Silver nanoparticles: A novel antibacterial agent for control of Cronobacter sakazakii.

Silver nanoparticles (AgNP) have been widely applied because of their broad spectrum of antimicrobial activities against bacteria, fungi, and viruses. However, little research has been done to evaluate their effects on Cronobacter sakazakii, an opportunistic pathogen usually infecting infants and having a high fatality rate. The aims of this work were to investigate the antibacterial property of novel, synthesized, positively charged silver nanoparticles against C. sakazakii and to discuss the potential antibacterial mechanisms involved. In this study, the spherical and face-centered cubic silver nanoparticles had a mean particle size of 31.2 nm and were synthesized by reducing Ag+ using citrate and dispersed by glycerol and polyvinylpyrrolidone (PVP) under alkaline conditions. Minimum inhibitory concentrations (MIC) and inhibition zone tests showed that the AgNP exhibited strong antibacterial activity against 4 tested C. sakazakii strains with mean MIC of 62.5 to 125 mg/L and average inhibition zone diameters of 13.8 to 16.3 mm. Silver nanoparticles caused cell membrane injury accompanied by adsorption of AgNP onto the cell surface, as shown by changes in cell morphology, cell membrane hyperpolarization, and accelerated leakage of intracellular reducing sugars and proteins outward from the cytoplasm. In addition, dysfunction of the respiratory chain was induced after treatment with AgNP, which was supported by a decrease in intracellular ATP and inhibition of related dehydrogenases. This research indicates that AgNP could be a novel and efficient antibacterial agent to control C. sakazakii contamination in environments producing powdered infant formulas from milk.

Analysis of the Listeria monocytogenes Population Structure among Isolates from 1931 to 2015 in Australia.

Listeriosis remains among the most important bacterial illnesses, with a high associated mortality rate. Efforts to control listeriosis require detailed knowledge of the epidemiology of the disease itself, and its etiological bacterium, Listeria monocytogenes. In this study we provide an in-depth analysis of the epidemiology of 224 L. monocytogenes isolates from Australian clinical and non-clinical sources. Non-human sources included meat, dairy, seafood, fruit, and vegetables, along with animal and environmental isolates. Serotyping, Multi-Locus Sequence Typing, and analysis of inlA gene sequence were performed. Serogroups IIA, IIB, and IVB comprised 94% of all isolates, with IVB over-represented among clinical isolates. Serogroup IIA was the most common among dairy and meat isolates. Lineage I isolates were most common among clinical isolates, and 52% of clinical isolates belonged to ST1. Overall 39 STs were identified in this study, with ST1 and ST3 containing the largest numbers of L. monocytogenes isolates. These STs comprised 40% of the total isolates (n = 90), and both harbored isolates from clinical and non-clinical sources. ST204 was the third most common ST. The high prevalence of this group among L. monocytogenes populations has not been reported outside Australia. Twenty-seven percent of the STs in this study contained exclusively clinical isolates. Analysis of the virulence protein InlA among isolates in this study identified a truncated form of the protein among isolates from ST121 and ST325. The ST325 group contained a previously unreported novel mutation leading to production of a 93 amino acid protein. This study provides insights in the population structure of L. monocytogenes isolated in Australia, which will contribute to public health knowledge relating to this important human pathogen.

Prevalence, seasonality, and growth of enterococci in raw and pasteurized milk in Victoria, Australia.

This study investigated the prevalence, seasonality, and species variety of enterococci present in raw milk factory silos and pasteurized milk in 3 dairying regions in Victoria, Australia, over a 1-yr period. Additionally, the growth ability of thermoduric enterococci isolated in this study (Enterococcus faecalis, E. faecium, E. hirae, and E. durans) was determined in milk at temperatures likely to occur during storage, transport, and distribution, and before domestic consumption (4 and 7°C). Enterococci were detected in 96% of 211 raw milk samples, with an average count of 2.48 log10 cfu/mL. Counts were significantly lower in winter than summer (average 1.84 log10 cfu/mL) and were different between factories but not regions. Enterococcus faecalis was the most prevalent species isolated from raw milk in every factory, comprising between 61.5 and 83.5% of enterococcal species across each season. Enterococci were detected in lower numbers in pasteurized milk than in raw milk and were below the limit of detection on spread plates (<10 cfu/mL) after factory pasteurization. Residual viable cells were only detected following enrichment using 100-mL samples of milk, with 20.8% of the samples testing positive; this equated to a decrease in the average raw milk enterococci count of >4 log10 cfu/mL following pasteurization. Although E. faecalis predominated in raw milk and E. durans was found in only 2.9% of raw milk samples, E. durans was the most prevalent species detected in pasteurized milk. The detection of enterococci in the pasteurized milk did not correlate with higher enterococci counts in the raw milk. This suggested that the main enterococci populations in raw milk were heat-sensitive and that thermoduric enterococci survived pasteurization in a small numbers of instances. All of the thermoduric enterococci that were assessed for growth at likely refrigeration temperatures were able to grow at both 4 and 7°C in sterile milk, with generation times of 35 to 41h and 16 to 22h, respectively. Thermoduric enterococci were detected in pasteurized milk stored at 4°C for 2 wk (typically 1 to 9 cells/100mL, up to 2.82 log10 cfu/mL), demonstrating the potential of enterococci to survive pasteurization and contribute to milk spoilage at refrigeration temperatures. This is particularly relevant for milk that is aseptically packaged to exclude gram-negative psychrotrophic bacteria and kept above the recommended storage temperature of ≤5°C.

Geographically distinct Escherichia coli O157 isolates differ by lineage, Shiga toxin genotype, and total shiga toxin production.

While the differential association of Escherichia coli O157 genotypes with animal and human hosts has recently been well documented, little is known about their distribution between countries and how this might affect regional disease rates. Here, we used a 48-plex single nucleotide polymorphism (SNP) assay to segregate 148 E. coli O157 isolates from Australia, Argentina, and the United States into 11 SNP lineages. We also investigated the relationship between SNP lineages, Shiga toxin (Stx) gene profiles, and total Stx production. E. coli O157 isolates clearly segregated into SNP lineages that were differentially associated with each country. Of the 11 SNP lineages, seven were detected among isolates from a single country, two were detected among isolates from all three countries, and another two were detected only among U.S. and Argentinean isolates. A number of Australian (30%) and Argentinean (14%) isolates were associated with novel, previously undescribed SNP lineages that were unique to each country. Isolates within SNP lineages that were strongly associated with the carriage of stx2a produced comparatively more Stx on average than did those lacking the stx2a subtype. Furthermore, the proportion of isolates in stx2a-associated SNP lineages was significantly higher in Argentina and the United States than Australia (P < 0.05). This study provides evidence for the geographic divergence of E. coli O157 and for a prominent role of stx2a in total Stx production. These results also highlight the need for more comprehensive studies of the global distribution of E. coli O157 lineages and the impacts of regionally predominant E. coli O157 lineages on the prevalence and severity of disease.

Escherichia coli strains expressing H12 antigens demonstrate an increased ability to attach to abiotic surfaces as compared with E. coli strains expressing H7 antigens.

The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.

Multilocus genotype analysis of Escherichia coli O157 isolates from Australia and the United States provides evidence of geographic divergence.

Escherichia coli O157 is a food-borne pathogen whose major reservoir has been identified as cattle. Recent genetic information has indicated that populations of E. coli O157 from cattle and humans can differ genetically and that this variation may have an impact on their ability to cause severe human disease. In addition, there is emerging evidence that E. coli O157 strains from different geographical regions may also be genetically divergent. To investigate the extent of this variation, we used Shiga toxin bacteriophage insertion sites (SBI), lineage-specific polymorphisms (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a tir 255T>A polymorphism to examine 606 isolates representing both Australian and U.S. cattle and human populations. Both uni- and multivariate analyses of these data show a strong association between the country of origin and multilocus genotypes (P < 0.0001). In addition, our results identify factors that may play a role in virulence that also differed in isolates from each country, including the carriage of stx1 in the argW locus uniquely observed in Australian isolates and the much higher frequency of stx2-positive (also referred to as stx2a) strains in the U.S. isolates (4% of Australian isolates versus 72% of U.S. isolates). LSPA-6 lineages differed between the two continents, with the majority of Australian isolates belonging to lineage I/II (LI/II) (LI, 2%; LI/II, 85%; LII, 13%) and the majority of U.S. isolates belonging to LI (LI, 60%; LI/II, 16%; LII, 25%). The results of this study provide strong evidence of phylogeographic structuring of E. coli O157 populations, suggesting divergent evolution of enterohemorrhagic E. coli O157 in Australia and the United States.

Pathogenic Escherichia coli and One Health implications.

Escherichia coli are common inhabitants in the intestinal tracts of warm blooded animals where they generally cause no harm to the host, although there are some types of E. coli which are able to cause disease. The most significant of these are enterohaemorrhagic E. coli (EHEC) which can cause severe human disease that can result in death. EHEC have an animal reservoir, particularly cattle, and are considered to be an important zoonotic pathogen having significant impact for One Health. EHEC can be transmitted from animals into humans, either from consumption of foods made from these animals, or from contact with foods which may have become contaminated directly or indirectly from animal wastes. Increasingly, EHEC have also been associated with uncooked leafy green vegetables and sprouts. Several large outbreaks of E. coli have highlighted the importance for addressing these organisms in a One Health perspective.

Phylogenetically related Argentinean and Australian Escherichia coli O157 isolates are distinguished by virulence clades and alternative Shiga toxin 1 and 2 prophages.

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.

Heat resistance of thermoduric enterococci isolated from milk.

Enterococci are reported to survive pasteurisation but the extent of their survival is unclear. Sixty-one thermoduric enterococci isolates were selected from laboratory pasteurised milk obtained from silos in six dairy factories. The isolates were screened to determine log(10) reductions incurred after pasteurisation (63°C/30 min) and ranked from highest to lowest log(10) reduction. Two isolates each of Enterococcus faecalis, Enterococcus faecium, Enterococcus durans and Enterococcus hirae, exhibiting the median and the greatest heat resistance, as well as E. faecalis ATCC 19433, were selected for further heat resistance determinations using an immersed coil apparatus. D values were calculated from survival curves plotted from viable counts obtained after heating isolates in Brain Heart Infusion Broth at 63, 69, 72, 75 and 78°C followed by rapid cooling. At 72°C, the temperature employed for High Temperature Short Time (HTST) pasteurisation (72°C/15s), the D values extended from 0.3 min to 5.1 min, depending on the isolate and species. These data were used to calculate z values, which ranged from 5.0 to 9.8°C. The most heat sensitive isolates were E. faecalis (z values 5.0, 5.7 and 7.5°C), while the most heat resistant isolates were E. durans (z values 8.7 and 8.8°C), E. faecium (z value 9.0°C) and E. hirae (z values 8.5 and 9.8°C). The data show that heat resistance in enterococci is highly variable.

Surface roughness of stainless steel influences attachment and detachment of Escherichia coli O157.

Determining the influence of surface roughness on Escherichia coli O157 attachment to and detachment from stainless steel (SS) is important for controlling this foodborne pathogen. The aim of this study was to evaluate the interactions of six E. coli strains (four O157:H7, one O157:H12, and one O1:H7) with SS type 304 finishes of various surface roughness: 2B (unpolished surface), 4 (common food grade SS), and 8 (polished smooth surface). In attachment assays (exposure to cell suspensions with periodic swirling), bacteria were enumerated by epifluorescence microscopy, and in detachment assays a blotting technique and atomic force microscopy (AFM) were used. Attachment data suggest that E. coli attach in greater numbers to significantly smoother SS8; however, detachment assays and AFM data suggest cells are more easily removed from this finish. Conversely, attachment to SS2B was lower, and AFM data suggest that E. coli O157 may adhere more strongly to this finish. Attachment and detachment data for SS4 was variable, suggesting complex attachment mechanisms to this type of SS. SS4 is the most common material used in food processing facilities. The data from this study indicate that bacterial interactions with SS4 are complex and less easily predicted than those with SS of other finishes, including 2B and 8. These differences in bacterial attachment may be of concern to the food industry and warrant further investigation.

The DNA protection during starvation protein (Dps) influences attachment of Escherichia coli to abiotic surfaces.

The attachment of bacterial species such as Escherichia coli to abiotic materials is of concern to the food industry. This study investigated the role of DNA protection during starvation protein (Dps) in cell surface hydrophobicity and attachment of E. coli to glass, stainless steel, and Teflon surfaces. The Dps was not found to influence hydrophobicity, but did have a putative role in attachment in a strain- and substrate-dependent manner.

International comparison of clinical, bovine, and environmental Escherichia coli O157 isolates on the basis of Shiga toxin-encoding bacteriophage insertion site genotypes.

Escherichia coli O157:H7 genotypes in the bovine reservoir may differ in virulence. The proportion of clinical genotypes among cattle isolates was weakly (P = 0.054) related to the international incidence of E. coli O157:H7-associated hemolytic-uremic syndrome, varied among clinical isolates internationally, and also differed along the putative cattle-hamburger-clinical case transmission chain.

EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation.

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC and z3948/ehaD) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Preliminary characterization revealed that ehaA, ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes (ehaA) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H(-) did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.

Diverse class 2 integrons in bacteria from beef cattle sources.

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.

Emergence and spread of two distinct clonal groups of multidrug-resistant Escherichia coli in a veterinary teaching hospital in Australia.

Multidrug-resistant Escherichia coli (MDREC) expressing AmpC beta-lactamases have emerged as a cause of opportunistic infections in dogs. Following a cluster of extraintestinal infections caused by two distinct clonal groups (CGs) of bla(CMY)-producing MDREC, a 12-month infection control study was undertaken at a veterinary teaching hospital in Brisbane, Australia. Swabs from the rectum of hospitalized dogs (n=780), hospital staff (n=16) and the hospital environment (n=220) were plated onto selective agar to obtain multidrug-resistant (MDR) coliforms. These were then tested by multiplex PCR for E. coli uspA, bla(CMY) and the class 1 integron-associated dfrA17-aadA5 gene cassette for rapid identification of MDREC CG 1 (positive for all three genes) and CG 2 (positive for uspA and bla(CMY) only). A total of 16.5 % of the dog rectal swabs and 4.1% of the hospital environmental swabs yielded MDREC, and on the basis of multiplex PCR, PFGE and plasmid profiling, these were confirmed to belong to either CG 1 or CG 2. Both CG 1 and CG 2 isolates were obtained from clinical cases of extraintestinal infection and rectal swabs from hospitalized dogs over the same period of time, whereas only CG 1 isolates were obtained from the hospital environment. Both CGs were prevalent during the first 6 months, but only CG 2 was isolated during the second 6 months of the study. Two isolates obtained from rectal swabs of staff working in the hospital belonged to CG 2, with one of the isolates possessing the same REDP as nine isolates from dogs, including six isolates associated with cases of extraintestinal infection. CG 1 isolates belonged to E. coli serotypes O162 : H-, OR : H- or Ont : H-, whereas CG 2 isolates belonged to O153 : HR, OR : HR or OR : H34. These results confirm that in this particular outbreak, canine MDREC were highly clonal and CG 2 MDREC may colonize both humans and dogs.

Shiga toxin-producing Escherichia coli in ground beef and lamb cuts: results of a one-year study.

Shiga toxin-producing Escherichia coli (STEC) have been associated with a broad spectrum of diarrhoeal syndromes. Some of these cases have been attributed to foods of bovine origin or other foods cross-contaminated by beef products or cow manure. The purpose of this study was to determine the pattern of STEC distribution in selected red meats over time. Samples of ground beef and lamb cuts were collected over a 52-week period from 31 different outlets and 25 g portions were assayed for STEC. STEC were isolated from 46/285 (16%) ground beef and 111/275 (40%) lamb samples using an stx PCR screen followed by colony hybridisation. All isolates were tested by PCR for additional STEC virulence markers with 95% of ground beef isolates shown to possess stx(2) and 80% of lamb cutlet isolates shown to possess stx(1) and stx(2). The enterohaemolysin gene (ehxA) was detected in 65% and 53% of ground beef and lamb isolates respectively. Putative enterohaemorrhagic E. coli (EHEC), i.e. STEC possessing the E. coli attaching and effacing gene (eae) were not isolated. The STEC isolates comprised 18 and 15 different serotypes from ground beef and lamb respectively. STEC of serotypes O157, O111 and O26 (common enterohaemorrhagic E. coli serotypes) were not isolated. Serotypes O174 and O91 were the most common serotypes isolated from ground beef samples and O128 and O91 the most common from lamb cutlet samples. The presence of STEC in retail red meats highlights the need for a clearer understanding of STEC in food and human illness to interpret the public health significance of these findings.

Identification of bla(CMY-7) and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia.

OBJECTIVES: To determine clonality and identify plasmid-mediated resistance genes in 11 multidrug-resistant Escherichia coli (MDREC) isolates associated with opportunistic infections in hospitalized dogs in Australia. METHODS: Phenotypic (MIC determinations, modified double-disc diffusion and isoelectric focusing) and genotypic methods (PFGE, plasmid analysis, PCR, sequencing, Southern hybridization, bacterial conjugation and transformation) were used to characterize, investigate the genetic relatedness of, and identify selected plasmid-mediated antimicrobial resistance genes, in the canine MDREC. RESULTS: Canine MDRECs were divided into two clonal groups (CG 1 and 2) with distinct restriction endonuclease digestion and plasmid profiles. All isolates possessed bla(CMY-7) on an approximately 93 kb plasmid. In CG 1 isolates, bla(TEM), catA1 and class 1 integron-associated dfrA17-aadA5 genes were located on an approximately 170 kb plasmid. In CG 2 isolates, a second approximately 93 kb plasmid contained bla(TEM) and unidentified class 1 integron genes, although a single CG 2 strain carried dfrA5. Antimicrobial susceptibility profiling of E. coli K12 transformed with CG 2 large plasmids confirmed that the bla(CMY-7)-carrying plasmid did not carry any other antimicrobial resistance genes, whereas the bla(TEM)/class 1 integron-carrying plasmid carried genes conferring resistance to tetracycline and streptomycin also. CONCLUSIONS: This is the first report on the detection of plasmid-mediated bla(CMY-7) in animal isolates in Australia. MDREC isolated from extraintestinal infections in dogs may be an important reservoir of plasmid-mediated resistance genes.

Isolation and characterization of integron-containing bacteria without antibiotic selection.

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.

Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources.

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c vha), stx(2c vhb), or stx(2d EH250). Subsequently, the stx(2c vha) and stx(2c vhb) operons were screened for the absence of a PstI site in the stx(2A) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.