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 (intI1andintI2, 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 asEscherichia coli,Aeromonasspp.,Proteusspp.,Morganella morganii,Shewanellaspp., and urea-positiveProvidencia stuartiiisolates 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.

Antimicrobial agents are used in cattle production systems for the prevention and control of bacteria associated with diseases. Australia is the world's 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.

Objectives: The purpose of this study was to determine the diversity of class 2 integrons in bacteria isolated from beef cattle sources. Methods: The variable regions of a subset of 11 class 2 integron-containing bacteria were analysed by PCR and DNA sequencing for the presence of novel rearrangements. Results: A total of six different class 2 integron arrays were identified and four of these were fully characterized. Three of the four arrays characterized have been previously described; however the remaining array is unlike previously described class 2 integrons. The novel class 2 integron was found in Providencia stuartii and contains an apparently functional class 2 integrase. Examination of the variable region of the P. stuartii integron identified nine open reading frames, mostly of unknown function, and represents the first report of a class 2 integron without inserted antibiotic resistance gene cassettes. Conclusions: This study has identified a novel class 2 integron found in P. stuartii that contains an apparently functional naturally occurring class 2 integrase. Further investigation of this novel class 2 integron is required to determine the impact of a functional class 2 integrase upon the evolution of class 2 integrons.

Shiga toxigenicEscherichia coliO157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies ofstxgenotypes 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. coliO157 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) typestx₂(locus of insertion,argW) in Argentinean isolates (P< 0.0001). In Argentinean LI/II strains,stx₂is carried by a prophage inserted atargW, whereas in Australian LI/II strains theargWlocus is occupied by the novelstx₁prophage. In both Argentinean and Australian LI/II strains,stx_2cis almost exclusively carried by a prophage inserted atsbcB. However, alternativeq₉₃₃- orq₂₁-related alleles were identified in the Australianstx_2cprophage. 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 thetir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.

The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n = 27), potential EHEC (pEHEC, n = 3), atypical enteropathogenic E. coli (aEPEC, n = 8) and non-toxigenic E. coli (NTEC, n = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated. Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don’t produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed. Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air-liquid interface under the conditions used in this study. Biofilms provided a protective effect to E. coli O26 strains against the three sanitizers, previously shown to successfully control the growth of their planktonic counterparts. Whether the characteristics of biofilm forming and non-biofilm forming strains observed in this study reflect their attributes within the food and meat-processing environments is unknown. Further studies that represent the food and meat-processing environments are required.