Molecular epidemiology of multi-resistant Escherichia coli.

In this case-control study multi-resistant Escherichia coli isolates were characterized on a molecular level and risk factors for their development were identified. Thirty-two multi-resistant E. coli strains were isolated from the urine of 13 patients attending a renal clinic for chronic urinary tract infection (UTI) and from different sites of 11 terminally ill patients with nosocomial infections hospitalized on five different wards. All 32 isolates were resistant to ciprofloxacin, cotrimoxazole and produced beta-lactamase. All strains contained plasmids of 2-110 MDa of which a 50 MDa and a 100 MDa plasmid were present in 81% of the strains. Pulse-field gel electrophoresis (PFGE) analysis demonstrated 17 genotypes among 32 strains which indicates a polyclonal outbreak with some geographic clustering. Monitoring of patients over the study period showed that either the resident genotype remained the same and that these retained strains underwent changes in their plasmid contents, or that they were replaced by a different genotype after several months of therapy for chronic UTI. Univariate analysis indicated that multi-resistant E. coli develop in the presence of long-term selective ciprofloxacin pressure at a dosing regimen of 250 mg bid for more than 20 days and that treatment with a broad spectrum antimicrobial for more than three days favours the selection of multi-resistant E. coli in the flora of terminally ill patients with multiple disorders.

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.

Identification of a conjugative plasmid carrying antibiotic resistance and salmonella plasmid virulence (spv) genes in epidemic strains of Salmonella typhimurium phage type 193.

A plasmid pDEP34 that codes for resistance to ampicillin, streptomycin, sulphonamides and tetracyclines has been identified in strains of Salmonella typhimurium phage type 193 which have become increasingly common in England and Wales since 1988. pDEP34 is also self-conjugative, carries the genes responsible for the virulence of host strains for BALB/c mice (spv genes) and is closely related to the Salm. typhimurium'serotype-specific' plasmid pSLT.

Molecular characterization of plasmids in Salmonella enteritidis phage types.

Plasmids in selected type strains of 26 of the Salmonella enteritidis phage types have been characterized by restriction enzyme fingerprinting and by DNA-DNA hybridization with oligonucleotide probes for Salmonella plasmid virulence (Spv) genes. With one exception, the fingerprints of the 38 MDa plasmids studied were homogeneous but there was heterogeneity in the fingerprints of 59 MDa plasmids found in 4 of the type strains. However all 38 MDa and 59 MDa plasmids were related as was a 45 MDa plasmid identified in the type strain of phage type 19. A 3.5 kb fragment homologous to SpvC was conserved in Hind III digests of all 38 MDa and 59 MDa plasmids, and in the related 45 MDa plasmid. In contrast a 65 MDa plasmid found in the type strain of phage type 10 was not related to these three plasmid molecular weight groups and did not carry the SpvC gene.

Use of plasmid profile typing for surveillance of Salmonella enteritidis phage type 4 from humans, poultry and eggs.

Plasmids were found in 1022 of 1089 (94%) of drug-sensitive strains of Salmonella enteritidis phage type 4 from humans (sporadic and outbreak cases), poultry (chickens) and eggs in England and Wales in the 5-year period 1988-92 and 25 plasmid profile patterns were identified. Strains characterized by a single plasmid of 38 MDa predominated (= plasmid profile type SE 38), comprising over 90% of isolates from humans, 70% from poultry and 92% from eggs. Eleven profile types were identified in strains from humans, 21 in strains from poultry and 3 in strains from eggs. Eight of the 11 patterns identified in human isolates were found in strains from poultry and 2 in strains from eggs. In contrast 15 patterns seen in poultry were not found in strains from humans. Four percent of strains from humans and 13% from poultry did not carry the 38 MDa plasmid but all strains from eggs were found to carry this plasmid. The second most common profile type in strains isolated between 1981 and 1988 was not identified in strains isolated from 1988-92. It is concluded that plasmid profile typing is a useful method for rapid differentiation within phage type 4 of S. enteritidis but that methods which can discriminate within the predominant profile type, SE 38, are now required.

Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe.

Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991-92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%). Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm. dublin serovar-specific plasmid. On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized. Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol. wt plasmid which coded for tetracycline resistance only. Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting. These results demonstrate that Salm. typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.

Molecular fingerprinting of multidrug-resistant Salmonella enterica serotype Typhi.

For epidemiologic investigations, the primary subdivision of Salmonella Typhi is vi-phage typing; 106 Vi-phage types are defined. For multidrug-resistant strains the most common types have been M1 (Pakistan) and E1 (India, Pakistan, Bangladesh, and the Arabian Gulf); a strain untypable with the Vi phages has been responsible for a major epidemic in Tajikistan. Most often, isolates from the Indian subcontinent have been resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracyclines, and trimethoprim; but in the 1997 Tajikistan outbreak, the epidemic strain was also resistant to ciprofloxacin. For multidrug-resistant strains, subdivision within phage type can be achieved by plasmid profile typing and pulsed-field gel electrophoresis.

Characterization of Salmonella virchow phage types by plasmid profile and IS200 distribution.

The type strains of the 57 phage types of Salmonella virchow have been characterized by plasmid profile and by distribution of the insertion sequence IS200. Thirty-two strains carried plasmids and 21 profile types were identified; 17 strains were resistant to antimicrobial agents. In contrast only six of the type strains carried IS200 elements and three patterns were identified. Within Salm. virchow phage type 31, five of 10 wild-type isolates carried plasmids and two plasmid profiles were identified; in contrast, an IS200 element was identified in the genome of only one of these strains. It is concluded that for Salm. virchow, IS200 is unlikely to significantly extend the degree of discrimination achieved by phage typing which may be supplemented when appropriate by plasmid profile typing.

Epidemiological application of differentiating multiresistant Salmonella typhimurium DT104 by plasmid profile.

Human isolates of multiresistant Salmonella typhimurium definitive phage type (DT) 104 in England and Wales are currently second in number only to those of S. enteritidis phage type 4. Differentiation of strains is essential in epidemiological investigations and the value of one method, plasmid profile typing, has been assessed in a study of 600 isolates of S. typhimurium DT 104 with multiresistant antibiograms (R-types) ACSSuT, ACSSuTCp and ACSSuTTm from humans, food animals, human food, pets, and animal feed made in England and Wales from January 1990 to April 1996. Twenty plasmid profile (PP) types have been identified in isolates of R-type ACSSuT and ACSSuTCp. One profile type, with a single plasmid of 60 megadaltons-PP type A-has predominated, but identification of PP type has proved useful in some epidemiological investigations. A further four PP types have been identified in isolates of DT 104 R-type ACSSuTTm, in which resistance to trimethoprim is encoded by a plasmid of 4.6 megadaltons and the two commonest PP types are related to those also common in DT 104 R-type ACSSuT. Methods of differentiating within the commonest profile type are now needed.

Subtyping of Listeria monocytogenes on the basis of plasmid profiles and arsenic and cadmium susceptibility.

The susceptibilities to arsenic and cadmium together with the detection of plasmid DNA were evaluated for use as epidemiological markers for the subtyping of Listeria monocytogenes. Plasmid DNA was detected in 34% of 322 apparently unrelated isolates of L. monocytogenes. The resistance to cadmium and arsenic differentiated 565 apparently unrelated cultures into four groups, the smallest being 5% of cultures resistant to both agents, and the largest (53%) being sensitive to cadmium and resistant to arsenic. The resistance patterns to these agents and the presence of plasmid DNA varied markedly between the serotypes of the cultures. The detection of plasmid DNA was strongly associated with cadmium resistance in serogroup 1/2 cultures, but not within those of serogroup 4. Arsenic resistance was not associated with plasmid DNA. All methods were sufficiently stable to be useful for epidemiology investigations. The techniques described here offer simple methods which can be easily utilized in laboratories without a specialized expertise for this bacterium.

Pulsed field gel electrophoresis identifies an outbreak of Salmonella enterica serotype Montevideo infection associated with a supermarket hot food outlet.

In February 1996 Salmonella enterica serotype Montevideo infection in a patient in the North Tyneside area was attributed to consumption of cooked chicken bought from a supermarket hot food outlet. Isolates from the patient, leftover food, and environmental samples were indistinguishable by pulsed field gel electrophoresis (PFGE). PFGE also demonstrated that an outbreak of infection with S. Montevideo associated with the hot food outlet had occurred in late 1995 and early 1996. This study shows the importance of microbial strain discrimination in outbreak investigations and illustrates the value of close liaison between microbiologists, epidemiologists, and environmental health officers in the control of salmonella outbreaks.

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.

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.

Collaborative investigation of an outbreak of Salmonella enterica serotype Newport in England and Wales in 2001 associated with ready-to-eat salad vegetables.

In June 2001, as part of a microbiological study of bagged, ready-to-eat salad products, Salmonella enterica serotype Newport was isolated from a sample of pre-packed green salad distributed by a major supermarket retailer. The strain was characterised by phage typing, plasmid profile typing and pulsed-field gel electrophoresis. Other isolates of S. Newport from cases of human infection in England and Wales in the first six months of 2001 were similarly characterised. Of 60 strains from cases of human infection, 19 were found to be indistinguishable from that isolated from the salad product. This study highlights the benefits of an integrated approach to outbreak investigations, involving the various elements of the PHLS and the Food Standards Agency, and acknowledges the full co-operation of the retailer in ensuring the rapid withdrawal of the contaminated product.