Comparison of the environmental survival characteristics of Salmonella Dublin and Salmonella Typhimurium.

To examine possible correlations in bovine Salmonella isolates between environmental survival and serovar-associated epidemiological patterns, bovine field isolates of Salmonella serovars Typhimurium and Dublin (two each) were inoculated into bovine faeces slurry and tested monthly by culture for survival during a six-month period of storage at a variable ambient temperature in a disused animal transporter. Low moisture conditions, where the slurry was dried onto wooden dowels, increased detectable survival of a low-level inoculum by up to five months, compared with wet slurry. A more modest increase of survival time was seen with storage of wet slurry under refrigeration at 4°C. Under both dry and wet conditions, the concentration of culturable Salmonella Typhimurium declined at a slower rate than did that of Salmonella Dublin. Salmonella that was naturally contaminating bovine faeces from farms with Salmonella Typhimurium did not show superior survival times compared with Salmonella Typhimurium that had been artificially inoculated into samples. The differing survival characteristics of the two serovars that was observed in environmental faeces may complement their different modes of infection in cattle. Salmonella Dublin, being a bovine host-adapted strain that establishes chronic infection in some animals, may have less need to survive for a prolonged period outside of its host than does Salmonella Typhimurium.

Drug-resistant Salmonella Typhimurium DT 120: use of PFGE and MLVA in a putative international outbreak investigation.

Isolates of Salmonella enterica serovar Typhimurium belonging to definitive phage type (DT) 120 (Salmonella Typhimurium DT 120) from simultaneous outbreaks of infection in the England and Denmark have been compared on the basis of antibiogram, pulsed-field gel electrophoresis (PFGE), and multiple locus variable number tandem repeat analysis (MLVA). Isolates from England had the resistance profile (ampicillin, streptomycin, sulfamethoxazole, and tetracycline), MLVA profiles 2-4-4-0-2, 2-4-5-0-2, and 2-4-0-0-2, and the PFGE type STYMXB.0083. Representative isolates from the Denmark outbreak were resistant to ampicillin only (A) and had the MLVA type 2-12-6-0-2 and the PFGE type STYMXB.0010. These results demonstrated that outbreak isolates from England and Denmark were not identical. Subsequently, comparison of outbreak isolates with contemporary animal isolates showed that an isolate with the same PFGE type and a similar MLVA type had been isolated in England before its identification in Denmark. These results confirmed the usefulness of MLVA in international outbreak investigations of multiresistant Salmonella Typhimurium and have demonstrated how new molecular strategies may be used to supplement existing methods such as PFGE to enable the accurate and rapid comparison of isolates from different countries. The data also indicate that MLVA proves a useful method for detection of specific Salmonella Typhimurium DTs from human and veterinary sources.

Distribution of Cryptosporidium species in sheep in the UK.

There have been few studies of the distribution of Cryptosporidium species and genotypes in sheep, and the anthropozoonotic potential has been questioned since one of the major human pathogens, Cryptosporidium parvum, is not always found. To investigate the situation in the UK we undertook three studies: a reactive sampling programme of flocks identified as exposures for human cases of cryptosporidiosis; investigation of neonatal cryptosporidiosis in lambs; and a screening programme of lambs at an open farm. C. parvum was the only species found in neonatal lambs with cryptosporidiosis and predominated in flocks sampled reactively to a human case of cryptosporidiosis. C. bovis was also found in the latter study but at a lower frequency than C. parvum. C. bovis and the cervine genotype were found in the orphan lambs under the screening programme. The results of these studies show that C. parvum is important in neonatal lamb diarrhoea and is widespread in sheep flocks in the UK, but that other Cryptosporidium species and genotypes are also present. Sheep, and young lambs in particular, must still be considered as a source of C. parvum infection for humans.

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.

Persistence of Escherichia coli O157 isolates on bovine farms in England and Wales.

We performed pulsed-field gel electrophoresis on Escherichia coli O157 isolates (n = 318) from 199 healthy animals in a longitudinal study carried out on nine farms. Investigation of the restriction types proved that at the farm level, the same clones can be detected on sampling occasions separated by as much as 17 months. The cohort animals were repeatedly sampled, and for some of these, the same clones were obtained on sampling occasions separated by as much as 8 months.

Characterization of beta-lactamases responsible for resistance to extended-spectrum cephalosporins in Escherichia coli and Salmonella enterica strains from food-producing animals in the United Kingdom.

Nine epidemiologically unrelated isolates [1 Salmonella Bredeney from turkeys, and 8 Escherichia coli [3 environmental isolates (2 from chickens, 1 from pigs), and 5 isolates from cattle with neonatal diarrhea]] were examined both pheno- and genotypically for extended-spectrum beta-lactam (ESBL) resistance. Resistance phenotypes (ampicillin, aztreonam, cefotaxime, cefpodoxime, ceftazidime, and ceftriaxone) suggested the presence of an ESBL enzyme, but cefoxitin MICs (>/= 32 mg/L) suggested the presence of an AmpC-like enzyme. Synergism experiments with benzo(b)thiophene-2-boronic acid (BZBTH2B) and isoelectric focusing (IEF) revealed the presence of an AmpC beta-lactamase with a pI >/= 9. amp C multiplex PCR, sequence, and Southern analyses indicated that only the Salmonella isolate had a plasmid-encoded AmpC beta-lactamase CMY-2 on a nonconjugative 60-MDa plasmid. PCR and sequence analysis of the E. coli ampC promoter identified mutations at positions -88(T), -82(G), -42(T), -18(A), -1(T) and +58(T) in all the isolates. In addition one strain had two extra-mutations at positions +23(A) and +49(G), and another strain had one extra-mutation at position +32(A). DNA fingerprinting revealed that all the E. coli isolates were different clones. It also showed that the U.K. Salmonella isolate was indistinguisable from a Canadian Salmonella isolate from turkeys; both had identical resistance phenotypes and produced CMY-2. This is the first report of a CMY-2 Salmonella isolate in the United Kingdom. These data imply that beta-lactam resistance in animal isolates can be generated de novo as evidenced by the E. coli strains, or in the case of the Salmonella strains be the result of intercontinental transmission due to an acquired resistance mechanism.

Multiple genetic typing of Salmonella Enteritidis phage-types 4, 6, 7, 8 and 13a isolates from animals and humans in the UK.

Salmonella enterica serovar Enteritidis is a common cause of salmonellosis in people in the UK. This study aimed to assess the degree of genetic diversity among animal and human isolates from UK, Wales and northern Ireland. A total of 250 isolates from humans (n = 59) and animals or their environment (n = 191), belonging to the most common phage-types, were fingerprinted by a combination of PFGE, PS ribotyping and plasmid profiling. The different techniques identified different degrees of polymorphism (PS ribotyping (52 types) > PFGE (22 types) > plasmid profiling (17 types)). A prevalent genomic clone, as well as a variety of less frequent clones are present for each of the phage-types. In most cases, the prevalent clones appeared within isolates from several animal species and from several geographical locations. The percentage of sporadic clones found in animal and human populations were very similar. There was not clear evidence of a higher degree of diversity for human or animal isolates. Some clones were found to be present in both human and animal.

Genetic diversity among Escherichia coli O157:H7 isolates from Bovines living on farms in England and Wales.

Pulsed-field gel electrophoresis of Escherichia coli O157:H7 isolates (n = 228) from 122 healthy animals on 11 farms discriminated 57 types. Most clones were found only on individual farms. Numerous clones were found within each farm, with a prevalent clone normally found in several animals. A variety of clones were found within the different phage types.

Multiple genetic typing of Salmonella enterica serotype typhimurium isolates of different phage types (DT104, U302, DT204b, and DT49) from animals and humans in England, Wales, and Northern Ireland.

Salmonella enterica serotype Typhimurium is a common cause of salmonellosis among humans and animals in England, Wales, and Northern Ireland. Phage types DT104 and U302 were the most prevalent types in both livestock and humans in 2001. In addition, Salmonella serotype Typhimurium DT204b was responsible for a recent international outbreak involving England. A total of 119 isolates from humans (n = 28) and animals or their environment (n = 91), belonging to DT104 (n = 66), U302 (n = 33), DT204b (n = 12), and DT49 (n = 8), were fingerprinted by a combination of well-established genetic methods (pulsed-field gel electrophoresis [PFGE], PstI/SphI [PS] ribotyping, and plasmid profiling). The different techniques identified different degrees of polymorphism (from greatest to least, plasmid profiling [40 types], PS ribotyping [34 types], and PFGE [23 types]). It seems clear that a prevalent genomic clone, as well as a variety of less frequent clones, is present for each of the phage types. In most cases, the prevalent clones appeared within isolates from several animal species and from several geographical locations. We did not find clear evidence of a higher degree of diversity for any of the animal species included, or of any link between isolates from particular animal species and humans. The data presented show the inaccuracy of drawing epidemiological conclusions based on a single fingerprinting method. Strains that share one of the markers do not necessarily belong to the same clone, and a multiple typing approach is required to enable enough discrimination to track strains for epidemiological investigations.

Production of attaching-effacing lesions in ligated large intestine loops of 6-month-old sheep by Escherichia coli O157:1H7.

Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) is associated with potentially fatal human disease, and a persistent reservoir of the organism is present in some farm animal species, especially cattle and sheep. The mechanisms of persistent colonisation of the ruminant intestine by STEC O157:H7 are poorly understood but may be associated with intimate adherence to eukaryotic cells. Intimate adherence, as evidenced by induction of attaching-effacing (AE) lesions by STEC O157, has been observed in 6-day-old conventional lambs after deliberate oral infection but not in older animals. Thus, the present study used a ligated intestinal loop technique to investigate whether STEC O157:H7 and other attaching-effacing E. coli may adhere intimately to the sheep large intestinal mucosa. To do this, four STEC O157:H7 strains, one STEC O26:K60:H11 and one Shiga toxin-negative E. coli O157:H7 strain, suspended in either phosphate-buffered saline or Dulbecco's modified Eagle's medium, were inoculated into ligated spiral colon loops of each of two lambs. The loops were removed 6 h after inoculation, fixed and examined by light and electron microscopy. AE lesions on the intestinal mucosa were produced by all the inoculated strains. However, the lesions were sparse and small, typically comprising bacterial cells intimately adhered to a single enterocyte, or a few adjacent enterocytes. There was little correlation between the extent of intimate adherence in this model and the bacterial cell density, pre-inoculation growth conditions of the bacteria or the strain tested.

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

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 >or=1 mg/liter, while 89.8% of isolates with mutations in codon 87 presented MICs of or=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.