Nola Leonard

Physiological and Transcriptional Characterization of Persistent and Nonpersistent Listeria monocytogenes Isolates

ABSTRACT This study aimed to characterize physiological differences between persistent and presumed nonpersistent Listeria monocytogenes strains isolated at processing facilities and to investigate the molecular basis for this by transcriptomic sequencing. Full metabolic profiles of two strains, one persistent and one nonpersistent, were initially screened using Biologs Phenotype MicroArray (PM) technology. Based on these results, in which major differences from selected antimicrobial agents were detected, another persistent strain and two nonpersistent strains were characterized using two antimicrobial PMs. Resistance to quaternary ammonium compounds (QACs) was shown to be higher among persistent strains. Growth of persistent and nonpersistent strains in various concentrations of the QACs benzethonium chloride (BZT) and cetylpyridinium chloride (CPC) was determined. Transcriptomic sequencing of a persistent and a presumed nonpersistent strain was performed to compare gene expression among these strains in the presence and absence of BZT. Two strains, designated “frequent persisters” because they were the most frequently isolated at the processing facility, showed overall higher resistance to QACs. Transcriptome analysis showed that BZT induced a complex peptidoglycan (PG) biosynthesis response, which may play a key role in BZT resistance. Comparison of persistent and nonpersistent strains indicated that transcription of many genes was upregulated among persistent strains. This included three gene operons: pdu, cob-cbi, and eut. These genes may play a role in the persistence of L. monocytogenes outside the human host.

Horizontal transmission of Escherichia coli O157:H7 during cattle housing.

Ruminant livestock, particularly cattle, is considered the primary reservoir of Escherichia coli O157:H7. This study examines the transmission of E. coli O157:H7 within groups of cattle during winter housing. Holstein Friesian steers were grouped in six pens of five animals. An animal inoculated with and proven to be shedding a marked strain of E. coli O157: H7 was introduced into each pen. Fecal (rectal swabs) and hide samples (900 cm2 from the right rump) were taken from the 36 animals throughout the study. Water, feed, and gate or partition samples from each pen were also examined. Within 24 h of introducing the inoculated animals into the pens, samples collected from the drinking water, pen barriers, and animal hides were positive for the pathogen. Within 48 h, the hides of 20 (66%) of 30 cohort animals from the six pens were contaminated with E. coli O157:H7. The first positive fecal samples from the noninoculated cohort animals were detected 3 days after the introduction of the inoculated steers. During the 23 days of the study, 15 of 30 cohort animals shed the marked E. coli O157:H7 strain in their feces on at least one occasion. Animal behavior in the pens was monitored during a 12-h period using closed circuit television cameras. The camera footage showed an average of 13 instances of animal grooming in each pen per hour. The study suggests that transmission of E. coli O157:H7 between animals may occur following ingestion of the pathogen at low levels and that animal hide may be an important source of transmission.

Survival of Escherichia coli O157:H7 in farm water: its role as a vector in the transmission of the organism within herds

Aims: The study aimed to investigate the survival characteristics of Escherichia coli O157:H7 in farm water (FW), and in sterile distilled municipal water (SDW), stored outdoors under field conditions, with or without the addition of faeces (1% w/v), in a farmyard shed and the laboratory at 15°C. 
Methods and Results: Water samples were inoculated with E. coli O157:H7 at 103 and 106 ml−1, and sampled over a 31‐day period. In FW stored outdoors in a field, E. coli O157:H7 survived for 14 days at temperatures <15°C, at both inoculation levels, while in the laboratory at 15°C, the organism was still detectable at low levels (<1 log10 cfu ml−1) after 31 days. The addition of bovine faeces to water outdoors (1% w/v) resulted in survival for 24 days. In SDW inoculated at 106 ml−1 and stored in the laboratory (15°C), only a 2·5 log reduction was observed after 31 days, while the organism could not be detected after 17 days in the field. Preliminary screening of water samples stored outdoors isolated a bacterium which exhibited antimicrobial activity towards E. coli O157:H7. 
Conclusions: The survival of E. coli O157:H7 observed in this study illustrates the potential of farm water to act as a vehicle in the transfer of the organism across a herd. 
Significance and Impact of the Study: The difficulty in extrapolating results from controlled laboratory situations to on‐farm conditions is also highlighted in this study.

A comparison of the survival in feces and water of Escherichia coli O157:H7 grown under laboratory conditions or obtained from cattle feces.

Escherichia coli O157:H7 is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic uremic syndrome. Cattle feces and fecally contaminated water are important in the transmission of this organism on the farm. In this study, the survival of E. coli O157:H7 in feces and water was compared following passage through the animal digestive tract or preparation in the laboratory. Feces were collected from steers before and after oral inoculation with a marked strain of E. coli O157:H7. Fecal samples collected before cattle inoculation were subsequently inoculated with the marked strain of E. coli O157:H7 prepared in the laboratory. Subsamples were taken from both animal and laboratory-inoculated feces to inoculate 5-liter volumes of water. E. coli O157:H7 in feces survived up to 97 days, and survival was not affected by the method used to prepare the inoculating strain. E. coli O157:H7 survived up to 109 days in water, and the bacteria collected from inoculated cattle were detected up to 10 weeks longer than the laboratory-prepared culture. This study suggests that pathogen survival in low-nutrient conditions may be enhanced by passage through the gastrointestinal tract.

Characterization of Multidrug-Resistant Escherichia coli Isolates from Animals Presenting at a University Veterinary Hospital

ABSTRACT In this study, we examined molecular mechanisms associated with multidrug resistance (MDR) in a collection of Escherichia coli isolates recovered from hospitalized animals in Ireland. PCR and DNA sequencing were used to identify genes associated with resistance. Class 1 integrons were prevalent (94.6%) and contained gene cassettes recognized previously and implicated mainly in resistance to aminoglycosides, β-lactams, and trimethoprim (aadA1, dfrA1-aadA1, dfrA17-aadA5, dfrA12-orfF-aadA2, bla OXA-30-aadA1, aacC1-orf1-orf2-aadA1, dfr7). Class 2 integrons (13.5%) contained the dfrA1-sat1-aadA1 gene array. The most frequently occurring phenotypes included resistance to ampicillin (97.3%), chloramphenicol (75.4%), florfenicol (40.5%), gentamicin (54%), neomycin (43.2%), streptomycin (97.3%), sulfonamide (98.6%), and tetracycline (100%). The associated resistance determinants detected included bla TEM, cat, floR, aadB, aphA1, strA-strB, sul2, and tet(B), respectively. The bla CTX-M-2 gene, encoding an extended-spectrum β-lactamase (ESβL), and bla CMY-2, encoding an AmpC-like enzyme, were identified in 8 and 18 isolates, respectively. The mobility of the resistance genes was demonstrated using conjugation assays with a representative selection of isolates. High-molecular-weight plasmids were found to be responsible for resistance to multiple antimicrobial compounds. The study demonstrated that animal-associated commensal E. coli isolates possess a diverse repertoire of transferable genetic determinants. Emergence of ESβLs and AmpC-like enzymes is particularly significant. To our knowledge, the bla CTX-M-2 gene has not previously been reported in Ireland.

Tracking the Salmonella status of pigs and pork from lairage through the slaughter process in the Republic of Ireland.

Salmonella Typhimurium is the predominant serotype isolated from humans in Europe. Pork and pork products are recognized vehicles of Salmonella and are responsible for outbreaks of human salmonellosis. Pigs can become infected with Salmonella on the breeding or fattening farm and during transport, lairage, and slaughter. The aim of this study was to investigate selected points of Salmonella contamination from the time pigs entered the lairage to the time the carcass was processed in the boning hall and to determine the importance of different sources of Salmonella along the Irish pork production chain. A second objective was to evaluate whether the serological status or category of a herd influenced the levels of bacteriological contamination detected on individual carcasses and pork cuts during slaughter and dressing operations. All samples were tested for the presence and numbers of Salmonella. Enterobacteriaceae numbers were also determined. Serotype, phage type, and pulsed-field gel electrophoresis were utilized to determine similarity among Salmonella isolates. Lairage was a major source of cross-contamination with Salmonella as were the hands of evisceration operatives, conveyor belts, and equipment in the boning hall. Cross-contamination within the slaughter plant environment accounted for up to 69 % of Salmonella carcass contamination. In general, herd category reflected the bacteriological status of carcasses and pork cuts. Major findings were a strong association (P < 0.01) between Enterobacteriaceae counts and Salmonella occurrence on prechill carcasses and a significant association (P < 0.05) between Enterobacteriaceae counts and Salmonella occurrence on pork cut samples.

Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Irish Cattle Farms

ABSTRACT This study describes the genotypic characteristics of a collection of 100 multidrug-resistant (MDR) Escherichia coli strains recovered from cattle and the farm environment in Ireland in 2007. The most prevalent antimicrobial resistance identified was to streptomycin (100%), followed by tetracycline (99%), sulfonamides (98%), ampicillin (82%), and neomycin (62%). Resistance was mediated predominantly by strA-strB (92%), tetA (67%), sul2 (90%), bla TEM (79%), and aphA1 (63%) gene markers, respectively. Twenty-seven isolates harbored a class 1 integrase (intI1), while qacEΔ1 and sul1 markers were identified in 25 and 26 isolates, respectively. The variable regions of these integrons contained aminoglycoside, trimethoprim, and β-lactam resistance determinants (aadA12, aadB-aadA1, bla OXA-30-aadA1, dfrA1-aadA1, dfrA7). Class 2 integrons were identified less frequently (4%) and contained the gene cassette array dfrA1-sat1-aadA1. Resistance to ampicillin, neomycin, streptomycin, sulfonamide, and tetracycline was associated with transferable high-molecular-weight plasmids, as demonstrated by conjugation assays. A panel of virulence markers was screened for by PCR, and genes identified included vt1, K5 in 2 isolates, papC in 10 isolates, and PAI IV536 in 37 isolates. MDR commensal E. coli isolates from Irish cattle displayed considerable diversity with respect to the genes identified. Our findings highlight the importance of the commensal microflora of food-producing animals as a reservoir of transferable MDR.

Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals.

ABSTRACT Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.

The characterization of Listeria spp. isolated from food products and the food‐processing environment

Aim:  To enhance the information pertaining to the epidemiology of a collection of 378 Listeria spp. isolates obtained from several food‐processing plants in Ireland over a 3‐ year period (2004–2007).

Characterization of antimicrobial resistance in Salmonella enterica food and animal isolates from Colombia: identification of a qnrB19-mediated quinolone resistance marker in two novel serovars.

Ninety-three Salmonella isolates recovered from commercial foods and exotic animals in Colombia were studied. The serotypes, resistance profiles and where applicable the quinolone resistance genes were determined. Salmonella Anatum (n=14), Uganda (19), Braenderup (10) and Newport (10) were the most prevalent serovars, and resistance to tetracycline (18.3%), ampicillin (17.2%) and nalidixic acid (14%) was most common. Nalidixic acid-resistant isolates displayed minimum inhibitory concentrations ranging from 32 to 1024 μg mL(-1) . A Thr57→Ser substitution in ParC was the most frequent (12 of the 13 isolates). Six isolates possessed an Asp87→Tyr substitution in GyrA. No alterations in GyrA in a further seven nalidixic acid-resistant isolates were observed. Of these, four serovars including two Uganda, one Infantis and a serovar designated 6,7:d:-, all carried qnrB19 genes associated with 2.7 kb plasmids, two of which were completely sequenced. These exhibited 97% (serovar 6,7:d:- isolate) and 100% (serovar Infantis isolate) nucleotide sequence identity with previously identified ColE-like plasmids. This study demonstrates the occurrence of the qnrB19 gene associated with small ColE plasmids hitherto unrecognized in various Salmonella serovars in Colombia. We also report unusual high-level quinolone resistance in the absence of any DNA gyrase mutations in serovars S. Carrau, Muenchen and Uganda.