Linda R. Ward

Bacteriophage-typing designations of Salmonella typhimurium

The phage-typing scheme of Callow (1959) has been extended. The original number of types was 34; this has now risen to 207. Tables are presented which show the provisional type designations and the definitive designations now being introduced.

A phage-typing scheme for Salmonella enteritidis

For many years phage typing has proved invaluable in epidemiological studies on Salmonella typhi, S. paratyphi A and B, S. typhimurium and a few other serotypes. A phage-typing scheme for S. enteritidis is described. This scheme to date differentiates 27 types using 10 typing phages.

Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods

Salmonella and Campylobacter continue to be major foodborne pathogens and raw poultry is considered to be an important source of these bacteria. In this study, the prevalence and numbers of Salmonella and Campylobacter spp. in relation to isolation/sampling methods were determined in 241 whole raw chickens purchased from retail outlets in England during the winters of 1998/1999 (101 chickens) and 1999/2000 (140 chickens). The packaging of the 140 chickens was also examined for the presence of the above pathogens. The prevalence and numbers of enterococci were examined in 21 of the 101 chickens. In total, Salmonella and Campylobacter spp. were present in 25% and 83% of the chickens, respectively. Salmonella were isolated from a sample representing both the inside and outside of the packaging in 19% of the chickens, while the corresponding figure for Campylobacter spp. was 56%. Both of these pathogens were isolated from the outside of the packaging in 6% of the chickens. Salmonella was more frequently isolated from samples containing chicken skin in comparison with those containing carcass-rinse fluid only. Two chickens (0.8%) were positive for Salmonella by direct enumeration methods with contamination levels of log10 3.8 and 4.5 colony forming units (cfu) per carcass, respectively. The most prevalent serotypes were S. Hadar, S. Enteritidis and S. Indiana and two different serotypes were identified in 5/20 salmonella-positive chickens. Resistance to at least one antibiotic was found in 70% of the strains, 46% were multiresistant (resistant to > or = four drugs) and 52% showed a lowered susceptibility to ciprofloxacin. The likelihood of isolating Campylobacter spp. from neck-skin, carcass-rinse or carcass-rinse plus whole skin samples was similar, Campylobacter spp. were found in higher levels in carcass-rinse or carcass-rinse plus whole skin samples than in neck-skin. The log10 cfu of Campylobacter spp. were 2.70-4.99 in 18% of the chickens and 5.00-6.99 in 20%. Campylobacter isolates (425) comprised Campylobacter jejuni (98%) and C. coli (2%) and 98 different sero/phagetypes of these two species were identified. Resistance to at least one antibiotic was found in 73% of the strains and 13% were multiresistant. Thirteen percent of the strains showed lowered susceptibility to ciprofloxacin, while 4.9% were resistant to erythromycin. Vancomycin-resistant enterococci (VRE), able to grow on agar containing 15 mg l(-1) vancomycin (VRE15), were present in 19 chickens. The log10 cfu of VRE15 was 2.90-3.99 in 10 chickens and between 4.00 and 4.99 in two chickens. The data presented here contribute to risk assessment and highlight the need to continue to emphasise the safe handling of raw retail poultry.

The emergence and spread of antibiotic resistance in food-borne bacteria.

Since the early 1990s there has been a dramatic increase in resistance to antimicrobial drugs in Salmonella enterica and Campylobacter spp., and to a lesser extent in Vero cytotoxin-producing Escherichia coli O157 from cases of human infection in developed countries. For S. Typhimurium a particularly important aspect of this increase has been the widespread dissemination of a multiply drug-resistant (MR) strain of definitive phage type (DT) 104 in food animals since the early 1990s. The use of antimicrobials for prophylaxis in food producing animals has been an important factor in the emergence of strains with resistance to certain antimicrobials. It is hoped that recently introduced Codes of Practice for the prophylactic use of antimicrobials in food animals will result in a decline in the occurrence of drug resistant strains in the food chain.

A national outbreak of multi-resistant Salmonella enterica serovar typhimurium definitive phage type (DT) 104 associated with consumption of lettuce

Between 1 August and 15 September 2000, 361 cases of Salmonella enterica serotype Typhimurium definitive phage type (DT) 104, resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, spectinomycin and tetracycline (R-type ACSSuSpT), were identified in England and Wales residents. Molecular typing of 258 isolates of S. Typhimurium DT104 R-type ACSSuSpT showed that, although isolates were indistinguishable by pulsed-field gel electrophoresis, 67% (174/258) were characterized by a particular plasmid profile. A statistically significant association between illness and consumption of lettuce away from home was demonstrated (OR = 7.28; 95% CI=2.25-23.57; P=0.0006) in an unmatched case-control study. Environmental investigations revealed that a number of food outlets implicated in the outbreak had common suppliers of salad vegetables. No implicated foods were available for microbiological testing. An environmental audit of three farms that might have supplied salad vegetables to the implicated outlets did not reveal any unsafe agricultural practices. The complexity of the food supply chain and the lack of identifying markers on salad stuffs made tracking salad vegetables back to their origin extremely difficult in most instances. This has implications for public health since food hazard warnings and product withdrawal are contingent on accurate identification of the suspect product.

Decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant Salmonella typhimurium DT104.

Dr Boltons present address is Food Safety Microbiology Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT DURING the past decade, multiresistant Salmonella typhimurium definitive phage type (DT) 104 (MR DT104) which is resistant to ampicillin, chloramphenicol, streptomycin/ spectinomycin, sulphonamides and tetracyclines (ACSSpSuT) has caused numerous infections in food animals and human beings in the UK. Infections have also been reported in many European countries as well as in the USA and Canada (Threlfall 2000). In the UK, human infections with MR DT104 are primarily zoonotic in origin (Threlfall and others 1994). Although there has been a decline in isolations from food animals since 1994, MR DT104 remains the most common salmonella strain in cattle, sheep and pigs in the UK, and is second only to Salmonella enteritidis in poultry (Anon 1999a). In human salmonellosis in England and Wales, MR DT104 has declined from a peak of 4006 isolations in 1996 (Threlfall and others 1999) to 1030 isolations in 1999 (PHLS, unpublished observations). Nevertheless, the organism remains second only to S enteritidis phage type 4 in human beings in England and Wales. Antimicrobial drugs are used under veterinary prescription for therapy and prophylaxis in food-producing animals. In 1997 over 70 per cent of isolates of S typhimurium from livestock were resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracyclines, and a substantial proportion of the resistant isolates were MR DT104 (Anon 1999b). Recently, particular concern has surrounded the isolation of an increasing proportion of MR DT104 with additional resistance to the first generation quinolone nalidixic acid (Nx) in isolations from food-producing animals (Davies and others 1999). Similarly, there have been reports of decreased susceptibility to the fluoroquinolone antimicrobial ciprofloxacin in isolations of MR DT104 from human beings (Threlfall and others 1998). Ciprofloxacin is the first-line drug for the treatment of invasive salmonellosis in humans. In 1994, 1 per cent of MR DT104 from human beings in England and Wales showed decreased susceptibility to ciprofloxacin, and by 1998 this figure had risen to 16 per cent (Threlfall and others 1999). Although the ciprofloxacin minimum inhibitory concentrations (MICs) are below the breakpoint concentration of 1 mg/litre as recommended by the British Society for Antimicrobial Chemotherapy (Anon 1996), S typhimurium isolates with similar MICs have been associated with treatment failure (Piddock and others 1990). In particular, in a recent outbreak of MR DT104 in Denmark, four of 11 patients did not respond to treatment with ciprofloxacin and there were two deaths (M0lbak and others 1999). The increase in the proportion of isolates of MR DT104 with decreased susceptibility to ciprofloxacin in England and Wales is, therefore, particularly disturbing. Between August 23 and September 6, 1998, a large community outbreak of MR DT104 (86 cases) occurred in northwest Lancashire (Anon 1998), and at least four patients were admitted to hospital. Epidemiological investigations revealed that 79 per cent of cases had consumed milk from a local dairy which received raw milk supplied by two farms. MR DT104 was isolated from the milk filter and failure of on-farm pasteurisation was thought to be the cause of the outbreak. On two occasions, in March and August 1998, S typhimurium, subsequently identified as MR DT104, was isolated from dairy cattle on one of the farms supplying the dairy. Strains from human beings, the dairy cattle and the milk filter were characterised. All were of R-type ACSSpSuTNXCPL (Nx nalidixic acid, CPL ciprofloxacin [L:MIC 0.5 mg/litre]) and possessed a single 90 kilobase (kb) plasmid. When a selection of isolates were typed by pulsed-field gel electrophoresis (PFGE), all gave a single PFGE pattern (XTmi) identical to that predominant in MR DT104 (Ridley and Threlfall 1998, Prager and others 1999). Long PCR (R. A. Walker, unpublished observations) demonstrated that these isolates also possessed the chromosomally-encoded 13 kb gene cluster that confers resistance to ACSSpSuT and is characteristic of MR DT104 (Briggs and Fratamico 1999). To investigate further the clonal nature of the strains, the mechanism of resistance to nalidixic acid and ciprofloxacin was determined in isolates from human beings, dairy cattle and the milk filter. The primary mechanism of resistance to nalidixic acid and ciprofloxacin commonly involves a single nucleotide mutation of the gyrA gene (Piddock and others 1998). Mutations in the gyrA gene were therefore characterised, using a rapid LightCycler assay (Gibson and others 1999). This involved amplifying a region of gyrA by PCR followed by the detection of ciprofloxacin resistance-associated mutations using hybridisation probes and thermal analysis. All the isolates were found to have a GAC-*GGC (aspartate to glycine) substitution at codon 87, subsequently confirmed by DNA sequencing. Ten different base pair substitutions in gyrA have been identified in ciprofloxacin-resistant salmonellas. However, although the GAC-*GGC substitution has been observed in MR DT104 (Ridley and Threlfall 1998), the most common mutation in strains with decreased susceptibility to ciprofloxacin involves a GAC->AAC (aspartate to asparagine) mutation at codon 87 (Ridley and Threlfall 1998, Molbak and others 1999, R. A. Walker, unpublished observations). In this outbreak, identification of the GAC->GGC gyrA mutation at codon 87 provided further confirmation of clonal identity of the strains involved. The emergence of decreased susceptibility to ciprofloxacin in MR DT104 is subsequent to the licensing in the UK, in November 1993, of the related fluoroquinolone enrofloxacin for use in food-producing animals. However, there is considerable controversy over the apparent association between enrofloxacin usage in food animals and the emergence of strains with decreased susceptibility to ciprofloxacin in human beings. Indeed, it has been stated that without evidence of scientific data, such an association cannot be substantiated (Jones 1998). However, this investigation has demonstrated a distinct mutation in the gyrA gene in isolates of MR DT104 from dairy cattle, a milk filter and human beings in a defined outbreak situation. An identical gyrA mutation was also identified in MR DT104 isolated from dairy cattle on one of the farms four months before the outbreak. The fluoroquinolone antimicrobial marbofloxacin was in use on the farm in the months preceding the outbreak (T. Leonard, personal communication). Although it is common for subclinical infection of cattle herds with MR DT104 to persist for several months or years (Davies 1997), it is possible

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.

MULTIRESISTANT SALMONELLA TYPHIMURIUM DT104 IN CATS : A PUBLIC HEALTH RISK

1Of particular importance in this increase has been the epidemic spread of a multiresistant strain of R type ACSSuT (A ampicillin; C, chloramphenicol; S, streptomycin, Su, sulphonamides; T, tetracyclines). 1 In 1995 over 87% of strains from human beings were resistant to these five antibiotics, with 26·9% and 6·2% of strains having additional resistances to trimethoprim and ciprofloxacin, respectively. 2

Variation in Clonality and Antibiotic-Resistance Genes Among Multiresistant Salmonella enterica Serotype typhimurium Phage-Type U302 (MR U302) from Humans, Animals, and Foods

Since 1990 multiresistant (MR) Salmonella enterica serotype Typhimurium definitive phage-type (DT) 104 (MR DT104) and closely related phage types have emerged as a worldwide health problem in humans and food animals. In this study the presence of the blaCARB-2 (ampicillin), cmlA (chloramphenicol), aadA2 (streptomycin/spectinomycin), sul1 (sulphonamide), and tetG (tetracycline) resistance genes in isolates of one such phage type, U302, have been determined. In addition blaTEM primers have been used for the detection of TEM-type beta-lactamases. Isolates have also been characterized by plasmid profile and pulsed field gel electrophoresis (PFGE). Thirty-three of 39 isolates were positive for blaCARB-2, cmlA, aadA2, sul1 and tetG, four for blaTEM, aadA2 and sul1, one for aadA2 and sul1, and one for blaTEM only. blaTEM-mediated ampicillin resistance was transferred to Escherichia coli K12 from three isolates along with other resistance markers, including resistance to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracyclines. Strains carried up to 6 plasmids and 34 plasmid profiles were identified. Although the majority of strains (33/39) produced a PFGE profile identical to that predominant in MR DT104, six different patterns were generated demonstrating the presence of various clones within MR U302. The results show that the majority of the MR U302 strains studied possessed the same antibiotic resistance genes as MR DT104. However, isolates with distinctive PFGE patterns can have different mechanisms of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. Such resistance genes may be borne on transmissible plasmids.

A Comparison of Antimicrobial Susceptibilities in Nontyphoidal Salmonellas from Humans and Food Animals in England and Wales in 2000

A joint study by the Public Health Laboratory Service and the Veterinary Laboratories Agency of resistance to antimicrobials in isolates of Salmonella enterica serotypes Enteritidis, Typhimurium, Hadar, and Virchow from humans and food-producing animals in England and Wales in 2000 has demonstrated that resistance was most common in Typhimurium, particularly in strains of definitive phage type (DT) 104. However resistance was also common in other phage types, particularly DTs 193 and 208 and phage type U302. Multiresistant strains of DT208 appeared to be predominantly associated with pigs; for the other phage types, the human/food-producing animal relationships of drug-resistant isolates were more complex. For Enteritidis, Virchow, and Hadar, there were substantial differences in the resistance spectra of isolates from humans and food-producing animals, suggesting that food-producing animals bred in England and Wales may not be the primary sources of drug-resistant strains of these serotypes causing infections in humans. Further phenotypic and molecular comparison of drug-resistant isolates of these serotypes may be required to ascertain the sources of strains responsible for infections in humans.