Multiple antimicrobial resistant Salmonella enterica serovar Paratyphi B variant Java in cattle: a case report.

An epidemiological investigation of a calf rearing premises and a closely associated dairy herd was carried out after the isolation of Salmonella enterica serovar Paratyphi B variant Java phage type 3b variant 2 from clinically diseased calves on the premises. The isolate was resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, tetracyclines, trimethoprim and cefoperazone. The organism was widespread on the calf unit and was also recovered from the dairy premises, mainly from groups of weaned calves. The investigation was extended to 10 epidemiologically linked farms but no S Java was isolated from any of the 40 to 60 samples collected from each premises. Molecular studies showed that the S Java isolates were genetically most similar to isolates from cases of human disease associated with ornamental fish tanks or feed. Long PCR and resistance gene profiling identified a resistance island which was indistinguishable from the human 'fish tank' strain of S Java and animal and human epidemic strains of S Typhimurium DT104. The isolates were clearly distinguished from multi-resistant S Java strains commonly associated with continental poultry. This is the first report of S Java with this resistance pattern in Great Britain.

Multiple drug resistance in salmonellae in England and Wales: a comparison between 1981 and 1988.

Each year from 1981 through to 1988 the most common serotypes isolated from man in England and Wales and identified at the Division of Enteric Pathogens were S typhimurium, S enteritidis, and S virchow. In 1981 these three serotypes accounted for 45%, 12%, and 7% of isolations. The remaining 35% comprised strains belonging to a further 188 different serotypes, none of which accounted for more than 1% of the total. In 1988 S typhimurium accounted for 24% of isolations, S enteritidis 57%, and S virchow 4%. The remaining 15% comprised strains of a further 184 serotypes. The resistances to the common antimicrobial drugs in non-typhoidal salmonellas isolated in England and Wales in 1981 and 1988 were reported with particular reference to resistance to four or more antimicrobial drugs (multiple resistance). For S typhimurium the overall percentage of resistant strains varied little, but multiple resistance more than doubled from 5% to 12%; in S enteritidis the incidence remained the same. In S virchow the percentages of strains resistant to all the antimicrobial drugs and in particular, to chloramphenicol, streptomycin, trimethoprim and furazolidone, rose from 0.2% to 10.4%. Salmonella enteritis in man is usually a self limiting disease and antimicrobial treatment is seldom required; but should spread beyond the intestine occur, effective antimicrobial treatment is essential. Under these circumstances a knowledge of the likelihood of resistances to commonly available drugs could be of considerable value to the clinician.

An immunoblotting procedure comprising O = 9,12 and H = d antigens as an alternative to the Widal agglutination assay.

AIMS: To compare the established Widal agglutination assay with an immunoblotting procedure. METHODS: 110 sera were used to compare the established Widal agglutination assay with an immunoblotting procedure incorporating lipopolysaccharide (LPS) (O = 9,12) and flagellar (H = d) antigens. RESULTS: Antibodies to the LPS antigens were detected in 18 sera by the Widal assay and in 37 by immunoblotting. Antibodies to the flagellar antigens were detected in 27 sera by Widal assay and in 25 by immunoblotting. CONCLUSIONS: An immunoblotting procedure incorporating O = 9,12 LPS and H = d flagellar antigens was rapid and more sensitive than the established Widal agglutination assay for providing evidence of infection with S typhi.

Resistance to ciprofloxacin in pathogenic Enterobacteriaceae in England and Wales in 1996.

In 1996, 6% of Escherichia coli from extraintestinal infections were resistant to ciprofloxacin with minimum inhibitory concentrations (MICs) > or = 2 mg/l (high level resistance). Low level resistance (MIC 0.125-1 mg/l) was also identified in 7% of Salmonella typhi, 4% of S paratyphi A, and 4% of non-typhoidal salmonellas. However, resistance to ciprofloxacin was rarely identified in shigellas. For E coli, physicians should be aware that treatment failures may occur when patients with invasive illness are treated with ciprofloxacin before the results of laboratory sensitivity tests are available. For salmonellas an increasing number of treatment failures have been recorded for patients infected with strains with low level resistance. Because of the increasing incidence of Enterobacteriaceae with low level resistance to ciprofloxacin, it is recommended that for this group of organisms a breakpoint of 0.125 mg/l should be included in laboratory sensitivity tests.

Antimicrobial drug resistance in non-typhoidal salmonellas from humans in England and Wales in 1999: decrease in multiple resistance in Salmonella enterica serotypes Typhimurium, Virchow, and Hadar.

In 1999 the incidence of multiple drug resistance (to four or more antimicrobials) in non-typhoidal salmonellas from humans in England and Wales fell in isolations of Salmonella enterica serotypes Typhimurium, Virchow, and Hadar. This fall has been most noticeable in S. Typhimurium, where 59% of isolates were multiresistant compared to 81% in 1996. The main reason for this has been a 75% decline in isolations of multiply-resistant S. Typhimurium definitive phage type (DT) 104 (MR DT104) since 1996. Nevertheless MR DT104 remains second to S. Enteritidis phage type 4 as the most common strain in cases of human salmonellosis in England and Wales. Multiple resistance has also remained high in S. Hadar, with 49% of isolates resistant to four drugs or more compared to 56% in 1996. Isolates with decreased sensitivity to ciprofloxacin (minimal inhibitory concentration: 0.25-1.0 mg/L) have increased in incidence in S. Enteritidis, S. Virchow, and S. Hadar; in S. Hadar 70% of isolates were resistant to ciprofloxacin at this level. It is hoped that Codes of Practice introduced by some pharmaceutical companies, governments, professional organisations, and others to combat the unnecessary prophylactic use of fluoroquinolones in animal husbandry will not result in a reduction in the incidence of resistance to ciprofloxacin in salmonella organisms causing infections in humans.

Increase in multiple antibiotic resistance in nontyphoidal salmonellas from humans in England and Wales: a comparison of data for 1994 and 1996.

The incidence of multiple drug resistance (to four or more antimicrobials) in salmonellas from humans in England and Wales in 1996 has been compared with corresponding data for 1994. For Salmonella enteritidis multiple resistance has remained rare, although a high proportion of isolates of phage type 6A have shown resistance to ampicillin. For S. typhimurium multiple resistance has continued to increase, with 81% of isolates now multiresistant. Of particular importance in S. typhimurium has been the continued epidemic of multiresistant DT 104 and the increasing occurrence of strains of this phage type with additional resistance to trimethoprim and/or ciprofloxacin. For S. virchow, a 10% increase in multiple resistance is mainly concentrated in two phage types common in returning travellers. For S. hadar, there has been a substantial increase in the incidence of multiple resistance with over 50% of isolates now multiresistant. Substantial increases in the incidence of resistance to ciprofloxacin in multiresistant S. typhimurium DT 104, S. virchow, and S. hadar since 1993, when the fluoroquinolone antibiotic enrofloxacin was licensed for veterinary use in the UK, are of particular concern.

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.

Harmonization of antibiotic susceptibility testing for Salmonella: results of a study by 18 national reference laboratories within the European Union-funded Enter-net group.

For the effective surveillance of antimicrobial drug resistance within Salmonella organisms from humans, harmonization of methods used for sensitivity testing by laboratories responsible for the typing of such organisms is essential. A study of resistance or sensitivity to a panel of 11 antimicrobials by the Enter-net international surveillance network was therefore undertaken. There are 18 national Salmonella reference laboratories within this European Union-funded network. Forty-eight strains of Salmonella enterica were distributed to each laboratory for testing for resistance or sensitivity to ampicillin, cefotaxime, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfonamides, tetracyclines, trimethoprim, nalidixic acid, and ciprofloxacin. Over 8,500 tests were assessed involving disk diffusion (DD), agar breakpoint (BP), or full minimum inhibitory concentration (MIC). Results indicated that whichever method was used, there was a high degree of concordance for the detection of resistance to most antimicrobials; only for decreased sensitivity to ciprofloxacin was there substantial nonconcordance. Because all isolates with decreased sensitivity to ciprofloxacin were resistant to nalidixic acid, it is suggested that, if required, MICs to ciprofloxacin could be determined for isolates resistant to nalidixic acid. For the detection of sensitivity, the main area of nonconcordance was in the detection of sensitivity to streptomycin. With the exception of decreased sensitivity to ciprofloxacin, we are confident that a database of antimicrobial susceptibilities can now be established and harmonized antibiogram data for Salmonella can now be exchanged for national Salmonella reference laboratories within the European Union.

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.

Molecular techniques in the study of Salmonella typhi in epidemiologic studies in endemic areas: comparison with Vi phage typing.

We examined 141 Salmonella typhi strains of known phage type isolated during ongoing epidemiologic studies in Santiago, Chile, and Lima, Peru. Plasmids were present in 12 (17%) of 70 S. typhi isolates from Santiago and 5 (7%) of 71 isolates from Lima; these plasmids were not associated with antimicrobial resistance. Identical 21 kilobase (kb) plasmids (as defined by restriction endonuclease digest pattern) were present in 13 of the 17 plasmid-containing isolates. Virtually identical digest patterns were identified when chromosomal DNA of selected strains from Santiago, Lima, and the United States was extracted and then digested with restriction endonucleases. The similarities among plasmids and chromosomal digest patterns emphasize the homogeneity and possible clonal origin of S. typhi isolates; these data also suggest that there is only a limited role for plasmid and chromosomal analysis as a substitute for phage typing in epidemiologic studies.

Conversion of Salmonella enteritidis phage type 4 to phage type 7 involves loss of lipopolysaccharide with concomitant loss of virulence.

Three strains of Salmonella enteritidis phage type 4 (PT4) and 33 strains of S. enteritidis phage type 7 (PT7) were examined for the ability to produce lipopolysaccharide (LPS) and for plasmid carriage. The LPS of all strains of PT4 gave a typical 'ladder' pattern by SDS-PAGE and silver staining, and on serotyping these strains were shown to express the O-antigens 9, 12. In contrast, strains of PT7 did not express long-chain LPS and were autoagglutinable. All strains of PT4 and the majority of strains of PT7 carried a single plasmid of 38 MDa, indistinguishable when characterised by restriction endonuclease fragmentation analysis. Epidemiological and experimental observations have demonstrated a relationship between strains of S. enteritidis PT4 and PT7, and our results, using mice, show that the loss of ability of strains of PT4 to snythesise LPS is responsible for the conversion of highly virulent strains of PT4 to avirulent strains of PT7. From epidemiological data of human infections in England and Wales, we suggest that strains of S. enteritidis PT7 may be less virulent for humans.

Insertion sequence IS200 can differentiate drug-resistant and drug-sensitive Salmonella typhi of Vi-phage types E1 and M1.

The type strains of Vi-phage types E1, M1 and A of Salmonella typhi, together with drug-resistant and drug-sensitive strains of phage types E1 and M1 isolated in 1992 from patients associated with India or Pakistan, and a drug-resistant strain of phage type A isolated in South Africa in 1991, were characterised with respect to the presence of plasmids conferring resistance to antimicrobial drugs and their chromosomal insertion sequence IS200 profiles. The three type strains, the drug-sensitive strains of Vi-phage types E1 and M1, and a strain of phage type M1 resistant to ampicillin and trimethoprim but not to chloramphenicol, did not contain plasmids. In contrast, for strains of phage types E1 and M1 resistant to chloramphenicol, ampicillin and trimethoprim, and for the drug-resistant strain of phage type A, the complete spectrum of resistance was encoded by high molecular mass plasmids belonging to the H1 incompatibility group. Characterisation of IS200 profiles demonstrated that at least 13 IS200 copies were distributed on the chromosome of all strains tested. Although the IS200 profiles of the type strains of Vi-phage types A, E1 and M1 were identical, it was possible to distinguish between drug-sensitive and drug-resistant strains of Vi-phage types E1 and M1 isolated from patients infected in India and Pakistan by this method. It was concluded that although IS200 typing is not as discriminatory as phage typing for the primary subdivision of S. typhi, it may be useful for certain epidemiological investigations and, in particular, for investigating the origins of strains with multiple drug resistance.

Combination of pulsed-field gel electrophoresis (PFGE) and single-enzyme amplified fragment length polymorphism (SAFLP) for differentiation of multiresistant Salmonella enterica serotype typhimurium.

OBJECTIVE: To assess the combined application of plasmid profile typing, pulsed-field gel electrophoresis (PFGE) and PCR-based single-enzyme amplified fragment length polymorphism (SAFLP) for the differentiation of 18 multiresistant (MR) and one drug-sensitive strain of Salmonella enterica serotype typhimurium from humans and food animals. METHODS: Strains were phage typed and tested for resistance to a panel of antimicrobial agents. Strains were also tested for the ability to transfer resistance either directly or by mobilization to standard strains of Escherichia coli K12. Plasmid DNA was extracted from both drug-resistant donor strains and from drug-resistant exconjugants. Total genomic DNA was characterized by PFGE following digestion with the restriction endonuclease XbaI. The resultant patterns were categorized and analyzed by dendrogram analysis using the Dice coefficient and by data clustering using unweighted pair-group arithmetic averaging (UPGMA). Isolates were also characterized and categorized by SAFLP. The levels of discrimination achieved by each method were assessed individually and in combination. RESULTS: Plasmid DNA was detected in all of the 18 MR isolates but, not in the drug-sensitive isolate. Using PFGE, 19 different profiles were identified, falling into eight major categories. However, by SAFLP, only eight profiles were observed. Subsequent investigations have demonstrated epidemiologic relationships within at least one of these SAFLP profile groupings. CONCLUSIONS: These studies have demonstrated that PFGE and SAFLP can be used independently for the differentiation of MR S. Typhimurium from humans and food animals. However, when used in combination, SAFLP can provide a format for broad epidemiologic groupings. These groupings can be further subdivided by PFGE to provide detailed information on putative strain relationships at the genotypic level.

Molecular characterisation of an outbreak strain of multiresistant Salmonella enterica serovar Typhimurium DT104 in the UK.

A major national outbreak of multiresistant Salmonella enterica serovar Typhimurium definitive phage type 104 (MR DT104) occurred in England and Wales in the summer of 2000. Isolates of MR DT104 were characterised by antimicrobial resistance type (R-type), pulsed-field gel electrophoresis (PFGE), plasmid profiling and fluorescent amplified fragment length polymorphism (fAFLP) analysis. Results of R-type, PFGE and fAFLP showed that summer 2000 outbreak-associated isolates were indistinguishable from most MR DT104 isolates collected in England and Wales during the 1980s and 1990s. However, outbreak-associated isolates all had an additional 2-MDa plasmid (PP D), and this distinct profile allowed outbreak cases to be distinguished from background MR DT104 infections, thereby facilitating the epidemiological investigation by improving the specificity of the case definition. The study demonstrated the highly clonal nature of MR DT104 and the importance of a hierarchical approach to molecular subtyping for outbreak investigations.

A European outbreak of Salmonella enterica serotype Typhimurium definitive phage type 204b in 2000.

OBJECTIVE: To describe the clinical, epidemiologic and microbiological features of a large outbreak of infection with a multiresistant Salmonella enterica serotype Typhimurium definitive type DT204b infection involving at least 392 people in five European countries. METHODS: Icelandic public-health doctors responded to a report on an Internet news site of an outbreak of infection with a multiresistant strain of Typhimurium DT104 in England by contacting the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC). An international alert was sent out through Enter-net. All strains from England & Wales, The Netherlands, Scotland and Germany, and 17 of the outbreak isolates from Iceland, were phage-typed, screened for antimicrobial resistance, and subjected to molecular typing. Hypothesis-generating interviews were conducted, followed by case-control studies performed in Iceland and England. RESULTS: Isolates from cases in Iceland, England and Wales, The Netherlands, Scotland and Germany were identified as Typhimurium DT204b. The antimicrobial resistance pattern was ACGNeKSSuTTmNxCpL. All strains tested displayed an identical plasmid profile. Strains from five cases in England & Wales and five cases in Iceland possessed identical pulsed-field profiles. Although a common source was suspected, only Iceland implicated imported lettuce as a vehicle, with an analytic epidemiologic study (OR = 40.8; P = 0.005; 95% CI 2.7-3175). CONCLUSION: The identification of international outbreaks, necessary for investigation and control, can be facilitated by standardized phage-typing techniques, the electronic transfer of molecular typing patterns, formal and informal links established through international surveillance networks, and the early reporting of national outbreaks to such networks.

LightCycler gyrA mutation assay (GAMA) identifies heterogeneity in GyrA in Salmonella enterica serotypes Typhi and Paratyphi A with decreased susceptibility to ciprofloxacin.

Mutations in gyrA in strains of Salmonella enterica serotypes Typhi and Paratyphi A have been characterised by a LightCycler-based PCR-hybridisation gyrA mutation assay (GAMA) and by DNA sequencing. Four mutations (Ser-83 to Phe, Asp-87 to Asn, Ser-83 to Tyr and Asp-87 to Gly) have been identified in 13 strains of Typhi and three strains of Paratyphi A resistant to nalidixic acid (=nal(r)) and with decreased susceptibility to ciprofloxacin (=Cp(L)), with the mutation Ser-83 to Phe predominating. The results have demonstrated heterogeneity in gyrA in nal(r) Cp(L) strains of Typhi and Paratyphi A and may be useful for epidemiological investigations. No mutations in gyrA were identified in four Cp(L) strains of S. Typhi that were sensitive to nalidixic acid. The mechanism of decreased susceptibility to ciprofloxacin in these strains is under investigation.

Outbreaks of salmonellosis in hospitals in England and Wales: 1992-1994.

Data from the surveillance scheme of general outbreaks of infectious intestinal disease in England and Wales were used to describe the epidemiology of outbreaks of salmonellosis in hospitals from 1992-1994. Outbreaks of infectious intestinal disease in hospitals accounted for 15% (189/1275) of all outbreaks. A salmonella was the implicated pathogen in 12% (22/189). The mode of transmission was described as mainly person to person in 12 outbreaks, mainly foodborne in eight and equal or unknown proportions of foodborne and person to person in two. The most common strain involved was Salmonella enteritidis PT4 (11 outbreaks). The mean duration of outbreaks was 16 days. The mean attack rate in patients was 25% but varied from 2-67%. Illness was reported in 260 patients, of whom 130 had a laboratory confirmed infection. Eight hundred and twenty-six asymptomatic patients were tested, 31 of whom were positive. The salmonella infection was believed to have contributed to the deaths of five patients. Ill staff (115) were tested and 68 were positive; 1508 well staff were tested and 33 were positive. Outbreaks of salmonellosis in hospitals are preventable. Attack rates can be high and outbreaks are often prolonged, with high morbidity and associated disruption of hospital services. There is need for effective infection control policies, appropriate training of staff, simple surveillance systems and readily available expert advice to ensure outbreaks are rapidly controlled.

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.

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.