Robert H. Davies

Molecular Typing of Salmonella Serotypes Prevalent in Animals in England: Assessment of Methodology

ABSTRACT Salmonella enterica serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no “gold standard” fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. BothPstI/SphI ribotyping andXbaI-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by XbaI-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations.

Longitudinal Farm Study of Extended-Spectrum β-Lactamase-Mediated Resistance

ABSTRACT Extended-spectrum β-lactamase (ESBL)-mediated resistance is of considerable importance in human medicine. Recently, such enzymes have been reported in bacteria from animals. We describe a longitudinal study of a dairy farm suffering calf scour with high mortality rates. In November 2004, two Escherichia coli isolates with resistance to a wide range of β-lactams (including amoxicillin-clavulanate and cefotaxime) were isolated from scouring calves. Testing by PCR and sequence analysis confirmed the isolates as being both blaCTX-M14/17 and blaTEM-35(IRT-4) positive. They had indistinguishable plasmid and pulsed-field gel electrophoresis (PFGE) profiles. Transferability studies demonstrated that blaCTX-M was located on a conjugative 65-MDa IncK plasmid. Following a farm visit in December 2004, 31/48 calves and 2/60 cows were positive for E. coli with blaCTX-M. Also, 5/48 calf and 28/60 cow samples yielded blaCTX- and blaTEM-negative E. coli isolates that were resistant to cefotaxime, and sequence analysis confirmed that these presented mutations in the promoter region of the chromosomal ampC gene. Fingerprinting showed 11 different PFGE types (seven in blaCTX-M-positive isolates). Six different PFGE clones conjugated the same blaCTX-M-positive IncK plasmid. One clone carried a different-sized, blaCTX-M-positive, transformable plasmid. This is the first report of blaCTX-M from livestock in the United Kingdom, and this report demonstrates the complexity of ESBL epidemiology. Results indicate that horizontal plasmid transfer between strains as well as horizontal gene transfer between plasmids have contributed to the spread of resistance. We have also shown that some clones can persist for months, suggesting that clonal spread also contributes to the perpetuation of resistance.

Diversity of Strains of Salmonella enterica Serotype Enteritidis from English Poultry Farms Assessed by Multiple Genetic Fingerprinting

ABSTRACT Reliable and sufficiently discriminative methods are needed for differentiating individual strains of Salmonella entericaserotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism wasPstI-SphI ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of XbaI-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins ofSalmonella serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.

Distribution of salmonella contamination in ten animal feedmills

Detailed sampling of spillage and dust from milling equipment was carried out in nine animal feedmills, three of which were sampled twice. The salmonella isolation rate ranged from 1.1% to 41.7% of the samples and the most contaminated mills were those where the inside of the cooling systems for pellet or mash had been colonised by salmonella. A wide range of salmonella serotypes were isolated which included Salmonella typhimurium and S. enteritidis. Limited sampling every two weeks for an 18-month period in another animal feedmill showed marked variation in the contamination rate of samples and range of salmonella serotypes found. Contamination of ingredient intake pits and outloading gantries for finished products by wild bird droppings containing salmonella was also found in four mills.

A longitudinal study of environmental salmonella contamination in caged and free-range layer flocks

The environmental contamination by salmonella was examined over a 12-month period in 74 commercial layer flocks from eight farms in the UK, which previously had been identified as being contaminated with salmonella. Samples of faeces, dust, litter, egg belt spillage and wildlife vectors were taken, plus swabs of cages, feeders, drinkers, floors, egg belts and boots. Some sampling was performed in each month of the year. Numerous serovars were detected but Salmonella enterica serotype Enteritidis was the only persistent serotype found among single-age flocks. There was a significant correlation between qualitative environmental samples and semi-quantitative faeces samples. The level of environmental contamination increased significantly over time. There were significant temperature and seasonal effects upon contamination. Wildlife vectors proved to be sensitive samples for the detection of salmonella. The efficacy of cleaning and disinfection upon residual salmonella contamination, and upon subsequent flock contamination, was highly variable between and within premises. The variability between detected prevalences over time and between flocks indicates a need for regular, sensitive monitoring of flocks for salmonella to permit targeting of control measures aimed at eliminating contamination of the layer environment by salmonella. There is substantial scope for improvement of cleaning and disinfection procedures.

Use of a LightCycler gyrA Mutation Assay for Rapid Identification of Mutations Conferring Decreased Susceptibility to Ciprofloxacin in Multiresistant Salmonella enterica Serotype Typhimurium DT104 Isolates

ABSTRACT A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella entericaserotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC→AAC) mutation, an Asp-87-to-Gly (GAC→GGC) mutation, and a Ser-83-to-Phe (TCC→TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore differentgyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC→TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC→TAC) substitution. One animal strain had nogyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according togyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Comparison of gyrA mutations, cyclohexane resistance, and the presence of class I integrons in Salmonella enterica from farm animals in England and Wales.

ABSTRACT This study is focused on real-time detection of gyrA mutations and of the presence of class I integrons in a panel of 100 veterinary isolates of Salmonella enterica from farm animals. The isolates were selected on the basis of resistance to nalidixic acid, representing a variety of the most prevalent serotypes in England and Wales. In addition, organic solvent (cyclohexane) resistance in these isolates was investigated in an attempt to elucidate the presence of efflux pump mechanisms. The most prevalent mutation among the isolates studied was Asp87-Asn (n = 42), followed by Ser83-Phe (n = 38), Ser83-Tyr (n = 12), Asp87-Tyr (n = 4), and Asp87-Gly (n = 3). Two distinct subpopulations were identified, separated at the 1-mg/liter breakpoint for ciprofloxacin: 86% of isolates with mutations in codon 83 showed MICs of ≥1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of ≤0.5 mg/liter. Cyclohexane resistance was more prevalent among Ser83 mutants than among Asp87 mutants (34.7 and 4%, respectively), and in 79% of isolates that presented both gyrA mutations and cyclohexane resistance, the level of ciprofloxacin resistance was ≥2.0 mg/liter. Thirty-four isolates contained class I integrons, with 71% of the S. enterica serovar Typhimurium isolates and 6.9% of isolates belonging to other serotypes containing such elements. The methods used represent sensitive ways of investigating the presence of gyrA mutations and of detecting class-I integrons in Salmonella isolates. The results can be obtained in less than 1 h from single colonies without the need for purifying DNA.

Chemical Treatment of Animal Feed and Water for the Control of Salmonella

The control of Salmonella in animal feedstuffs is important, principally to protect the human food chain from contamination by Salmonella derived from infected animals. The transmission of Salmonella from animal feeds to animals, and onward to human food products, has been convincingly documented. This is especially important for chicken breeding and laying flocks and pigs, in view of the consequences of recent or imminent control legislation in the European Union. Animal feed ingredients, particularly animal and plant-derived protein meals, are frequently contaminated with Salmonella either from source or from processing plant, and recontamination in compounding mills is an additional problem. Several complementary strategies have been used to control this feed contamination, and these include a range of chemical treatments. The principal agents used are as follows: organic acids and their salts, formaldehyde, and bacterial membrane disruptors such as terpenes and essential oils. Experimental agents include chlorate compounds. Many products use blends of agents from the same or different chemical groups to achieve synergistic or combination effects. The present review draws upon published and company data to describe the various modes of action and efficacies of different chemical agents delivered in feed or in drinking water against Salmonella occurring in feed or in livestock environments. Reasons for the failure of protection are explored, along with problems in usage such as corrosion and reduced palatability. Given the wide array of products available with contrasting modes of action, the need for standardized tests of efficacy is also discussed.

Observations on Salmonella contamination of eggs from infected commercial laying flocks where vaccination for Salmonella enterica serovar Enteritidis had been used

Eggs were collected monthly from 12 cage-layer flocks on four farms where Salmonella Enteritidis was present in vaccinated flocks despite vaccination with an S. Enteritidis bacterin. Where possible, hens were also taken for culture at the end of the laying period, and faecal and environmental samples were taken from the laying houses before and after cleaning and disinfection. Twenty-four batches of six egg shells from the 13u2008652 tested (0.18% [0.11 to 0.26 CI95] single egg equivalent) were positive for S. Enteritidis and 54 (0.40% [0.30 to 0.52 CI95] single egg equivalent) for other serovars. Six batches of 13u2008640 (0.04% [0.02 to 0.10 CI95] single egg equivalent) egg contents, bulked in six egg pools, contained S. Enteritidis and three batches contained other serovars. In addition three further batches contained S. Enteritidis in both contents and shells, and two other batches contained other serovars in both. The total level of contamination by S. Enteritidis of both contents and shells found in vaccinated flocks was therefore 33 batches/13u2008682 eggs(0.24% [0.17 to 0.34 CI95] single egg equivalent). The total of contamination for any Salmonella serovar was 92 batches/13u2008682 eggs (0.68% [0.55 to 0.84 CI95] single egg equivalent). These results contrast with the findings of testing of eggs from three unvaccinated flocks prior to this study where 21 batches of egg shells from a total of 2101 eggs (1.0% [0.63 to 1.56 CI95] single egg equivalent) and six batches of contents from 2051 eggs (0.29% [0.11 to 0.64 CI95] single egg equivalent) were contaminated with S. Enteritidis. S. Enteritidis was found in 67/699 (9.6%) of vaccinated spent hens and 64/562 (11.4%) of bulked fresh faecal samples taken from laying houses. Failure to adequately clean and disinfect laying houses and to control mice appeared to be a common feature on the farms. Résumé Surveillance de la contamination, par Salmonella , des œufs provenant dun troupeau de pondeuses infectées chez lesquelles la vaccination Salmonella enterica serovar Enteritidis a été réalisée Tous les mois, des œufs ont été collectés dans 12 troupeaux de pondeuses en cage répartis dans quatre élevages, où Salmonella Enteritidis a été isolée malgré la vaccination avec un vaccin inactivé S. Enteritidis. En fonction des possibilités, les pondeuses ont fait lobjet de culture à la fin de la période de ponte et des échantillons de lenvironnement et des fèces ont été réalisés dans les bâtiments avant et après nettoyage et désinfection. Vingt-quatre lots de six œufs en coquille sur 13 652 testés se sont révélés positifs vis-à-vis de S. Enteritidis (0.18%u2005[0.11–0.26 Cl95] équivalent dun œuf) et 54 pour les autres sérovars (0.40%u2005[0.30–0.52 Cl95] équivalent dun œuf). Six lots de 13 640 contenus d’œuf, vrac dun mélange de 6 œufs, étaient contaminés par S. Enteritidis (0.04%u2005[0.02–0.10 Cl95] équivalent dun œuf) et trois lots contenant dautres sérovars. De plus, trois autres lots ont été trouvés contaminés par S. Enteritidis à la fois dans les contenus et dans les œufs en coquille et deux autres lots contenaient dautres sérovars dans les deux types d’échantillons. Le niveau total de contamination, par S. Enteritidis dans les contenus et à partir des œufs en coquille, trouvé dans les troupeaux vaccinés a été de 33u2005lots/13 682 œufs (0.24%u2005[0.17–0.34 Cl95] équivalent dun œuf). Le total des contaminations, quel que soit le sérovar de Salmonella a été de 92u2005lots/13 682u2005œufs (0.68%u2005[0.55–0.84 Cl95] équivalent dun œuf). Ces résultats contrastent avec ceux des testages des œufs de trois troupeaux non vaccinés réalisés avant cette étude où une contamination par S. Enteritidis avait été observée dans 21 lots dœufs en coquille sur un total de 2 101 œufs (1.0%u2005[0.63–1.56 Cl95] équivalent dun œuf) et dans six lots de contenus d’œufs sur 2 051 œufs (0.29%u2005[0.11–0.64 Cl95] équivalent dun œuf). S. Enteritidis a été trouvé dans 67/699 (9.6%) de pondeuses de réforme vaccinées et dans 64/562 (11u2005.4%) échantillons de fèces prélevés dans les bâtiments. Les caractéristiques communes de ces fermes semblent être l’échec du lavage et de la désinfection des bâtiments ainsi que celui du contrôle des souris. Zusammenfassung Beobachtungen zur Salmonellenkontamination bei Eiern aus infizierten kommerziellen Legehennenherden nach Vakzination gegen Salmonella enterica Serovar Enteritidis Von 12 Käfiglegehennenherden auf vier Farmen, bei denen Salmonella enteritidis trotz Vakzination mit einem S. enteritidis-Bakterin auftrat, wurden monatlich Eier eingesammelt. Wo es möglich war, wurden am Ende der Legeperiode auch Hennen für die Untersuchung entnommen und Umgebungsproben wurden in den Stallgebäuden vor und nach der Reinigung und Desinfektion gezogen. 24 Proben bestehend aus jeweils 6 Eischalen von insgesamt 13.652 getesteten (0.18% (0.11–0.26 CI95) Einzeleiäquivalent) waren positiv für S. enteritidis und 54 (0.40% (0.30–0.52 CI95) Einzeleiäquivalent) für andere Serovare. 6 Proben von 13.640 Eiinhalten (0.04% (0.02–0.10 CI95) Einzeleiäquivalent) in Pools von jeweils 6 Eiern beinhalteten S. enteritidis und 3 Proben hatten andere Serovare. Außerdem waren bei drei weiteren Proben Inhalt und Schalen positiv für S. enteritidis und zwei andere Proben enthielten andere Serovare in beiden Anteilen. Insgesamt waren in den vakzinierten Herden 33 Proben/13.682 Eiern (0.24% (0.17–0.34 CI95) Einzeleiäquivalent) mit S. enteritidis kontaminiert. Die totale Kontamination für andere Salmonella-Serovare betrug 92 Proben/13.682 (0.68% (0.55–0.84 CI95) Einzeleiäquivalent). Diese Resultate stehen im Gegensatz zu vorherigen Ei-Untersuchungsergebnissen aus drei nicht-vakzinierten Herden, wo 21 Proben aus Eischalen von insgesamt 2.101 Eiern (1.0% (0.63–1.56 CI95) Einzeleiäquivalent) und 6 Eiinhaltsproben von 2.051 Eiern (0.29% (0.11–0.64 CI95) Einzeleiäquivalent) mit S. enteritidis kontaminiert waren. S. enteritidis wurde in 67/699 (9.6%) untersuchten vakzinierten Schlachthennen und in 64/562 (11.4%) gepoolten frischen Faezesproben aus Legehennenställen gefunden. Misserfolge beim adäquaten Reinigen und Desinfizieren von Legehennenställen und bei der Mäusebekämpfung schienen ein gemeinsames Merkmal auf diesen Farmen zu sein. Resumen Observaciones sobre la contaminación de huevos por Salmonella procedentes de lotes de ponedoras comerciales vacunados frente a Salmonella enterica serovar EnteritidisSe recogieron, mensualmente, huevos de 12 lotes de gallinas de puesta en batería provenientes de 4 granjas en las cuales Salmonella Enteritidis estaba presente en los lotes vacunados a pesar de la vacunación con una bacterina de S. Enteritidis. También se tomaron gallinas al final del período de puesta para cultivo cuando esto fue posible, y se tomaron muestras del medio ambiente y de heces de las jaulas antes y después de su limpieza y desinfección. 24 lotes de 6 cáscaras de huevo de los 13.652 testados (0.18% [0.11–0.26 CI95] un huevo equivalente) fueron positivos para S. Enteritidis y 54 (0.40% [0.30–0.52 CI 95] un huevo equivalente) lo fueron para otros serovares. 6 lotes de los 13.640 (0.04% [0.02–0.10 CI95 un huevo equivalente) contenidos procedentes de huevos, repartidos en grupos de 6 huevos, contenían S. Enteritidis y 3 lotes contenían otros serovares. Además, otros 3 lotes más contenían S. Enteritidis tanto en la cáscara como en su contenido y otros 2 lotes contenían otros serovares en ambas partes. Por lo tanto, el nivel total de contaminación por S. Enteritidis tanto en la cáscara como en el contenido que se encontró en las manadas vacunadas fue de 33 lotes/13,682 huevos (0.24% [0.17–0.34 CI95] un huevo equivalente). El grado de contaminación total por cualquier serovar de Salmonella fue de 92 lotes/13,682 huevos (0.68% [0.55–0.84 CI95] un huevo equivalente). Estos resultados contrastan con los resultados del análisis de huevos procedentes de 3 manadas no vacunadas previos a este estudio en el cual 21 lotes de cáscaras de huevos de un total de 2,101 huevos (1.0% [0.63–1.56 CI95] un huevo equivalente) y 6 lotes de contenido de un total de 2,051 huevos (0.29% [0.11–0.64 CI95] un huevo equivalente) estaban contaminados con S. Enteritidis. S. Enteritidis se encontró en 67/699 (9.6%) de las gallinas de desvieje y en 64/562 (11.4%) de las muestras de heces frescas que se tomaron de las jaulas. Fallos en la limpieza y desinfección de las jaulas y en el control de ratones parece ser un hecho común en las granjas.