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.

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 β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-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 β-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.

1 genes among Escherichia coli from humans, animals and food in Germany, 2 Netherlands and United Kingdom 3 4 Michaela J. Day, Irene Rodríguez, Alieda van Essen-Zandbergen, Cindy Dierikx, 5 Kristina Kadlec, Anne-Kathrin Schink, Guanghui Wu, Marie A. Chattaway, Vivienne 6 DoNascimento, John Wain, Reiner Helmuth, Beatriz Guerra, Stefan Schwarz, John 7 Threlfall, Martin J. Woodward, Nick Coldham, Dik Mevius and Neil Woodford 8 9 10 11 1 Public Health England, London, United Kingdom, 2 Federal Institute for Risk Assessment (BfR), 12 Berlin, Germany, 3 University Hospital Ramón y Cajal, and Instituto Ramón y Cajal de Investigación 13 Sanitaria (IRYCIS), Madrid, Spain, 4 Central Veterinary Institute (CVI) of Wageningen UR, Lelystad, 14 The Netherlands, 5 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt15 Mariensee, Germany, 6 Animal and Plant Health Agency (APHA, Weybridge), Addlestone, United 16 Kingdom, 7 University of East Anglia, Norwich, United Kingdom, 8 The University of Reading, 17 Whiteknights, Reading, United Kingdom, 9 Utrecht University, Utrecht, The Netherlands. 18 19 20 Running title: E. coli diversity in animals, humans and food in Western Europe 21 22

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.

The aim of this study was to investigate the bacterial strains and farm environment that may contribute to the persistence of ESBL-producing E. coli on a single UK dairy farm. A longitudinal study was conducted comprising 6 visits, between August and October 2010, followed by a further visit at approximately 69weeks after the initial visit. Faecal and environmental samples were collected from different parts of the farm. The persistence and extent of faecal shedding of ESBL E. coli by individual calves was also determined. Twenty two different PFGE types were identified. Four of these were persistent during the study period and were associated with serotypes: O98, O55, O141 and O33. The counts suggest that shedding in calf faeces was an important factor for the persistence of strains, and the data will be useful for parameterising mathematical models of the spread and persistence of ESBL strains within a dairy farm.

To the Editor: Several clones of extended-spectrum β-lactamase (ESBL)–producing extraintestinal pathogenic Escherichia coli (ExPEC) have globally expanded their distribution, including multilocus sequence types (MLSTs) ST38, ST131, ST405, and ST648 (1). ExPEC infections often originate from the patient’s own intestinal flora, although the degree of overlap between diarrheagenic E. coli and ExPEC pathotypes is unclear. Relatively little is known about antimicrobial drug resistance in the most common diarrheagenic E. coli groups, including enteroaggregative E. coli (EAEC), and bacterial gastroenteritis is generally managed without use of antimicrobial drugs. The ability of diarrheagenic E. coli to cause extraintestinal infections has been shown in previous studies: a study among children in Nigeria linked EAEC to uropathogenic clonal group A (2), and a study in Brazil showed that EAEC markers were present in 7.1% of the E. coli isolates from urinary tract infections (3). Neither of these studies identified clonal lineages of EAEC specifically associated with extraintestinal infections. We conducted this study to establish the presence and characteristics of ESBL-producing EAEC in a well-defined collection of ESBL-producing isolates (4). The isolates were from human and animal sources in Germany, the Netherlands, and the United Kingdom. The study was conducted at Public Health England during January–April 2013. DNA from 359 ESBL isolates (4) was screened for the presence of the EAEC transport regulator gene (aggR), located on the EAEC plasmid, by using a real-time PCR assay and the following primers and probe: AggR_F 5′-CCATTTATCGCAATCAGATTAA-3′ AggR_R 5′-CAAGCATCTACTTTTGATATTCC-3′, AggR_P Cy5-CAGCGATACATTAAGACGCCTAAAGGA-BHQ. The amplification parameters were 50°C for 2 min, 95°C for 2 min, and 40 cycles at 95°C for 10 s and at 60°C for 20 s. Isolates positive for aggR were confirmed to be E. coli by using the Omnilog GenIII MicroPlate (Biolog, Hayward, CA, USA). Serotyping was done by using standard methods (5). The phylogroup was determined for each isolate, and isolates were then assigned to 1 of the 4 major E. coli groups: A, B1, B2, and D (6). A microarray was used to detect ESBL genes, such as blaCTX-M, at the group level, as previously described (4). The antimicrobial drug susceptibilities of EAEC isolates were determined by using the agar incorporation method, as described in the British Society for Antimicrobial Chemotherapy guidelines (7). Virulence factors associated with intestinal and extraintestinal infection (8) and with EAEC were investigated as previously described (9). We assigned a virulence score (total number of virulence factor genes detected; maximum possible score 22) and a resistance score (total number of drug classes; maximum score 11) to each isolate. We isolated 11 EAEC from humans. Eight of the EAEC were isolated from urine specimens, and 1 was isolated from a blood culture; 63% belonged to phylogroup D (Table). EAEC ST38, the most common (55%) ST, was significantly associated with extraintestinal sites in the subset of 140 human isolates (Fisher exact test, p<0.0001). Table Characteristics of human-derived ESBL-producing enteroaggregative Escherichia coli isolates from sources in Germany, the Netherlands, and the United Kingdom* In this study, we identified multidrug-resistant EAEC isolates belonging to ST38; the isolates had various somatic antigens and blaCTX-M genes (Table). The multiple somatic antigens, variety of antimicrobial drug–resistance scores, and variety of gene complements in this successful ST indicate multiple acquisitions of virulence markers, rather than clonal expansion from a single source (Table; Technical Appendix Figure). In the MLST public database, which contained 5,143 E. coli entries in June 2013, ST38 is predominantly associated with urinary tract infections, but in-house MLST studies at the Gastrointestinal Bacteria Reference Unit, Public Health England, have shown that ST38 is a successful EAEC group. The presence of EAEC virulence factors, such as aggregative adherence fimbria AAFI and aggR, can mediate adherence of E. coli to bladder epithelial cells, but the virulence factors do not impart uropathogenic properties to all EAEC isolates (10). The ST38 strain described here probably originated from the gut and independently acquired the 2 phenotypes (uropathogenic E. coli [UPEC] and EAEC), which would suggest the emergence of a UPEC/EAEC hybrid strain. It seems likely that an ST38 E. coli strain adapted to EAEC plasmid carriage (a change that would help survival in the gut through increased adherence) has acquired UPEC virulence factors, facilitating the exploitation of an extraintestinal niche, the urinary tract. Despite the characterization of numerous virulence factors, no single genetic feature currently defines EAEC or UPEC isolates. Because the EAEC ST38 strain had 4–7 ExPEC-associated virulence factors, we suggest that, on the basis of epidemiologic, microbiological, and molecular characteristics, the EAEC ST38 described in this study should be considered an ExPEC associated with uropathogenic infections. It is possible that the multidrug-resistant EAEC ExPEC group has expanded globally but is currently underreported. We therefore urge testing for the EAEC genotype in all clinical studies of E. coli pathotypes. Our findings show the potential for EAEC, previously considered a gut pathogen, to cause extraintestinal infection. We suggest that the UPEC/EAEC pathotype may be an evolving clonal group. In particular, a single sequence type, ST38, was associated with multidrug resistance and with urinary tract infection in humans. Technical Appendix: Virulence factors and antimicrobial drug resistance gene content of enteroaggregative Escherichia coli isolates, grouped by phylogroup. Click here to view.(173K, pdf)

Over 230 serovars of Leptospira interrogans have been identified; however few have been completely characterised. The aim of this study was to characterise the proteome of serovar Canicola and to compare this against the serovars of Copenhageni and Pomona. 2D-LC/MS analysis identified 1653 Leptospira proteins in serovar Canicola; 60 of these proteins were common to Copenhageni and Pomona, 16 of which are known to be immunogenic. This study provides the first reported proteome for serovar Canicola and suggests that proteomic comparison of different serovars could be used as a tool for identification of novel target molecules for vaccine development.

Waste milk samples from 103 farms in England and Wales were examined for the presence of β-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.

The Leptospira interrogans vaccines currently available are serovar specific and require regular booster immunizations to maintain protection of the host. In addition, a hamster challenge batch potency test is necessary to evaluate these vaccines prior to market release, requiring the use of a large number of animals, which is ethically and financially undesirable. Our previous work showed that the N terminus of the outer membrane protein LipL32 was altered in Leptospira interrogans serovar Canicola vaccines that fail the hamster challenge test, suggesting that it may be involved in the protective immune response. The aim of this study was to determine if vaccination with LipL32 protein alone could provide a protective response against challenge with L. interrogans serovar Canicola to hamsters. Recombinant LipL32, purified from an Escherichia coli expression system, was assessed for protective immunity in five groups of hamsters (n = 5) following a challenge with the virulent L. interrogans serovar Canicola strain Kito as a challenge strain. However, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. Subsequent histological analysis revealed reduced amounts of L. interrogans in the kidneys from the hamsters vaccinated with recombinant LipL32 protein prior to challenge; however, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. This finding corresponded to a noticeably reduced severity of renal lesions. This study provides evidence that LipL32 is involved in the protective response against L. interrogans serovar Canicola in hamsters and is the first reported link to LipL32-induced protection against kidney invasion.

Previously described methicillin-resistant Staphylococcus aureus (MRSA) ST398 strains revealed a high frequency of phenotypic resistance to spectinomycin. However, only a few were found to carry the spc resistance determinant. The aim of this study was to identify the genetic mechanism of spectinomycin resistance among spc-negative MRSA ST398 strains.Nine spectinomycin-resistant, but spc-negative, MRSA ST398 strains were analysed. The strains were screened for carriage of the spw gene and tested for the presence of transferrable spectinomycin resistance. Plasmid DNA was isolated from all strains and used in transformation assays. The plasmid identified as mediating resistance to spectinomycin was fully sequenced. The function of the novel spectinomycin resistance gene was confirmed by restriction digest inactivation and its distribution was determined using a PCR assay.A single MRSA ST398 strain was spw positive. The remaining strains carried a plasmid that mediated resistance to spectinomycin. Sequence analysis of a single plasmid, termed pDJ91S, revealed that it was 3928 bp in size and contained three open reading frames: a novel spectinomycin resistance gene, designated spd, as well as a repN gene and a rec gene. The XmnI digest inactivation of the spd gene resulted in a 4-fold decrease in spectinomycin MIC. The spd gene was detected in seven other spectinomycin-resistant MRSA ST398 strains that carried a plasmid comparable in size to pDJ91S.A novel gene, designated spd, that confers resistance to spectinomycin has been identified on a small plasmid in MRSA ST398.

The objective of this study was to investigate the hypothesis that some sporadic Salmonella infections in domesticated animals may be associated with Salmonella infections originating from garden birds. Phage type and antimicrobial resistance details of isolates of S. Typhimurium obtained from wild birds were comparable with those from S. Typhimurium infections from domesticated animals or livestock between 2002 and 2010. A small panel of S. Typhimurium isolates (n=37) were characterised by multilocus variable number of tandem repeats analysis (MLVA), pulsed field gel electrophoresis (PFGE) and phage type. The MLVA-PFGE data clustered the strains according to phage type (DT40 or DT56). Within each group there were strains from wild birds and domesticated animals or livestock with MLVA profiles having up to 100% similarity. The results from this study therefore lend support to the hypothesis that Salmonella infections in domesticated animals could be caused by infections carried by wild birds.

ABSTRACT The same plasmid carrying bla CTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.

Enteric bacteria with resistance to third and fourth generation cephalosporin antibiotics, especially Escherichia coli bearing the blaCTX-M gene, have been detected in a wide range of food producing animals. However, commercial vaccines for these organisms are not currently available. An autogenous vaccine was prepared from E. coli bearing the blaCTX-M-14 gene and evaluated as a potential control measure to reduce shedding and dissemination of these organisms in cattle. Calves (n = 30) received either an autogenous vaccine prepared from E. coli serotype O33 bearing the blaCTX-M-14 gene or a placebo by intramuscular injection on three separate occasions. Two weeks after the final vaccination, all calves were challenged by oral gavage with the O33 CTX-M-14 strain of E. coli (1 × 1010 CFU). Faeces, intestinal mucosa and blood samples were taken for enumeration of total and CTX-M-14 E. coli and for assessment of the humoral immune response. The cumulative number of total E. coli excreted at 7 days post-challenge was significantly (p = 0.006) lower in the vaccinated group than the placebo group. However, there was no significant difference in the shedding of either CTX-M-14 E. coli or total E. coli between vaccinated and placebo calves throughout the study period. The systemic immune response to E. coli O33 antigen was tested by ELISA and was significantly higher (p < 0.001) in vaccinated than placebo calves. However, there was no significant difference in the mucosal immune response. These findings do not support the use of autogenous vaccination for the control of CTX-M-14 E. coli in calves.

The cause for the high prevalence of cefotaximase-producing Escherichia coli reported in dairy calves is unknown but may be partly due to the selective pressure of antimicrobial residues in waste milk (milk unfit for human consumption) fed to the calves. Antimicrobial use and waste milk feeding practices were investigated in 557 dairy farms in 2010/2011 that responded to a randomised stratified postal survey. The mean number of cases of mastitis per herd in the previous year was 47, and 93 per cent of respondents used antibiotic intra-mammary tubes to treat mastitis. The most frequently used lactating cow antibiotic tubes contained dihydrostreptomycin, neomycin, novobiocin, and procaine penicillin (37 per cent), and cefquinome (29 per cent). Ninety-six per cent of respondents used antibiotic tubes at the cessation of lactation (‘drying off’). The most frequently used dry cow antibiotic tube (43 per cent) contained cefalonium. Frequently used injectable antibiotics included tylosin (27 per cent), dihydrostreptomycin and procaine penicillin (20 per cent) and ceftiofur (13 per cent). Eighty-three per cent of respondents (413) fed waste milk to calves. Of these 413, 87 per cent fed waste milk from cows with mastitis, and only one-third discarded the first milk after antibiotic treatment. This survey has shown that on more than 90 per cent of the farms that feed waste milk to calves, waste milk can contain milk from cows undergoing antibiotic treatment. On some farms, this includes treatment with third- and fourth-generation cephalosporins. Further work is underway to investigate the presence of these antimicrobials in waste milk.

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.

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.

ABSTRACTThe current batch potency test forLeptospira interrogansserovar Canicola vaccines requires the use of a large number of hamsters and has severe effects (i.e., hepatic and renal failure resulting in death); while this vaccine is effective, a safer, cheaper, more ethical replacement is desired. The aim of this study was to analyze vaccine proteomes and identify target molecules common to allL. interrogansserovar Canicola vaccines which could be used to design anin vitropotency test. Initial analysis ofL. interrogansserovar Canicola vaccines (A to E) from different manufacturers, using theLimulusamebocyte lysate assay and silver-stained sodium dodecyl sulfate polyacrylamide gels, indicated that lipopolysaccharide was not present in all vaccines, preventing it from being a suitable target molecule. The protein contents of vaccines A to E were therefore determined by two-dimensional liquid chromatography mass spectrometry ([2D-LC/MS] 221 ± 31, 9 ± 8, 34 ± 4, 21 ± 5, and 34 ± 17 proteins [mean ± 1 standard deviation] found, respectively). The outer membrane protein LipL32 was established to be common to all and to be present at a significantly higher (P≤ 0.05) relative spectral abundance in a batch of vaccine which passed thein vivopotency test than in one which had failed. Further analysis using multiple reaction monitoring revealed that the concentration of the N terminus of LipL32 was significantly lower (P≤ 0.01) in failed batches (n= 2) of vaccine than in passed batches (n= 2); the concentration of the C terminus between the two batches was approximately the same. Anin vitro Leptospiravaccine potency test, based on N-terminal amino acid quantification of LipL32, was subsequently developed.

OBJECTIVES:To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales.METHODS:UK CTX-M-14-producing E. coli (n=70) from cattle (n=33), turkeys (n=9), sheep (n=2) and humans (n=26) were screened using multiplex PCR for the detection of a previously characterized plasmid, pCT. Isolates found to be carrying two or more pCT genetic markers were further analysed using PFGE. Their antimicrobial-resistance genes and virulence genes were also determined. These plasmids were transferred to Salmonella enterica serotype Typhimurium 26R and further examined for incompatibility type, genetic environment of the bla(CTX-M-14) gene, size, restriction fragment length polymorphism (RFLP) and nikB sequence.RESULTS:The 25 E. coli isolates carrying pCT genetic markers generated 19 different PFGE profiles, and 23 isolates had different virulence and antimicrobial-resistance gene patterns. One isolate from cattle was a verotoxigenic E. coli ('VTEC'); the rest were commensal or extra-intestinal pathogenic E. coli. pCT-like plasmids with similar molecular characteristics (size, replicon type, RFLP pattern, pCT markers and genetic environment of the bla(CTX-M-14) gene) were detected in 21/25 of the field isolates, which comprised those from cattle (n=9), turkeys (n=8) and humans (n=4). All pCT-like plasmids were conjugative, and most were IncK (n=21) and had the same local genetic environment flanking the bla(CTX-M-14) gene (n=23). RFLP analysis demonstrated ≥ 75% similarity among most plasmids (n=22).CONCLUSIONS:pCT-like plasmids were common vectors for horizontal dissemination of 30% of the bla(CTX-M-14) genes to different E. coli isolates from humans, cattle and turkeys.

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.