Robert S. Barlow

Isolation and Characterization of Integron-Containing Bacteria without Antibiotic Selection

ABSTRACT The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

Phylogenetically Related Argentinean and Australian Escherichia coli O157 Isolates Are Distinguished by Virulence Clades and Alternative Shiga Toxin 1 and 2 Prophages

ABSTRACT Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx 2 (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx 2 is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx 1 prophage. In both Argentinean and Australian LI/II strains, stx 2c is almost exclusively carried by a prophage inserted at sbcB. However, alternative q 933- or q 21-related alleles were identified in the Australian stx 2c prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.

Prevalence of Enterohemorrhagic Escherichia coli Serotypes in Australian Beef Cattle

OBJECTIVES Beef cattle are known reservoirs of Escherichia coli O157; therefore, it is possible that they may be reservoirs for other enterohemorrhagic E. coli (EHEC) serotypes. This study investigated the prevalence of EHEC serotypes O26, O45, O91, O103, O111, O121, O145, and O157 in 300 beef cattle fecal samples. MATERIALS A combination of 300 (140 grain-fed and 160 grass-fed) cattle fecal samples were collected post-evisceration from Australian abattoirs. Enriched samples were tested for the putative virulence markers stx₁, stx₂, and eae using real-time PCR. Samples that tested positive for stx and eae were then tested for the target serotypes. Isolation was performed on any sample testing positive to stx and eae, and a target serotype using immunomagnetic separation for serotypes O26, O103, O111, O145, and O157 or colony hybridization for serotypes where immunomagnetic separation beads were not commercially available (O45, O91, and O121). Resulting isolates were characterized for the presence of stx₁, stx₂, eae, and ehxA using a multiplex PCR. RESULTS Seventy-eight of the 300 samples (26%) were shown to contain stx and eae with 30 of these subsequently testing positive for the presence of at least one EHEC serotype. Of the 27 E. coli of EHEC serotypes isolated during the study, only 1 E. coli O91, 1 E. coli O26, and 5 E. coli O157 tested positive for the presence of any EHEC virulence markers. CONCLUSIONS This study found that the overall prevalence of EHEC in Australian beef cattle is very low. APPLICATIONS Testing for the presence of virulence determinants and O-specific genes alone will overestimate the presence of pathogenic serotypes in beef. Isolation of strains of interest and confirming the presence of virulence determinants in those strains should be an essential part of any test protocol.

The Effects of Transport and Lairage on Counts of Escherichia coli O157 in the Feces and on the Hides of Individual Cattle

OBJECTIVES The main objective of this study was to determine the impact of transport and lairage on the isolation rate and the number of Escherichia coli O157 on cattle. MATERIALS Ninety animals, divided into three groups (A, B, and C) of 30 animals each, were used in this study. Individual animals were tagged, and samples were collected from the hides and feces of each at a feedlot and again after slaughter. The carcass of each animal was also sampled. Samples were also collected from the feedlot pens, the sides and floors of the transport trucks, and abattoir holding pens. The isolation rate and the number of E. coli O157 were estimated using a combination of immunomagnetic separation and the Most Probable Number technique. RESULTS Cattle hides were more likely to be contaminated with E. coli O157 at the feedlot (31%) than at the abattoir (4%). E. coli O157 was detected in 18% and 12% of cattle feces collected at the feedlot and after slaughter, respectively. E. coli O157 was isolated from truck floors (26%), truck sides (11%), abattoir pen rails (47%), and pen floors (42%). The mean count of E. coli O157 in positive feces was log(10) 1.17 and 2.37 MPN/g at the feedlot and slaughter, respectively. A 3 log(10) increase in the number of E. coli O157 was observed between the feedlot (2.66 MPN/g) and slaughter (5.66 MPN/g) in the feces of one animal in group B. E. coli O157 was isolated from the hide and carcass of this animal. CONCLUSIONS Transport and lairage did not lead to an increase in the number or isolation rate of E. coli O157 from cattle. APPLICATIONS Intervention strategies for reducing E. coli O157 contamination of cattle carcasses should target mechanisms that limit the impact of animals shedding a high number throughout production and processing.

Prevalence and Antimicrobial Resistance of Salmonella and Escherichia coli from Australian Cattle Populations at Slaughter.

Antimicrobial agents are used in cattle production systems for the prevention and control of bacteria associated with diseases. Australia is the worlds third largest exporter of beef; however, this country does not have an ongoing surveillance system for antimicrobial resistance (AMR) in cattle or in foods derived from these animals. In this study, 910 beef cattle, 290 dairy cattle, and 300 veal calf fecal samples collected at slaughter were examined for the presence of Escherichia coli and Salmonella, and the phenotypic AMR of 800 E. coli and 217 Salmonella isolates was determined. E. coli was readily isolated from all types of samples (92.3% of total samples), whereas Salmonella was recovered from only 14.4% of samples and was more likely to be isolated from dairy cattle samples than from beef cattle or veal calf samples. The results of AMR testing corroborate previous Australian animal and retail food surveys, which have indicated a low level of AMR. Multidrug resistance in Salmonella isolates from beef cattle was detected infrequently; however, the resistance was to antimicrobials of low importance in human medicine. Although some differences in AMR between isolates from the different types of animals were observed, there is minimal evidence that specific production practices are responsible for disproportionate contributions to AMR development. In general, resistance to antimicrobials of critical and high importance in human medicine was low regardless of the isolate source. The low level of AMR in bacteria from Australian cattle is likely a result of strict regulation of antimicrobials in food animals in Australia and animal management systems that do not favor bacterial disease.

Escherichia coli O157 Somatic Antigen Is Present in an Isolate of E. fergusonii

A bacterium that tested positive with antibodies specific for Escherichia coli O157 was isolated from beef during routine screening procedures. The bacterium was identified as E. fergusonii by biochemical testing and partial sequencing of 16S rRNA. The isolate was tested for the presence of genes encoding Shiga toxins, the E. coli attaching and effacing factor, enterohemolysin, and the O157 O antigen. The isolate tested negative for Shiga toxins and other E. coli O157 virulence markers but was found to harbor the genes encoding the O157 antigen. These results suggest genetic transfer of the O antigen gene cluster between E. coli O157:H7 and E. fergusonii.