Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups.

This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 108 colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 106 CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine's value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars.

Salmonella phages and prophages--genomics and practical aspects.

Numerous bacteriophages specific to Salmonella have been isolated or identified as part of host genome sequencing projects. Phylogenetic analysis of the sequenced phages, based on related protein content using CoreGenes, reveals that these viruses fall into five groupings (P27-like, P2-like, lambdoid, P22-like, and T7-like) and three outliers (epsilon15, KS7, and Felix O1). The P27 group is only represented by ST64B; the P2 group contains Fels-2, SopEphi, and PSP3; the lambdoid Salmonella phages include Gifsy-1, Gifsy-2, and Fels-1. The P22-like viruses include epsilon34, ES18, P22, ST104, and ST64T. The only member of the T7-like group is SP6. The properties of each of these phages are discussed, along with their role as agents of genetic exchange and as therapeutic agents and their involvement in phage typing.

Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada.

Multi-drug-resistant (MDR) Salmonella enterica serovar Newport strains are increasingly isolated from animals and food products of animal origin and have caused septicemic illness in animals and humans. The purpose of this study was to determine the occurrence and the epidemiologic, phenotypic, and genotypic characteristics of S. Newport of animal origin that may infect humans, either via the food chain or directly. During the 1993-2002 period, the Office International des Epizooties Reference Laboratory for Salmonellosis in Guelph, Ontario, received 36 841 Salmonella strains for serotyping that had been isolated from animals, environmental sources, and food of animal origin in Canada. Of these, 119 (0.3%) were S. Newport. Before 2000, none of 49 S. Newport strains was resistant to more than 3 antimicrobials. In contrast, between January 2000 and December 2002, 35 of 70 isolates, primarily of bovine origin, were resistant to at least 11 antimicrobials, including the extended-spectrum cephalosporins. The blaCMY-2', flo(st'), strA, strB, sulII, and tetA resistance genes were located on plasmids of 80 to 90 MDa that were self-transmissible in 25% of the strains. Conserved segments of the integron 1 gene were found on the large MDR-encoding plasmids in 3 of 35 strains additionally resistant to gentamicin and spectinomycin or to spectinomycin, sulfamethoxazole-trimethoprim, and trimethoprim. Resistance to kanamycin and neomycin was encoded by the aphA-1 gene, located on small plasmids (2.3 to 6 MDa). The increase in bovine-associated MDR S. Newport infections is cause for concern since it indicates an increased risk of human acquisition of the infection via the food chain.

Prevalence of Salmonella spp. on Canadian pig farms using liquid or dry-feeding.

The objective of this study was to determine whether the shedding and antibody titre to Salmonella was lower for pig herds provided liquid-feed compared to those on traditional dry rations. Twenty liquid-feeding farms and 61 dry-feeding farms were selected. The amount of antibodies to Salmonella in sera from 15 finisher pigs on each of 80 Ontario swine farms was analyzed by means of enzyme-linked immunosorbent assay (ELISA). In addition, the presence of Salmonella on the 20 liquid-feeding farms and 21 of the dry-feeding farms was assessed by culture of 15 fecal samples taken directly from finisher pigs and five pooled pen-fecal samples at each farm. A cut-off of OD% 10 was used. The Salmonella sero-prevalence differed between the two groups of farms. At least one pig tested sero-positive on 98% of the dry-feeding farms and 84% of the liquid-feeding farms (P<0.05). A multi-variable mixed linear regression model with the farm as a random variable and farm factors as the fixed effects was fitted. Crude optical density (OD) of the individual pig was considered as the continuous dependent variable. Dry-feeding and antimicrobial daily usage was associated with crude OD (P<0.05). In addition, crude OD increased with increasing herd size (P<0.05). Salmonella was isolated from 25 out of 420 fecal samples (6%) from dry-feeding farms compared to three out of 400 samples (0.8%) from liquid-feeding farms. Eight of the dry-feeding farms (38%) tested positive compared to only three of the liquid-feeding farms (15%). Salmonella was also recovered from the pen environment on five dry-feeding farms but were not isolated from the facilities using liquid-feeding. Salmonella Typhimurium was isolated from four farms in the dry-feed group and on one farm with liquid-feeding. The one S. Typhimurium isolate from the liquid-feeding farm exhibited no antimicrobial resistance, but those from dry-feeding farms were resistant to four or more antimicrobial agents. The results of the logistic regression, with farm as a random effect showed that dry-feeding [OR=2.7 (1.1-15.1)] and continuous flow system [OR=2.3 (1.2-12.7)] increased risk of finding Salmonella in the individual pig. These findings indicate that liquid-feeding and all-in all-out management of the grower-finisher barns can reduce the Salmonella prevalence.

Antimicrobial resistance of Salmonella isolated from finishing swine and the environment of 60 Alberta swine farms.

The study objective was to describe and evaluate antimicrobial resistance profiles in Salmonella isolated from Alberta swine finishing farms. Salmonella isolates (n = 322) were obtained from 192 fecal and 84 environmental samples of the 60 Salmonella-positive swine finishing farms. Isolates were classified susceptible, intermediate or resistant based on NCCLS guidelines. More than half of the isolates (53.4%) were susceptible to all of the 18 antimicrobials in the testing panel. No resistance was observed to amikacin, amoxicillin/clavulanic acid, cefoxitin, ceftiofur, ceftriaxone, cephalothin, ciprofloxacin, imipenem or nalidixic acid. Less than 1% of isolates were resistant to apramycin, gentamicin and trimethoprim/sulfamethoxazole. Higher frequencies of resistance were observed for chloramphenicol (4.7%), ampicillin (7.8%), kanamycin (11.8%), sulfamethoxazole (21.1%), streptomycin (25.5%) and tetracycline (38.8%). Eleven Salmonella serovars had isolates with resistance to > or =3 antimicrobials. The most frequently resistant serovar was Salmonella Derby, with 27 (38.0%) isolates resistant to > or =3 antimicrobials, including resistance to five and six antimicrobials. An absence of resistance to cephalosporins and fluoroquniolones and a low proportion of isolates resistant to amikacin, amoxicillin/clavulanic acid, apramycin, gentamicin and trimethoprim/sulfamethoxazole are encouraging findings from public health and animal health perspectives. Frequent resistance observed for ampicillin, kanamycin, sulfamethoxazole, streptomycin and tetracycline, antimicrobials commonly used in veterinary medicine for decades, indicates an urgent need to utilize these antimicrobials more prudently if their benefits are to be preserved.

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.

Recovery of cephalosporin-resistant Escherichia coli and Salmonella from pork, beef and chicken marketed in Nova Scotia.

BACKGROUND: Antimicrobial use in farm animals is a potentially important contributor to the emergence of antimicrobial resistance. Resistant Salmonella may lead to serious human infections and resistant Escherichia coli may transfer plasmid-encoded resistance genes to other pathogens. OBJECTIVE: To determine the prevalence of E coli and Salmonella species resistant to the third generation of cephalosporins in retail meat products in Halifax, Nova Scotia in 2002. METHODS: Ground beef, ground pork and chicken wings were tested for E coli and Salmonella. E coli were selected on ceftriaxonecontaining media. Beta-lactamases were characterised by isoelectric focusing, polymerase chain reaction and sequencing. Pulsed field gel electrophoresis was performed to determine the relationship of strains. The transferability of plasmids and location of resistance genes was also determined. RESULTS: Forty-three of 75 packages of chicken wings contained ceftriaxone-resistant E coli; 42 of these contained beta-lactamases with isoelectric points at approximately 8.7. Six of seven CMY primer amplicons that were sequenced contained plasmid-mediated Citrobacter freundii-derived blaCMY-2; the other contained a CMY-2- like beta-lactamase. Pulsed field gel electrophoresis patterns demonstrated that strains were not clonal in nature. Four chicken samples contained Salmonella, one of which contained bla CMY-2-mediated resistance and an E coli bearing the same gene, but on different plasmids. Four of 100 beef samples contained blaCMY-2-bearing E coli; none contained Salmonella. Two of 75 pork samples contained ceftriaxone resistant E coli, one of which encoded for CMY-2. One susceptible Salmonella strain was recovered from pork. CONCLUSIONS: Chicken from retail outlets located in Halifax, Nova Scotia, commonly contained blaCMY-2-bearing E coli. The relationship antibiotics used in food-producing animals and its effect on resistance of commensals and pathogens needs to be determined.

Occurrence and characterization of resistance to extended-spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella isolated from food-producing animals in Canada.

Resistance of Salmonella to extended-spectrum cephalosporins (ESCs) is being reported with increasing frequency. In humans, infections with Salmonella resistant to ESCs threaten the efficacy of ceftriaxone, the drug of choice for treating salmonellosis in children. To determine the occurrence of resistance to ESCs, we examined 8426 strains isolated from food-producing animals in Canada in 1994-99 for reduced susceptibility or resistance to ceftriaxone. Of the 8 such strains identified (7 from turkeys and 1 from cattle), 5 had reduced susceptibility, and 3 were resistant; 2 were isolated in 1995, 1 was isolated in each of 1996 and 1997, and 4 were isolated in 1999. Isoelectric focusing showed that all 8 isolates produced a beta-lactamase with a pI > or = 9. The strains were resistant to cefoxitin and not inhibited by clavulanic acid. Primers specific for the Citrobacter freundii blaAmpC gene produced the expected product in the polymerase chain reaction. DNA sequencing showed that all isolates possessed the blaCMY-2 gene. Plasmid DNA from all 8 isolates transformed Escherichia coli DH10B, whereas only 1 isolate transferred blaCMY-2 conjugally. All transformants and the transconjugant were resistant to ampicillin, cefoxitin, ceftiofur, cephalothin, streptomycin, sulfisoxazole, and tetracycline. Southern blots of plasmids from the isolates, the transformants, and the transconjugant showed that blaCMY-2 was located on similar-sized plasmids (60 or 90 MDa) in the transformants and the transconjugant. In the S. Typhimurium DT104 and S. Ohio isolates, the floSt gene was found on the same plasmid. Class 1 integrons with the aadB gene cassette were detected in the S. Bredeney isolates but not in their transformants or the transconjugant. Pulsed-field gel electrophoresis and plasmid profiles indicated that both clonal dispersion and horizontal transfer of blaCMY-2 may have caused dissemination of the resistance determinant.

Phenotypic and genotypic characterization of food animal isolates of Salmonella with reduced sensitivity to ciprofloxacin.

Reports of nontyphoidal Salmonella enterica subsp. enterica showing reduced sensitivity to ciprofloxacin (RSC) have increased rapidly during the past decade. Infection in humans with Salmonella possessing RSC may compromise the effectiveness of ciprofloxacin therapy. Nineteen among 4,357 Salmonella strains isolated from food animals in Canada from 1998 to 1999 showed RSC; 17 were from turkeys and 2 from chickens. All were resistant to nalidixic acid and sulfisoxazole and possessed RSC at a level of 0.125-0.5 microg/ml. PCR-RFLP of the gyrA quinolone resistance-determining region (QRDR) with Hinfl revealed that S. Bredeney and S. Heidelberg isolates possessed a mutation in this region. Single-strand conformational polymorphism (SSCP) analysis showed that S. Schwarzengrund and S. Senftenberg isolates also possessed a point mutation in the QRDR. DNA sequencing confirmed the findings and showed that all isolates possessed a base substitution in the gyrA QRDR. Sequencing revealed no mutations in the gyrB and silent wobble mutations in the parC QRDR. Reserpine, a known efflux pump inhibitor, did not effect the MICs for ciprofloxacin, nalidixic acid, and tetracycline. The mar operon could be induced in all isolates at 37 degrees C and in 18 of 19 at 30 degrees C; induction resulted in a two- to four-fold increase in the MIC of ciprofloxacin. In 14 of the 19 isolates, the mutation rate was two-fold or higher than in a ciprofloxacin sensitive S. Bredeney and S. Typhimurium LT2 control strain. Examination of clonal relatedness using pulsed-field gel electrophoresis (PFGE) and plasmid profiles indicated that some degree of clonal dispersion may have occurred, but the majority of isolates may have arisen from de novo mutations.