Longitudinal study of CTX-M ESBL-producing E. coli strains on a UK dairy farm.

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.

Characterisation of the Proteome of Leptospira interrogans Serovar Canicola as a Resource for the Identification of Common Serovar Immunogenic Proteins.

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.

Vaccination with leptospiral outer membrane lipoprotein LipL32 reduces kidney invasion of Leptospira interrogans serovar canicola in hamsters.

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.

Identification of a novel plasmid-associated spectinomycin adenyltransferase gene spd in methicillin-resistant Staphylococcus aureus ST398 isolated from animal and human sources.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: A novel gene, designated spd, that confers resistance to spectinomycin has been identified on a small plasmid in MRSA ST398.

Complete sequence of pSAM7, an IncX4 plasmid carrying a novel blaCTX-M-14b transposition unit isolated from Escherichia coli and Enterobacter cloacae from cattle.

The same plasmid carrying blaCTX-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.

Wild birds carry similar Salmonella enterica serovar Typhimurium strains to those found in domestic animals and livestock.

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.

Evaluation of an autogenous vaccine in cattle against Escherichia coli bearing the CTX-M-14 plasmid.

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 (1×10(10) 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.

A survey of antimicrobial usage on dairy farms and waste milk feeding practices in England and Wales.

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.

Risk factors associated with extended spectrum beta-lactamase Escherichia coli (CTX-M) on dairy farms in North West England and North Wales.

This study investigated the potential spread of CTX-M-14 Escherichia coli from a known ESBL E. coli positive farm and risk factors for the presence of CTX-M E. coli on dairy farms. Between November 2009 and March 2010, 65 farms in North West England and North Wales were visited and animals sampled for E. coli producing CTX-M ESBLs. Seventeen of these were known to have received animals from a known ESBL E. coli positive 'index' farm since 2005 (linked farms). The prevalence of CTX-M E. coli in the population of linked farms was 58.8% (10/17; CI(95%) 32.9-81.6%) and in the randomly selected control population was 35.4% (17/48; CI(95%) 22.2-50.5%). There was no significant (p>0.05) linkage for the detection of any CTX-M E. coli or specifically a CTX-M-14 E. coli to the index farm. Group 1 (CTX-M-15, CTX-M-55, CTX-M-1, CTX-M-32), group 2 (CTX-M-2) and group 9 (CTX-M-14, CTX-M-14 B, CTX-M-27) CTX-M E. coli were identified on the study farms. Molecular analysis revealed that three plasmids from linked farms had similar sizes (95 kbp), replicon type (IncK) and backbone genes as that from the index farm. Logistic regression analysis revealed that farms that had used a 3rd or 4th generation cephalosporin (ceftiofur, cefoperazone and cefquinome) in livestock in the last 12 months were nearly 4 times more likely to have ESBL E. coli present (p=0.037; OR=3.93). There was no significant association between presence of CTX-M E. coli and the use of any 1st or 2nd generation cephalosporins. Several other risk factors for the presence of CTX-M E. coli were identified, such as storage of slurry in a pit, operating an open herd policy and infrequent cleaning of calf feeding equipment.

Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales.

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.

Analysis of multiple Leptospira interrogans serovar Canicola vaccine proteomes and identification of LipL32 as a biomarker for potency.

The current batch potency test for Leptospira interrogans serovar 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 all L. interrogans serovar Canicola vaccines which could be used to design an in vitro potency test. Initial analysis of L. interrogans serovar Canicola vaccines (A to E) from different manufacturers, using the Limulus amebocyte 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 the in vivo potency 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. An in vitro Leptospira vaccine potency test, based on N-terminal amino acid quantification of LipL32, was subsequently developed.

Investigation of the presence of ESBL-producing Escherichia coli in the North Wales and West Midlands areas of the UK in 2007 to 2008 using scanning surveillance.

Between November 5, 2007 and November 4, 2008, faecal samples from cattle and sheep submitted for diagnostic purposes to the Aberystwyth and Shrewsbury Veterinary Laboratories Agency (VLA) (now AHVLA) regional laboratories (covering North Wales and the West Midlands) were screened for the presence of Escherichia coli that produces CTX-M extended-spectrum ß-lactamase (ESBL) using the selective medium CHROMagar CTX. Samples from 113 farms were tested and eight ESBL-positive farms identified. Of these, six farms were identified via submissions of cattle faeces and two from sheep. Gene sequencing revealed both group 1 and group 9 CTX-M enzymes corresponding to CTX-M-14, CTX-M-14B (group 9) and CTX-M-15/28 (group 1). Analysis of these isolates by nanoarray revealed that some were carrying a range of virulence genes including ireA, iroN and prfB, which have been associated with extraintestinal pathogenic E coli, and were multidrug resistant. Geographical analysis with choropleth maps suggested that these CTX-M genes are relatively widespread in the North Wales and West Midlands study area. This work was carried out concurrently with the running of a VLA ESBL surveillance system, which has subsequently identified many more CTX-M positive farms in the UK.

Fecal carriage and shedding density of CTX-M extended-spectrum {beta}-lactamase-producing escherichia coli in cattle, chickens, and pigs: implications for environmental contamination and food production.

The number and proportion of CTX-M positive Escherichia coli organisms were determined in feces from cattle, chickens, and pigs in the United Kingdom to provide a better understanding of the risk of the dissemination of extended-spectrum ß-lactamase (ESBL) bacteria to humans from food animal sources. Samples of bovine (n = 35) and swine (n = 20) feces were collected from farms, and chicken cecal contents (n = 32) were collected from abattoirs. There was wide variation in the number of CTX-M-positive E. coli organisms detected; the median (range) CFU/g were 100 (100 × 10(6) to 1 × 10(6)), 5,350 (100 × 10(6) to 3.1 × 10(6)), and 2,800 (100 × 10(5) to 4.7 × 10(5)) for cattle, chickens, and pigs, respectively. The percentages of E. coli isolates that were CTX-M positive also varied widely; median (range) values were 0.013% (0.001 to 1%) for cattle, 0.0197% (0.00001 to 28.18%) for chickens, and 0.121% (0.0002 to 5.88%) for pigs. The proportion of animals designated high-density shedders (≥1 × 10(4) CFU/g) of CTX-M E. coli was 3/35, 15/32, and 8/20 for cattle, chickens, and pigs, respectively. We postulate that high levels of CTX-M E. coli in feces facilitate the dissemination of bla(CTX-M) genes during the rearing of animals for food, and that the absolute numbers of CTX-M bacteria should be given greater consideration in epidemiological studies when assessing the risks of food-borne transmission.

Prevalence of Escherichia coli carrying extended-spectrum ß-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009.

OBJECTIVES: to determine the prevalence of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli from poultry in Great Britain (GB). METHODS: E. coli was isolated from 388 broiler chicken caecal samples from 22 abattoirs and from boot swabs from 442 turkey flocks over successive 1 year periods. CHROMagar ECC with and without cephalosporin antibiotics was used as isolation medium and the chicken study also used CHROMagar CTX. ESBL phenotype isolates were tested for the presence of bla(CTX-M,) bla(OXA), bla(SHV), bla(TEM) and ampC genes(.) CTX-M isolates were tested for O25 serogroup, replicon, CTX-M sequence, multilocus sequence type (MLST), PFGE type, plasmid transfer and qnrA, qnrB, qnrS, qepA and aac(6')-Ib genes. RESULTS: CTX-M-carrying E. coli were isolated from 54.5% of the broiler abattoirs and from 3.6% of individual broiler caecal samples and were CTX-M sequence types 1 (mainly), 3 and 15 with replicon types I1-γ, A/C and P/F, and I1-γ, respectively. CTX-M-carrying E. coli were isolated from 5.2% of turkey meat production farms and 6.9% of turkey breeder farms and were CTX-M sequence types 1, 14 (mainly), 15 and 55 with mainly replicon types F, FIA, K and I1-γ, respectively. None of the CTX-M isolates was serogroup O25. PFGE/MLST showed the CTX-M isolates to be clonally diverse, although MLST 156 with CTX-M-15 was isolated from both chickens and turkeys and has been previously reported in gulls. CTX-M-negative, ESBL- and bla(TEM)-positive strains were mainly TEM-52C. CONCLUSIONS: poultry-derived CTX-M E. coli in GB are different from major CTX-M sequence types causing disease in humans.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella enterica serovar Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin-α was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols.

AIMS: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. METHODS AND RESULTS: Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 10(7) -1 × 10(9 ) CFU g(-1) Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 µg ml(-1) ciprofloxacin, MICs of 16 µg ml(-1) and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. CONCLUSIONS: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.

Evaluation of CHROMagar CTX, a novel medium for isolating CTX-M-ESBL-positive Enterobacteriaceae while inhibiting AmpC-producing strains.

OBJECTIVES: The aim of this study was to evaluate CHROMagar CTX (CHROMagar France), a novel agar for the selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene in the presence of enteric bacteria expressing AmpC enzymes. METHODS: A panel of 150 Gram-negative bacteria (mainly Escherichia coli, Enterobacter, Klebsiella, Pseudomonas and Salmonella) isolated from humans and animals were assembled for the purpose of evaluating CHROMagar CTX and comparing it with CHROMagar ECC with the addition of 1, 2, 4 and 8 mg/L cefotaxime or ceftazidime and with bioMérieux extended-spectrum beta-lactamase (ESBL)-Bx agar. CHROMagar CTX was also assessed for its ability to isolate bla(CTX-M) strains from farm animal faeces (n = 342). RESULTS: The panel contained CTX-M-positive (n = 70) strains (CTX-M types 1, 9, 14 and 15), ESBLs (n = 31) belonging to other families (OXA, PER, SHV, TEM, VEB), strains positive for ampC genes (n = 31), strains that overexpressed ampC (n = 6), non-ESBL/AmpC strains (n = 11) and Klebsiella oxytoca (n = 1). CHROMagar CTX was superior to other agars tested for selective isolation of Enterobacteriaceae expressing the bla(CTX-M) gene with 100% sensitivity and 64.2% specificity for CTX-M strains in the panel and 90.1% of the colonies from animal faeces plated on CHROMagar CTX were CTX-M strains. CONCLUSIONS: CHROMagar CTX is a valuable agar in situations where it is important to isolate bla(CTX-M) strains in the presence of AmpC strains. The agar may be particularly useful in veterinary studies, where AmpC-producing commensal E. coli can be encountered reasonably frequently in the enteric flora of some animal species and may also be useful, following further evaluation, for samples from humans.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence.

OBJECTIVES: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. METHODS: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. RESULTS: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. CONCLUSIONS: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation.

AIMS: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). METHODS: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. RESULTS: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. CONCLUSIONS: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

Concentrations of parabens in human breast tumours.

Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 +/- 4.2 ng x g(-1) tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 +/- 2.2 ng x g(-1) tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues.