacter calcoaceticus was the most frequently isolated microorganism during exposure to the saturate fraction of crude oil. Regardless of substrate, the culture medium supported a greater variety of organisms during the latter portions of cultures. Understanding the community structure and dynamics of mixed bacterial cultures involved in treatment of heterogeneous waste substrates may assist in process development and optimization studies.

Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems.

The performances of five automated microbial identification systems, relative to that of a reference identification system, for their ability to accurately and repeatedly identify six common food-borne pathogens were assessed. The systems assessed were the MicroLog system (Biolog Inc., Hayward, Calif.), the Microbial Identification System (MIS; MIDI Inc., Newark, Del.), the VITEK system (bioMérieux Vitek, Hazelwood, Mo.), the MicroScan WalkAway 40 system (Dade-MicroScan International, West Sacramento, Calif.), and the Replianalyzer system (Oxoid Inc., Nepean, Ontario, Canada). The sensitivities and specificities of these systems for the identification of food-borne isolates of Bacillus cereus, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and verotoxigenic Escherichia coli were determined with 40 reference positive isolates and 40 reference negative isolates for each pathogen. The sensitivities of these systems for the identification of these pathogens ranged from 42.5 to 100%, and the specificities of these systems for the identification of these pathogens ranged from 32.5 to 100%. Some of the systems had difficulty correctly identifying the reference isolates when the results were compared to those from the reference identification tests. The sensitivity of MIS for the identification of S. aureus, B. cereus, E. coli, and C. jejuni, for example, ranged from 47.5 to 72. 5%. The sensitivity of the Microlog system for the identification of E. coli was 72.5%, and the sensitivity of the VITEK system for the identification of B. cereus was 42.5%. The specificities of four of the five systems for the identification of all of the species tested with the available databases were greater than or equal to 97.5%; the exception was MIS for the identification of C. jejuni, which displayed a specificity of 32.5% when it was tested with reference negative isolates including Campylobacter coli and other Campylobacter species. All systems had >80% sensitivities for the identification of Salmonella species and Listeria species at the genus level. The repeatability of these systems for the identification of test isolates ranged from 30 to 100%. Not all systems included all six pathogens in their databases; thus, some species could not be tested with all systems. The choice of automated microbial identification system for the identification of a food-borne pathogen would depend on the availability of identification libraries within the systems and the performance of the systems for the identification of the pathogen.

Evaluation of universal preenrichment broth for the recovery of foodborne pathogens from milk and cheese.

The use of universal preenrichment broth for the recovery of verotoxigenic Escherichia coli, Salmonella spp., and Listeria monocytogenes from milk and cheese was examined. Universal preenrichment broth supported the growth of low inoculum levels (10 cfu/ml) of these organisms in pure cultures and in mixed cultures containing higher levels of other pathogens or bacterial flora from raw milk. This medium also supported the recovery and growth of heat-injured Salmonella spp., L. monocytogenes, and verotoxigenic E. coli at inoculum levels of 10(2) cfu/ml to yield cell levels of 10(8) cfu/ml in pure cultures and at least 10(5) cfu/ml in the presence of high levels of known competitive pathogens or microflora of cheese samples after 24 h of incubation. Universal preenrichment broth performed better than Listeria enrichment broth in supporting the recovery and growth of heat-injured L. monocytogenes and equally as well as buffered peptone water or trypticase soy broth in supporting the growth of uninjured L. monocytogenes, Salmonella spp., and verotoxigenic E. coli. Coenrichment of these pathogens in universal preenrichment broth reduced the quantity of milk or cheese samples that were required for analysis and also reduced the cost and labor involved in preparing and processing separate preenrichment media.

Identification of contamination sources of Bacillus cereus in pasteurized milk.

In order to determine the sources of Bacillus cereus in pasteurized milk, a total of 232 milk samples from various sampling points along milk processing lines and 122 environmental swabs were collected in two dairy plants between March and September, 1996. The incidence of B. cereus vegetative cells in raw milk from the plants was low (< or = 10%). However, the incidence and the average counts of B. cereus spores in the raw milk were very high and similar to those of B. cereus vegetative cells in pasteurized milk or final products after enrichment (> 80% and 1.1 x 10(5) cfu ml(-1), respectively). The incidence and average count of both vegetative cells and spores of B. cereus in environmental swabs was low. Using the microbial identification system (MIDI), a library of B. cereus fatty acid profiles comprising 229 B. cereus isolates from milk samples and environmental swabs was constructed using a critical Euclidian distance of 6.0 units as the cut-off value. Using this library, the relationship between 546 B. cereus isolates from the different sampling points along the milk processing lines and the environmental swabs was determined. Most B. cereus isolates obtained from the pasteurized milk and final products belonged to the same sub-groups as the B. cereus strains germinated from spores in raw milk. Furthermore, specific sub-groups were found in pasteurized milk, different dairy plants and at different sampling times. The results suggested that B. cereus spores in raw milk were the major source of B. cereus in pasteurized milk and that post-pasteurization contamination along the milk processing lines was possibly a minor source of B. cereus in pasteurized milk.

Epidemiological typing of Campylobacter isolates from meat processing plants by pulsed-field gel electrophoresis, fatty acid profile typing, serotyping, and biotyping.

Campylobacter spp. are a leading cause of bacterial gastroenteritis. Foods of animal origin, particularly under-cooked poultry, are common sources of Campylobacter species associated with disease in humans. A collection of 110 Campylobacter jejuni and 31 C. coli human and environmental isolates from different Ontario, Canada, abattoirs were analyzed by pulsed-field gel electrophoresis, fatty acid profile typing, and biotyping. Previously collected serotyping data for the same isolates were also analyzed in this study. Pulsed-field gel electrophoresis was found to be the most discriminatory of the typing methods, followed by serotyping, fatty acid profile typing, and biotyping. A wide variety of typing profiles were observed within the isolates, suggesting that several different Campylobacter sp. strains were present within the abattoirs.

Analysis of whole-cell fatty acid profiles of verotoxigenic Escherichia coli and Salmonella enteritidis with the microbial identification system.

Differentiation of strains within bacterial species, based on gas chromatographic analysis of whole-cell fatty acid profiles, was assessed with 115 strains of verotoxigenic Escherichia coli and 315 strains of Salmonella enteritidis. Fatty acid-based subgroups within each of the two species were generated. Variability of fatty acid profiles observed in repeat preparations from the same strain approached that observed between subgroups, limiting the usefulness of using fatty acid profiles to subgroup verotoxigenic E. coli and S. enteritidis strains.

Production of monoclonal antibodies specific for Haemophilus ducreyi: a screening method to discriminate specific and cross-reacting antibodies.

Haemophilus ducreyi is the etiological agent of chancroid. The organism shares extensive immunological cross-reactivity with other Haemophilus species. This presents substantial difficulties for the production of specific monoclonal antibodies (MAbs). A competition ELISA was devised for hybridoma screening which allowed the detection of H. ducreyi-specific antibody-producing hybridoma cultures during the initial screening process. With this screening method, seven MAbs specific for H. ducreyi were obtained in a single cell fusion exercise. The specificities of the 7 MAbs were demonstrated by direct ELISA and dot immunobinding assays against several strains each of H. influenzae, H. parainfluenzae and Neisseria gonorrhoeae. Five of the MAbs reacted against all ten strains of H. ducreyi. These MAbs may permit the development of rapid and efficient immunodiagnostics for chancroid. The principle of the competition ELISA for hybridoma screening should be widely applicable to the development of specific MAbs to other organisms in which immunological cross-reactivity is an impediment to hybridoma screening by conventional methods.

Relationship between lipopolysaccharide composition and virulence of Haemophilus ducreyi.

The relationship between lipopolysaccharide (LPS) composition and virulence of Haemophilus ducreyi strains was investigated. Glycoses identified in LPS by gas-liquid chromatography were glucose, galactose, and their amino derivatives glucosamine and galactosamine. Fucose was found in trace amounts but mannose and rhamnose, characteristic of the O-side chain of LPS in many species, were not detected. Qualitatively, the LPS composition of the eight strains examined was similar and differences were mainly quantitative. The total glycose:KDO ratio of the LPS of virulent strains exceeded that of avirulent strains. All strains had similar fatty-acid composition but lacked lauric acid. SDS-polyacrylamide gel electrophoresis of the LPS of virulent and avirulent strains also revealed differences in their electrophoretic mobilities. The LPS profiles of avirulent strains were similar, but differed from those of virulent strains. These profiles lacked high mol. wt bands representing O-side chain repeating units. Thus, differences in the electrophoretic mobilities of the LPS of virulent and avirulent strains may reflect differences in the amount of carbohydrates associated with the core polysaccharide.

Role of lipopolysaccharide and complement in susceptibility of Haemophilus ducreyi to human serum.

The role of lipopolysaccharide (LPS) in the susceptibility of Haemophilus ducreyi to human serum and the mechanism of complement activation by serum-susceptible (Sers) strains were investigated. Serum treated with 2 mM Mg2+ and 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was nonbactericidal, but inulin-treated serum remained bactericidal. Absorption of serum with heat-killed whole cells of an Sers strain removed its bactericidal activity against the absorbing strain and also against other Sers strains. LPS obtained from Sers strains inhibited the bactericidal activity of serum against all Sers strains, whereas LPS from serum-resistant (Serr) strains and an Serr isogenic strain did not. However, high concentrations of LPS from the Serr strain inhibited the bactericidal activity of serum, an indication that part of the structural site involved in serum susceptibility is retained in the LPS of this strain. The LPS of Sers strains exhibited higher anticomplement activity than the LPS of Serr strains. These findings suggest that the classical pathway of complement activation is involved in the serum killing of H. ducreyi and that LPS composition may contribute to their susceptibility to complement-mediated serum bactericidal activity.

Virulence factors of Haemophilus ducreyi.

We investigated the susceptibility of virulent and avirulent strains of Haemophilus ducreyi to the bactericidal activity of normal human serum and to phagocytosis and killing by human polymorphonuclear leukocytes (PMNL). Strains were defined as virulent if intradermal inoculation into a rabbit produced a typical necrotic lesion. Nonvirulent strains produced no cutaneous lesions in rabbits. Virulent strains were resistant to the complement-mediated lethal action of normal human and rabbit sera, whereas avirulent strains were susceptible (greater than 95% kill, 60 min). Virulent strains were relatively resistant to phagocytosis and killing by human PMNL, in contrast to the avirulent strains. In past studies polymyxin resistance has been correlated with virulence in H. ducreyi. In our studies, polymyxin resistance could not be correlated with virulence, since polymyxin-sensitive mutants obtained from polymyxin-resistant parent strains remained virulent for rabbits and resistant to bactericidal action of normal serum and phagocytosis and killing by human PMNL. Similarly, polymyxin-resistant mutants obtained from polymyxin-sensitive parent strains remained avirulent for rabbits and susceptible to bactericidal action of normal serum and PMNL. The acquisition of polymyxin resistance was accompanied by the loss of a 47,000-molecular-weight protein. The association of serum resistance and resistance to phagocytosis and killing by human PMNL with virulent strains, as defined by the rabbit intradermal test, suggests that these factors may mediate the pathogenicity of H. ducreyi.

Characterization of cell proteins of Haemophilus ducreyi by polyacrylamide gel electrophoresis.

The whole-cell proteins of 105 clinical isolates of Haemophilus ducreyi from several geographic sources (North America, Africa, Asia, and Europe) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein profiles were reproducible and unaffected by repeated subculturing or age of culture. At least seven different subtypes were determined by proteins in the molecular weight range of 24,000-50,000. These proteins are located in the outer membrane of the cell, as determined by SDS-PAGE of the sarcosinate-insoluble membrane preparations of these strains. Thirteen isolates from a Winnipeg, Manitoba, Canada, outbreak of chancroid had identical patterns, suggesting a common origin. Although H ducreyi shares a number of proteins in common with other Haemophilus species, the protein profiles appear to be species specific. Heterogeneity in the protein composition of H ducreyi has provided a basis for subtyping, which could be of value in future epidemiologic studies.