Diversity of STs, plasmids and ESBL genes among Escherichia coli from humans, animals and food in Germany, the Netherlands and the UK.

OBJECTIVES: This study aimed to compare ESBL-producing Escherichia coli causing infections in humans with infecting or commensal isolates from animals and isolates from food of animal origin in terms of the strain types, the ESBL gene present and the plasmids that carry the respective ESBL genes. METHODS: A collection of 353 ESBL-positive E. coli isolates from the UK, the Netherlands and Germany were studied by MLST and ESBL genes were identified. Characterization of ESBL gene-carrying plasmids was performed using PCR-based replicon typing. Moreover, IncI1-Iγ and IncN plasmids were characterized by plasmid MLST. RESULTS: The ESBL-producing E. coli represented 158 different STs with ST131, ST10 and ST88 being the most common. Overall, blaCTX-M-1 was the most frequently detected ESBL gene, followed by blaCTX-M-15, which was the most common ESBL gene in the human isolates. The most common plasmid replicon type overall was IncI1-Iγ followed by multiple IncF replicons. CONCLUSIONS: ESBL genes were present in a wide variety of E. coli STs. IncI1-Iγ plasmids that carried the blaCTX-M-1 gene were widely disseminated amongst STs in isolates from animals and humans, whereas other plasmids and STs appeared to be more restricted to isolates from specific hosts.

Complete sequence and molecular epidemiology of IncK epidemic plasmid encoding blaCTX-M-14.

Antimicrobial drug resistance is a global challenge for the 21st century with the emergence of resistant bacterial strains worldwide. Transferable resistance to ß-lactam antimicrobial drugs, mediated by production of extended-spectrum ß-lactamases (ESBLs), is of particular concern. In 2004, an ESBL-carrying IncK plasmid (pCT) was isolated from cattle in the United Kingdom. The sequence was a 93,629-bp plasmid encoding a single antimicrobial drug resistance gene, blaCTX-M-14. From this information, PCRs identifying novel features of pCT were designed and applied to isolates from several countries, showing that the plasmid has disseminated worldwide in bacteria from humans and animals. Complete DNA sequences can be used as a platform to develop rapid epidemiologic tools to identify and trace the spread of plasmids in clinically relevant pathogens, thus facilitating a better understanding of their distribution and ability to transfer between bacteria of humans and animals.

Exposure to isothiocyanates suppresses urinary mutagenicity in rats treated with heterocyclic amine IQ: lack of association with CYP1 activity.

The principal objectives of this study were to evaluate whether exposure of rats to low doses of isothiocyanates modulates the overall metabolism of heterocyclic amine 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ), as exemplified by urinary mutagenicity, a food carcinogen, and to relate any modifications in metabolism to changes in CYP1 and glutathione S-transferase activities. Animals were exposed to isothiocyanates either for 2 wk (long-term) or 1 day (short-term), and all animals were then treated with a single oral dose of IQ, and urine was collected daily for 3 days; animals continued to receive the isothiocyanates during this period. Urinary mutagenic activity was determined using the Ames mutagenicity assay in the presence of an activation system from Aroclor 1254-treated rats. At the end of the study, animals were killed and hepatic methoxy- and ethoxyresorufin dealkylations were determined as well as glutathione S-transferase activity. All isothiocyanates studied, namely sulforaphane, erucin, and phenethyl isothiocyanate, decreased urinary mutagenic activity, implying enhanced IQ metabolism, but only after long-term intake. Changes in mutagenic activity were not related to changes of any of the enzyme activities determined. It is concluded that long-term intake of isothiocyanates may stimulate the metabolism of IQ, but this effect is not linked to changes in hepatic CYP1A2 and glutathione S-transferase activities.

A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli.

OBJECTIVES: Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. METHODS: The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. RESULTS: The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. CONCLUSIONS: The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification.

OBJECTIVES: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-TolC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. METHODS: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. RESULTS: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. CONCLUSIONS: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Proteomic analysis of triclosan resistance in Salmonella enterica serovar Typhimurium.

OBJECTIVES: The aim of this study was to determine and compare the proteomes of three triclosan-resistant mutants of Salmonella enterica serovar Typhimurium in order to identify proteins involved in triclosan resistance. METHODS: The proteomes of three distinct but isogenic triclosan-resistant mutants were determined using two-dimensional liquid chromatography mass separation. Bioinformatics was then used to identify and quantify tryptic peptides in order to determine protein expression. RESULTS: Proteomic analysis of the triclosan-resistant mutants identified a common set of proteins involved in production of pyruvate or fatty acid with differential expression in all mutants, but also demonstrated specific patterns of expression associated with each phenotype. CONCLUSIONS: These data show that triclosan resistance can occur via distinct pathways in Salmonella, and demonstrate a novel triclosan resistance network that is likely to have relevance to other pathogenic bacteria subject to triclosan exposure and may provide new targets for development of antimicrobial agents.

Fitness and dissemination of disinfectant-selected multiple-antibiotic-resistant (MAR) strains of Salmonella enterica serovar Typhimurium in chickens.

OBJECTIVES: The aims of this study were to determine whether strains of Salmonella enterica serovar Typhimurium which had acquired low-level multiple antibiotic resistance (MAR) through repeated exposure to farm disinfectants were able to colonize and transmit between chicks as easily as the parent strain and, if such strains were less susceptible to fluoroquinolones, would high-level resistance be selected after fluoroquinolone treatment. METHODS: Two mutants were compared with the isogenic parent. In the first experiment, day-old chicks were co-infected with both the parent and a mutant to determine their relative fitness. In the second experiment, parent and mutant strains (in separate groups of chicks) were assessed for their ability to transmit from infected (contact) to non-infected (naive) birds and with respect to their susceptibility to fluoroquinolone treatment. Birds were regularly monitored for the presence of Salmonella in caecal contents. Replica plating was used to monitor for the selection of antibiotic-resistant strains. RESULTS: The parent strain was shown to be significantly fitter than the two mutants and was more rapidly disseminated to naive birds. Antibiotic treatment did not preferentially select for the two mutants or for resistant strains. CONCLUSIONS: The disinfectant-exposed strains, although MAR, were less fit, less able to disseminate than the parent strain and were not preferentially selected by therapeutic antibiotic treatment. As such, these strains are unlikely to present a greater problem than other salmonellae in chickens.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella enterica serovar Typhimurium selected following exposure to disinfectants.

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-TolC for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

Up-regulation of the CYP1 family in rat and human liver by the aliphatic isothiocyanates erucin and sulforaphane.

On the basis of animal studies, the chemopreventive activity of isothiocyanates has been linked to their ability to modulate carcinogen-metabolising enzyme systems, including cytochrome P450. However, the potential of isothiocyanates to influence these enzyme systems in human liver has not been investigated. We have evaluated the modulation of cytochrome P450 expression in two human liver samples by erucin and sulforaphane, in comparison to rat, following the incubation of precision-cut human and rat liver slices with the two isothiocyanates. Both compounds failed to influence cytochrome P450 activity, as exemplified by the dealkylations of methoxy-, ethoxy- and pentoxyresorufin, and benzyloxyquinoline, in either human or rat liver. Impairment of activity was, however, observed in some activities at high concentrations (50microM), which was attributed to toxicity. At the apoprotein level, however, both compounds markedly elevated CYP1A2/1B1 levels in rat liver, but in human liver only a modest increase was evident, and only in one of the livers. CYP3A2 apoprotein levels were modestly elevated in rat liver by both isothiocyanates both of which, however, failed to influence CYP3A4 expression in human liver. Neither isothiocyanate, in either rat or human liver, modulated CYP2B apoprotein levels. It may be inferred that (a) human and rat liver differ in their response to erucin and sulforaphane, (b) erucin and sulforaphane, despite being small molecular weight aliphatic compounds, up-regulate the CYP1 family but no increase in activity is observed as a result of mechanism-based inhibition, and (c) the chemopreventive effect of isothiocyanates, at dietary levels of intake, is unlikely to be due to inhibition of the cytochrome P450-mediated bioactivation of carcinogens.

Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-blaCTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 µM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying blaCTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 µM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

Translational Inhibition of CTX-M Extended Spectrum ß-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.

Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and -17 to -5, respectively) close to the translational initiation site of the extended-spectrum ß-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit ß-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a bla CTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 ß-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of bla CTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

Cytochrome P450 expression and testosterone metabolism in the liver of deer.

Cytochrome P450 expression in cervine liver was investigated using chemical probes and Western blot analysis, and compared with the rat. Deer liver, when compared with rat liver, was characterised by high ethoxyresorufin O-deethylase, coumarin 7-hydroxylase and, to a lesser extent, erythromycin N-demethylase activities; in contrast, deer liver exhibited low debrisoquine 4-hydroxylase, chlorzoxazone 6-hydroxylase and, particularly, lauric acid hydroxylase activities. Ethoxyresorufin O-deethylase activity in deer was markedly inhibited by alpha-naphthoflavone, but was relatively resistant to inhibition by furafylline. Coumarin 7-hydroxylase was inhibited by 8-methoxypsoralen. Western blot analysis using antibodies to rat CYP1A recognised a single, highly expressed protein. Kinetic analysis indicated that a single enzyme is likely to be responsible for the high ethoxyresorufin O-deethylase activity in deer liver. Probing of cervine hepatic microsomes with antibodies to rat CYP2A2 showed that apoprotein levels were higher in the deer compared with the rat. Eadie-Hofstee plot analysis indicated that more than one enzyme catalyses the 7-hydroxylation of coumarin. Western blot analysis using antibodies to rat CYP2B, rat CYP2C11, human CYP2D6, rat CYP3A and rat CYP4A1 revealed in each case the presence of single, poorly expressed, proteins in deer liver. In contrast, when antibodies to rat CYP2E1 were used, a highly expressed single protein was observed. Cervine hepatic microsomes metabolised testosterone to generate androstenedione and a number of hydroxylated products, the major hydroxylation sites being the 2beta-, 6beta- and possibly the 12-position. In summary, this is the first study showing that deer liver expresses all xenobiotic-metabolising cytochrome P450 families, but the level of expression differs from that of the rat.

Absolute bioavailability of [14C] genistein in the rat; plasma pharmacokinetics of parent compound, genistein glucuronide and total radioactivity.

The systemic plasma pharmacokinetics of genistein were determined in rats to evaluate the absolute oral bioavailability and make comparison with similar data in the literature derived from humans subjects. The plasma concentrations of genistein, genistein glucuronide and carbon-14 were determined by LC-MS/MS and liquid scintillation counting following oral and intravenous dosing with [14C]genistein (4 mg kg(-1) body weight). The absorption of total radioactivity from the gut, (parent compound and metabolites), was 56 and 111% in male and female rats, respectively. In contrast, the absolute oral bioavailability of genistein in male and female rats was 7 and 15%. There was a significant (P<0.001) difference between Cmax of genistein after intravenous (6921 and 4392 ng/ml) and oral (21 and 22 ng/ml) dosing in male and female rats, respectively. After oral administration, the concentration profile of genistein glucuronide in plasma greatly exceeded that of parent compound during the absorption/distribution phase suggesting extensive first pass metabolism, and provided evidence of entero-hepatic circulation. Selective plasma analysis by LC-MS/MS, without prior enzymatic hydrolysis, enabled ready discrimination between parent and conjugated metabolites and prevented gross overestimation of genistein bioavailability. Pharmacokinetic parameters Cmax, Tmax and AUC were similar to those reported in humans, which supports the use of the rat model for genistein toxicity studies.

Detection of antibiotic residues and association of cefquinome residues with the occurrence of Extended-Spectrum ß-Lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011.

Waste milk samples from 103 farms in England and Wales were examined for the presence of ß-lactam antibiotics and ESBL-producing Enterobacteriaceae. Approximately 10 months after the initial sampling, further waste milk, environmental and faecal samples from farms shown to be positive for CTX-M Escherichia coli were investigated further. Isolates with an ESBL phenotype were tested by PCR for the presence of blaCTX-M, blaOXA, blaSHV and blaTEM genes. Isolates positive for blaCTX-M were sequenced to determine CTX-M type. Representative isolates were further examined by PFGE, plasmid replicon typing and serotyping. Of particular interest, 21.4% of waste milk samples contained residues of the cephalosporin cefquinome, which was significantly associated with CTX-M bacteria. Such bacteria occurred in 5.8% of the waste milk samples (including 3.9% CTX-M E. coli). CTX-M types identified were 1, 14, 14b and 15, but none of the E. coli were serotype O25, the serotype of the human pandemic strain.

Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany.

The putative virulence and antimicrobial resistance gene contents of extended spectrum ß-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla(CTX-M-group-1) dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla(CTX-M-group-1) and bla(OXA-1-like) genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6')-Ib, catB3, bla(OXA-1-like) and bla(CTX-M-group-1). forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Virulence genes in bla(CTX-M) Escherichia coli isolates from chickens and humans.

The aim of this study was to determine the presence of virulence genes in isolates of CTX-M Escherichia coli from diseased chickens, from healthy chickens and from urinary tract infections in people. Three CTX-M E. coli strains from three different instances of disease in poultry (two of which were E. coli related) were tested for bla(CTX-M) sequence type and replicon type. Additionally, they were tested for the presence of 56 virulence genes (encoding fimbriae, adhesins, toxins, microcins and iron acquisition genes) using a micro-array. Results were compared to the virulence genes present in isolates from 26 healthy chickens and from 10 people with urinary tract infections. All genes found in isolates from diseased birds, including the astA (heat stable toxin) and tsh (temperature sensitive haemagglutinin) genes which have previously been associated with colibacillosis in chickens, were also present in isolates from healthy birds. However, 6/10 of the virulence genes found were exclusive to isolates from humans. Genes exclusive to chicken isolates included ireA (sidephore receptor), lpfA (long polar fimbriae), mchF (microcin transporter protein) and tsh whilst genes exclusive to human isolates included ctdB (cytolethal distending toxin), nfaE (non-fimbrial adhesion), senB (plasmid encoded enterotoxin) and toxB (toxin B). The results support previous findings that CTX-M E. coli strains in chickens are generally different from those causing disease in humans, but genes such as astA and tsh in isolates from diseased birds with colisepticaemia were also present in isolates from healthy birds.

Molecular and phenotypic characterisation of extended spectrum ß-lactamase CTX-M Escherichia coli from farm animals in Great Britain.

The aim of this study was to characterise CTX-M Escherichia coli isolates from cattle, chickens and turkeys in Great Britain with respect to CTX-M sequence type, replicon type, ability to transfer plasmids, and for the presence of antibiotic resistance, fitness and virulence genes as determined by micro-arrays. The main CTX-M enzymes identified in E. coli from cattle, chicken and turkeys were 14 and 15, 1 and 15, and 1 and 14 respectively. Most isolates from different animal species transferred their plasmids with similar frequencies. The plasmid replicon type I1-λ was most common and seen in 23%, 95% and 50% of the isolates tested from cattle, chickens and turkeys respectively, whilst types F, FIA, FIB and K were common to isolates from cattle and turkeys only. Thirty-eight different antibiotic resistance genes were detected by micro-array including aad genes, blaCTX-M, blaTEM, cat genes dfrA, floR, strA, strB, sul, sul2 tetA and tetB. Thirty-nine different fitness and virulence genes were also detected by-micro-array, including espP, ireA, lpfA, mchF, prfB and tsh. Fisher exact test and hierarchical clustering of the antibiotic resistance and virulence gene results showed some genes were more commonly associated with isolates from chickens or cattle. This study provides a baseline of the characteristics of CTX-M E. coli isolates from animals in Great Britain and suggests that chicken and cattle CTX-M E. coli represent different populations.

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans.

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.

Epidemiology of extended spectrum beta-lactamase E. coli (CTX-M-15) on a commercial dairy farm.

The epidemiology of an extended spectrum beta-lactamase Escherichia coli (CTX-M-15) was observed and described on a commercial dairy farm located in the United Kingdom. During 2008 longitudinal sampling of faecal pat samples from different cattle groups comprising milking and non-milking cows, calving cows, calves, and the environment was carried out. The proportion of CTX-M-15 E. coli positive samples was significantly (p<0.0.01) higher in milking cows (30.3%, CI(95%) 26.8; 33.8) than in the herd as a whole (17.0%, CI(95%) 14.9; 19.0). In 2008 95.6% of sampled calves tested positive for CTX-M-15 E. coli at two days of age. A more detailed investigation in 2009 revealed that cows and heifers were approximately eight times more likely to test positive in the 10 days after calving than the 9 days before (OR 7.6, CI(95%) 2.32; 24.9). The CTX-M15 E. coli was also readily isolated from the immediate calving pen environment, including the water troughs. A cyclic pattern was apparent where cows immediately after calving and as high yielders were highly positive, but where the prevalence decreased during the dry period. The increased prevalence of the CTX-M-15 E. coli in certain cattle groups and farm environments including calving pens suggested that husbandry, antimicrobial usage and hygiene may play a significant role on a farm with regards to the epidemiology of CTX-M-15. This may offer a practical opportunity to reduce further dissemination through good practice and hygiene around calving.