Farm level risk factors for fluoroquinolone resistance in E. coli and thermophilic Campylobacter spp. on poultry farms.

Data on husbandry practices, performance, disease and drug use were collected during a cross-sectional survey of 89 poultry meat farms in England and Wales to provide information on possible risk factors for the occurrence of fluoroquinolone (FQ)-resistant bacteria. Faeces samples were used to classify farms as "affected" or "not affected" by FQ-resistant (FQr) Escherichia coli or Campylobacter spp. Risk factor analysis identified the use of FQ on the farms as having by far the strongest association, among the factors considered, with the occurrence of FQr bacteria. Resistant E. coli and/or Campylobacter spp. were found on 86% of the farms with a history of FQ use. However, a substantial proportion of farms with no history of FQ use also yielded FQr organisms, suggesting that resistant bacteria may transfer between farms. Further analysis suggested that for Campylobacter spp., on-farm hygiene, cleaning and disinfection between batches of birds and wildlife control were of most significance. By contrast, for E. coli biosecurity from external contamination was of particular importance, although the modelling indicated that other factors were likely to be involved. Detailed studies on a small number of sites showed that FQr E. coli can survive routine cleaning and disinfection. It appears difficult to avoid the occurrence of resistant bacteria when FQ are used on a farm, but the present findings provide evidence to support recommendations to reduce the substantial risk of the incidental acquisition of such resistance by farms where FQ are not used.

Influence of production system on the rate of onset of Campylobacter colonization in chicken flocks reared extensively in the United Kingdom.

1. Because thermophilic Campylobacter spp. are common in chicken flocks reared extensively, cross-sectional and longitudinal studies were carried out on organic and free-range farms to determine the onset of colonisation (lag phase) and likely sources of flock infection. 2. For 14 organic and 14 free range flocks, there was a difference in lag phases, with the former being colonized at a mean of 14·1 d in comparison with 31·6 d for the latter. Whereas most free-range flocks became colonized when released on to pasture, those reared organically were usually colonized at the housed brooding stage. 3. Further study of organic flocks on three farms over 7 successive crop cycles confirmed that colonisation was strongly influenced by the prevailing husbandry conditions and was not a consequence of the length of the rearing period. 4. Molecular epidemiological investigations on a farm showing the shortest lag phase, using PFGE typing with two different restriction enzymes (SmaI and KpnI) and flaA SVR sequence typing, revealed that potential sources of colonisation for organic chickens were already present on the farm at the time of chick placement. Such sources included the ante area of the brooding house, surrounding pasture and other livestock being kept on the farm. 5. Overall, the study demonstrated that, under UK conditions, the prevalence of colonisation was greater in extensive flocks (95-100%) than it was for conventional broilers (55%), similar to the situation in other countries, but all three management systems showed comparable levels of caecal carriage in positive birds (log(10)/g 6·2-6·7).

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.

Sources and spread of thermophilic Campylobacter spp. during partial depopulation of broiler chicken flocks.

The practice of partial depopulation or thinning (early removal of a portion of birds from a commercial broiler flock) is a reported risk factor for Campylobacter colonization of residual birds because of the difficulty in maintaining biosecurity during the thinning process. The effect of this practice was studied in detail for 51 target flocks, each at a different growing farm belonging to one of seven major poultry companies throughout the United Kingdom. On 21 of these farms, the target flock was already colonized by Campylobacter, and at slaughter all cecal samples examined were positive, with a mean of 8 log CFU/g. An additional 27 flocks became positive within 2 to 6 days of the start of thinning and had similarly high levels of cecal carriage at slaughter. Just before the thinning process, Campylobacter was isolated frequently from the farm driveways, transport vehicles, equipment, and personnel. Strains from seven farms on which flocks became colonized after thinning were examined by pulsed-field gel electrophoresis typing. An association was found between strains occurring at specific sampling sites and those isolated subsequently from the thinned flocks. The results indicated that particular strains had spread from one farm to another when the farms were jointly owned by the same company and employed the same bird-catching teams and/or vehicles. These results highlight the need for better hygiene control in relation to catching equipment and personnel and more effective cleaning and disinfection of vehicles and bird-transport crates.

Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine.

AIMS: A panel of pulsed field gel electrophoresis (PFGE) type variants of Campylobacter jejuni, previously identified as of clonal origin, were investigated to determine whether genomic instability could be observed during competitive growth. METHODS AND RESULTS: Upon recovery from frozen storage, some variants had undergone alterations in PFGE profiles, but subsequent culture produced constant genotypes. Individual variants did not display differences in colonization potential when tested in orally challenged 1-day-old chickens. However, competitive colonization using mixtures of two or three PFGE types generally resulted, by 4 weeks postchallenge, in one predominant PFGE type in all birds. For some variant mixtures, a minor population of novel PFGE types was detected in individual birds. The creation of new variants appeared to be dependent on the extent of competition and of the individual host. Genomic rearrangements most likely explain this increase in genetic diversity, apparently without the involvement of natural transformation or plasmid acquisition. In vitro cultivation of mixed inoculations were again selected for particular variants; but genetic diversity was not generated, suggesting that the selection pressures in vitro differed from those active in vivo. CONCLUSION: These observations support the hypothesis that by generating genetic diversity, C. jejuni can improve its phenotypic fitness to survive and colonize subsequent hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: The consequences of such observations for the development of campylobacter control strategies for poultry may be substantial.

Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience.

AIMS: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. METHODS AND RESULTS: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98-100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. CONCLUSIONS: Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. SIGNIFICANCE AND IMPACT OF THE STUDY: This work should facilitate progress towards inter-laboratory standardization of fla typing.

Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals.

AIMS: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. MATERIALS AND METHODS: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. RESULTS: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P < or = 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. CONCLUSIONS: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.

Clusters of new tuberculosis cases in North-west London: a survey from three hospitals based on IS6110 RFLP typing.

OBJECTIVES: The relative contributions of reactivation of latent infection and clusters of new infections to the overall incidence of tuberculosis in the U.K. is unknown. A study was carried out in North-West London to determine the feasibility of IS6110 RFLP strain typing as a tool to investigate the relative contributions of these two sources. METHODS: All available isolates of M. tuberculosis from specimens collected over a calendar year at three participating hospitals were typed by RFLP using an IS6110 probe. Isolates exhibiting a single band pattern were subject to further typing using an oligonucleotide direct repeat probe. Demographic and clinical information on cases was obtained from the National Survey of Tuberculosis Notifications in England and Wales and further information sought on clustered cases as identified by RFLP typing. RESULTS: Twenty-seven (23%) of the 118 cases had shared IS6110 RFLP patterns. Strains from nine cases had single band patterns, but these were all distinguishable from each other when subjected to further typing by direct repeat probe. The remaining 18 cases belonged to eight clusters. Epidemiological links were established between all the patients in each cluster. The likelihood of being in a cluster was increased in cases with pulmonary smear-positive disease. It was lower in cases of Indian Sub-continent ethnic origin. For 10 of the 18 clustered cases epidemiological links had not been established by conventional contact tracing. CONCLUSIONS: Investigation of the relative contributions of reactivation of latent infection and new infection is feasible in a UJK population, using IS6110 RFLP typing of M. tuberculosis isolates and epidemiological enquiries. This study in London identified clustered, presumably new cases, the majority of whom had not been linked epidemiologically. Comprehensive IS6110 RFLP typing of UK isolates would probably identify many clusters of incident tubercular infection.

Molecular fingerprinting defines a strain of Salmonella enterica serotype Anatum responsible for an international outbreak associated with formula-dried milk.

Molecular analyses based on plasmid profile typing and pulsed-field gel electrophoresis have defined a strain of Salmonella enterica serotype Anatum associated with the consumption of a particular brand of formula-dried milk responsible for an outbreak in late 1996/early 1997 involving 15 infants and 2 relatives in the UK, and 2 infants in France. The study has demonstrated the value of laboratory-based surveillance involving identification of the outbreak strain at the molecular level coupled with food microbiology and targeted epidemiological investigations, and has highlighted the importance of rapid communication and subsequent international collaboration through the European Union-funded Salm-Net salmonella surveillance network.

Genotypic characterization of Salmonella enteritidis phage types by plasmid analysis, ribotyping, and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.

Genomic Fingerprinting by Application of rep-PCR.

Several families of short repetitive DNA sequences, widely distributed in the genome, have been identified in bacteria (1). They have an intercistronic location, are not translated, and their function is unclear, although they may be involved in transcription termination, mRNA stability or chromosomal organization. Repetitive extragenic palindrome (REP) elements (2), also known as palindromic units (PU) (3), and enterobacterial repetitive intergeinc consensus (ERIC) sequences (4) are the best characterized of these elements and were mitially identified in Salmonella typhimurium and Eschenchla colt, respectively. The REP consensus sequence was formulated through DNA sequence comparisons of intercistronic regions of different operons (3,5) and comprises a 38 nucleotide palindromic sequence that can form a stable stem-loop structure with a 5-bp variable loop m the central region (2). There may be 50-1000 copies of the REP sequence in the genome, frequently present in complex clusters (2), with each cluster comprising as many as 10 copies (6). REP sequences have been located between genes within an operon or at the end of an operon, in different orientations and in tandem arrays, and in operons distributed throughout the genome (2,3). The REP sequence has been identified in intergenic regions within operons from different bacterial species (7,8). REP like sequences have been shown to exist throughout the eubacterial kingdom, although the consensus sequences may differ among different bacteria 9 11).

Application of Molecular Methods to the Study of Infections Caused by Salmonella spp.

Disease caused by any member of the genus Salmonella is termed salmonellosis. The type of disease and its symptoms are generally related to the mfecting species and reflect the invasiveness and virulence of the organism. For example, enteric fevers are systemic diseases usually resulting from infection with Salmonella typhi, S paratyphi A, B, or C. Salmonellosis is caused by more than 2200 different salmonella serotypes, which can be classified into three groups according to their adaptation to human and animal hosts. One group of serotypes can be regarded as those as organisms that cause enteric fever only in humans and higher primates. Members of this group, which includes S. typhi, S paratyphi A, B, and C are restricted to humans and higher primates and are not found in food animals. A second group causes diseases in specific animals (e.g., S. dublin-cattle, S. pullorum--poultry, S choleraesuis-pigs). However, when some members of this group cause infections in humans the disease is frequently invasive and can be life-threatening (e.g., S. cholerae-suls, S dublin). The third group, which includes the great majority of the remaining 2000+ serotypes, typically causes mild-to-moderate enteritis in humans, which is often self-limiting, but which can be severe in the young, the elderly, and in patients with other underlying complications This group includes the four serotypes most common in humans in England and Wales at the present time: S. enteritidis, S, typhimurium, S. virchow, and S. hadar. The great majority of serotypes of this third group are zoonotic in origin and have as their reservoirs animals used for food, particularly cattle, poultry, and pigs.

Pulsed-field electrophoretic fingerprinting of Salmonella indiana and its epidemiological applicability.

Eight Xba I-generated pulsed-field profile (PFP) types and four subtypes within one of the most common PFP types have been identified in Salmonella indiana from patients, poultry and human food in England and Wales in the three-year period from January 1994 to December 1996. Two PFP types have predominated, PFP X1 and PFP X2. Although the PFP X1 type was identified throughout the study period, the PFP X2 type was not identified until late 1995, subsequently becoming the most common PFP type in humans in the first six months of 1996 with a significant distribution in elderly patients. It is concluded that PFGE can be used in support of epidemiological investigations for the subdivision of Salm. indiana. Furthermore, as both conditions and interpretation criteria can be easily standardized, it is suggested that for many salmonella serotypes, PFGE can provide the basis for a definitive scheme of genotypic subtyping suitable for epidemiological investigations at both a national and international level.

Application of molecular methods to a nosocomial outbreak of Salmonella enteritidis phage type 4.

A nosocomial outbreak of Salmonella enteritidis phage type 4 occurred in July 1995. Seven definite cases were identified over 13 days affecting four wards in a London hospital. The outbreak strain was characterized by plasmid profile typing and pulsed-field gel electrophoresis (PFGE), and was unusual in that it did not possess a 38 MDa plasmid common to most isolates of S. enteritidis PT 4 made from humans and food animals in England and Wales. Seven asymptomatic excreters were identified on screening. No additional cases occurred on wards after standard isolation procedures were implemented. No common or continuing food or dietary source was identified. Results of epidemiological, microbiological and environmental investigations suggested that the outbreak was due to person-to-person transmission within the hospital. The source of the outbreak was not established but was probably due to admission of a patient with an unrecognized infection of S. enteritidis PT 4. The study highlights the importance of close collaboration between hospital staff, epidemiologists and microbiologists, and demonstrates the value of molecular techniques for strain subdivision in outbreak investigations.

Plasmid characterization and pulsed-field electrophoretic analysis demonstrate that ampicillin-resistant strains of Salmonella enteritidis phage type 6a are derived from Salm. enteritidis phage type 4.

Plasmid incompatibility studies have demonstrated that strains of Salmonella enteritidis phage type (PT) 6a resistant to ampicillin possess a 36 megadalton incompatibility group (Inc) X plasmid coding for resistance to ampicillin which is capable of converting strains of Salm. enteritidis belonging to PTs 1 and 4 to PT 6a, and PT 8 to PT 13. However, pulsed-field gel electrophoresis (PFGE) has demonstrated that all clinical isolates of PT 6a have a characteristic XbaI pulsed-field profile which is distinct from that of PT 1 and which can only be differentiated from that of PT 4 by the presence of plasmid-associated fragments of less than 45 kb. It is concluded that ampicillin-resistant strains of Salm. enteritidis PT 6a are derived from strains of Salm. enteritidis PT 4 by acquisition of an Inc X ampicillin resistance plasmid.

Marshmallows cause an outbreak of infection with Salmonella enteritidis phage type 4.

Thirty-six cases of Salmonella enteritidis phage type (PT) 4 infection, mainly in children, were notified in one local authority in the two weeks beginning on 23 October 1995. Twenty-four cases and 42 controls nominated by cases were included in a case control study, which showed a significant association between illness and the consumption of marshmallow confectionery from a bakery. S. enteritidis PT4 was isolated from samples of the marshmallow, and all isolates had the same plasmid and pulsed field gel electrophoresis profiles. The marshmallow had been made using raw egg white, which shows that not all caterers follow the Department of Health's advice.

Four outbreaks of Salmonella enteritidis phage type 4 food poisoning linked to a single baker.

An outbreak of gastrointestinal illness occurred among a party of diners at a hotel in South Yorkshire. A case control study identified a gateau, from an outside supplier, as the likeliest vehicle of infection. Further gateaux from the same baker's premises and other outlets were examined microbiologically. Three other outbreaks of food poisoning in neighbouring districts were recognised and found to be associated with gateaux supplied by the same baker. A total of 32 cases were identified. Stool specimens from 24 cases grew Salmonella enteritidis phage type (PT) 4. The same organism was also grown from gateau in the domestic refrigerator of the chef of one hotel, cheesecake made by the same baker, and a gateau and ingredients from the baker's premises. The isolates of S. enteritidis PT4 were all fully sensitive to antibiotics, and had the same plasmid and pulsed field gel electrophoresis profiles. It is most likely that cross contamination occurred from infected raw eggs on the baker's premises. The control measures instituted probably prevented two further outbreaks, and the baker now uses only pasteurised eggs. The benefits of close cooperation between different local and health authority districts in the investigation of the outbreaks are discussed.

Evaluation of a restriction fragment length polymorphism typing method for Listeria monocytogenes.

The results of an evaluation of restriction fragment length polymorphism (RFLP) typing for Listeria monocytogenes are presented. The method depends on the use of cloned DNA fragments from an L. monocytogenes (serovar 4b) strain to probe Southern blotted NciI restriction fragments derived from L. monocytogenes strains. Analyses of 862 isolates of serogroups 1/2, 3 and 4 were performed and a total of 32 RFLP patterns were observed. Interstrain RFLPs were common within serogroup 1/2 and the numerical index of discriminatory power for this group was 0.883. Serogroup 4 strains were divided into two major and three minor RFLP types, suggesting a high level of genotypic homogeneity, and the numerical index of discriminatory power was correspondingly low. The technique was found to be highly reproducible, and the stability of patterns was demonstrated by analyses of passaged strains and multiple isolates of the same strain from related specimens. RFLP typing is of value in epidemiological investigations involving strains of serogroup 1/2.

The use of monoclonal antibodies against Listeria monocytogenes in a direct immunofluorescence technique for the rapid presumptive identification and direct demonstration of Listeria in food.

Two monoclonal antibodies which recognise serogroup specific cell surface antigens of Listeria monocytogenes are described. The antibodies were specific for Listeria (i.e. did not react with 87 strains of bacteria from 14 other genera). In an indirect immunofluorescent test the antibodies had similar specificities when tested against strains of L. monocytogenes from human infections grown under a number of different growth conditions, and serological reference strains. A direct immunofluorescent antibody test was developed, and similar specificities of the antibodies found with strains of Listeria which had been isolated from food and grown under 2 different growth conditions. This technique is also used to directly demonstrate Listeria in soft cheese.