Dublin Isolates Research Articles

Salmonella enterica Serovar Dublin from Cattle in California from 1993 to 2019: Characterization and Analysis of Antimicrobial Resistance Diversity.

For this study, antimicrobial susceptibility data for Salmonella enterica subsp. enterica serovar Dublin (S. Dublin)-a well-known cattle-adapted pathogen with current concerns for multidrug resistance-were recovered from cattle at the California Animal Health and Food Safety Laboratory System (CAHFS) over the last three decades (1993-2019) and were evaluated using tools to capture diversity in antimicrobial resistance. For this purpose, minimum inhibitory concentration (MIC) testing was conducted for 247 clinical S. Dublin isolates. Antimicrobial resistance (AMR) profiles revealed a predominant core multidrug-resistant pattern in the three most common AMR profiles observed. Antimicrobial resistance richness, diversity, and similarity analysis revealed patterns for changes in AMR profiles for different age groups. Discriminant analysis using MIC log2-transformed data revealed changes in MIC for year groups, with a time-sequence pattern observed. Drivers for reduced susceptibility were observed for 3rd generation cephalosporins and quinolones observed for more recent year groups (2011-2015 and 2016-2019) when compared to older year groups (1993-1999 and 2000-2005). Together, these results highlight the changes in the diversity of AMR profiles, as well as changes in susceptibility of S. Dublin over time for critical antimicrobials of importance to both animals and humans, and support the need for continued monitoring and efforts that will support judicious use of antimicrobials, especially for these two drug classes.

Trends in Salmonella Dublin over time in Denmark from food and animal related isolates.

Salmonella enterica serovar Dublin is highly adapted to cattle and a relatively rare cause of human infections. In Denmark S. Dublin has been endemic in the cattle population for many years. A national surveillance program in the cattle population was established at herd-level to reduce the occurrence of S. Dublin. In this study, we analyzed 421S. Dublin genomes from cattle and food in order to determine the trend of S. Dublin's population size over time in Denmark and the impact of intervention in the cattle industry on the bacterial population size. A phylogenetic tree based on SNPs exhibited two major clades and one small cluster. All isolates were ST10. The temporal phylogenetic tree for the S. Dublin isolates showed that the most recent common ancestor was estimated to be in ∼1980 for the two major clades. An effective population size over time based on a Bayesian skyline plot showed that the population size of S. Dublin decreased significantly between 2014 and 2019 in both major clades. This result was concordant with the decrease of infected human cases by S. Dublin in Denmark. The strengthening of a surveillance program in Denmark could be the cause for the reduction of S. Dublin's effective population size. This study showed that whole genome sequencing combined with computer intensive phylogenetic analysis estimating the effective size of the S. Dublin's population over time is a strongly relevant measure with respect to assessing the impact of control measures aiming to reduce the bacterial population in the reservoir and the risk for human infection.

Salmonella enterica Serovar Dublin from Cattle in California from 1993-2019: Antimicrobial Resistance Trends of Clinical Relevance.

Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) is a cattle-adapted pathogen that has emerged as one of the most commonly isolated and multidrug resistant (MDR) serovars in cattle. S. Dublin may be shed in feces, milk, and colostrum and persist in asymptomatic cattle, leading to spread and outbreaks in herds. Though infections with S. Dublin in humans are rare, they are frequently severe, with extraintestinal spread that requires hospitalization and antimicrobial therapy. To determine minimum inhibitory concentration (MIC) and antimicrobial resistance (AMR) patterns and trends in cattle in California, broth microdilution testing was performed on 247 clinical S. Dublin isolates recovered from cattle at the California Animal Health and Food Safety Laboratory System (CAHFS) over the last three decades (1993–2019). Mean MICs and classification of resistance to antimicrobial drugs using a clinical livestock panel and the National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative drug panels were utilized to assess prevalence and trends in AMR. Findings indicate an increase in AMR for the years 1993 to 2015. Notably, compared to the baseline year interval (1993–1999), there was an increase in resistance among quinolone and cephalosporin drugs, as well as an increased number of isolates with an MDR profile.

A retrospective and regional approach assessing the genomic diversity of Salmonella Dublin.

From a historically rare serotype, Salmonella enterica subsp. enterica Dublin slowly became one of the most prevalent Salmonella in cattle and raw milk cheese in some regions of France. We present a retrospective genomic analysis of 480 S. Dublin isolates to address the context, evolutionary dynamics, local diversity and the genesis processes of regional S. Dublin outbreaks events between 2015 and 2017. Samples were clustered and assessed for correlation against metadata including isolation date, isolation matrices, geographical origin and epidemiological hypotheses. Significant findings can be drawn from this work. We found that the geographical distance was a major factor explaining genetic groups in the early stages of the cheese production processes (animals, farms) while down-the-line transformation steps were more likely to host genomic diversity. This supports the hypothesis of a generalised local persistence of strains from animal to finished products, with occasional migration. We also observed that the bacterial surveillance is representative of diversity, while targeted investigations without genomics evidence often included unrelated isolates. Combining both approaches in phylogeography methods allows a better representation of the dynamics, of outbreaks.

Antimicrobial resistance and genomic characterization of Salmonella Dublin isolates in cattle from the United States.

Salmonella enterica subspecies enterica serotype Dublin is a host-adapted serotype in cattle, associated with enteritis and systemic disease. The primary clinical manifestation of Salmonella Dublin infection in cattle, especially calves, is respiratory disease. While rare in humans, it can cause severe illness, including bacteremia, with hospitalization and death. In the United States, S. Dublin has become one of the most multidrug-resistant serotypes. The objective of this study was to characterize S. Dublin isolates from sick cattle by analyzing phenotypic and genotypic antimicrobial resistance (AMR) profiles, the presence of plasmids, and phylogenetic relationships. S. Dublin isolates (n = 140) were selected from submissions to the NVSL for Salmonella serotyping (2014–2017) from 21 states. Isolates were tested for susceptibility against 14 class-representative antimicrobial drugs. Resistance profiles were determined using the ABRicate with Resfinder and NCBI databases, AMRFinder and PointFinder. Plasmids were detected using ABRicate with PlasmidFinder. Phylogeny was determined using vSNP. We found 98% of the isolates were resistant to more than 4 antimicrobials. Only 1 isolate was pan-susceptible and had no predicted AMR genes. All S. Dublin isolates were susceptible to azithromycin and meropenem. They showed 96% resistance to sulfonamides, 97% to tetracyclines, 95% to aminoglycosides and 85% to beta-lactams. The most common AMR genes were: sulf2 and tetA (98.6%), aph(6)-Id (97.9%), aph(3’’)-Ib, (97.1%), floR (94.3%), and blaCMY-2 (85.7%). All quinolone resistant isolates presented mutations in gyrA. Ten plasmid types were identified among all isolates with IncA/C2, IncX1, and IncFII(S) being the most frequent. The S. Dublin isolates show low genomic genetic diversity. This study provided antimicrobial susceptibility and genomic insight into S. Dublin clinical isolates from cattle in the U.S. Further sequence analysis integrating food and human origin S. Dublin isolates may provide valuable insight on increased virulence observed in humans.

Assessment of beef carcass contamination with Salmonella and E. coli O 157 in slaughterhouses in Bishoftu, Ethiopia

BackgroundSalmonella and E. coli O157 are common causes of foodborne diseases. Evisceration and de-hiding steps can lead to carcass contamination during slaughter operation. In Ethiopia, information on the association between the presence of these pathogens in the rectal content and/or on the hide of cattle and their presence on the carcass is lacking.MethodsThe aim of this study was to assess the sources of beef carcass contamination with Salmonella and E. coli O157 during slaughter. Rectal contents and hide- and carcass-swabs (from three sites: foreleg, brisket and hind leg) were collected from 70 beef cattle at two small scale slaughterhouses. Isolates were genotyped by the Pulsed Field Gel Electrophoresis method and tested for resistance against 14 microbial drugs.ResultsSalmonella was detected at equal proportions (7.1%) in rectal content samples and hide swabs. E. coli O157 was detected in 8.6% of the rectal contents and 4.3% of the hide swabs. The proportion of contaminated carcasses was 8.6% for Salmonella and 7.1% for E. coli O157. Genetic linkage between the Salmonella and E. coli O157 isolates from the rectal contents and/or hides and carcasses were observed only in a few cases (2 and 1 carcasses, respectively) indicating the limited direct transfer of the pathogens from the feces and/or hide to the carcass during slaughter. Most carcasses became positive by cross contamination. All the S. Typhimurium isolates (n = 8) were multidrug resistant being resistant to ampicillin, chloramphenicol, sulfamethoxazole and tetracycline. The two S. Dublin isolates were resistant to colistin. All E. coli O157 isolates were susceptible to the antimicrobials tested.ConclusionThe results indicated that cross contamination may be an important source for carcass contamination.

Detection of Cephalosporin and Fluoroquinolone Resistance Genes via Novel Multiplex qPCR in Fecal Salmonella Isolates From Northern Californian Dairy Cattle, 2002-2016.

The objectives of this study were to evaluate the prevalence of extended spectrum β-lactamase (ESBL) genes, AmpC-type β-lactamase (ACBL) genes, and plasmid mediated quinolone resistance (PMQR) genes in Salmonella isolated at a Veterinary Medical Teaching Hospital microbiology laboratory, examine trends in presence of these resistance genes, and to explore the correlation between phenotypic resistance and presence of specific genes. The presence of ESBL, ACBL, and PMQR genes were detected using a single, novel multiplex qPCR. Only the genes blaCMY–2 and blaTEM were detected in the 110 Salmonella isolates tested. PMQR genes were not detected in isolates screened. Of 94 third-generation cephalosporin resistant isolates, representing eight serotypes, 48% (n = 45) were positive for blaCMY–2 only and 50% (n = 47) were simultaneously positive for blaCMY–2 and blaTEM. Two third-generation cephalosporin resistant isolates were tested negative for all β-lactamase genes in our qPCR assay and likely house ESBL genes not screened for by our qPCR assay. A logistic regression model revealed that for serotype Dublin isolates (n = 38) the odds ratio for testing positive for blaTEM when compared to all other serotypes was 51.6 (95% CI: 4.01–664.03, p = 0.0029). For serotype Typhimurium (n = 9) the odds ratio for testing positive for blaTEM when compared to all other serotypes was 43.3 (95% CI: 1.76–1000, p = 0.0216). Overall, our results suggest that the prevalence of resistance to cephalosporins and fluoroquinolones due to ESBLs, ACBLs, and PMQR genes present in bovine nontyphoidal Salmonella enterica isolates has remained relatively constant in the isolates screened over a 14-year period.

Genome Sequences of Multidrug-Resistant Salmonella enterica Serovar Dublin Isolates.

Salmonella enterica serovar Dublin, which can cause enteritis and systemic infections in humans, has been associated with antimicrobial resistance. Here, we report draft genome sequences of seven multidrug-resistant S Dublin isolates from human samples. These sequences will contribute to an understanding of pathogenesis and resistance determinants in this serovar.

Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods.

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed.

A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties.

Salmonella enterica serovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to prevent Salmonella dissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) of S Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory properties in vitro and in vivo The aim of this work was to elucidate the underlying cause of the absence of flagella in S Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in the fliE gene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only when fliA was artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences in fliE adjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected in S Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

The invasome of Salmonella Dublin as revealed by whole genome sequencing

BackgroundSalmonella enterica serovar Dublin is a zoonotic infection that can be transmitted from cattle to humans through consumption of contaminated milk and milk products. Outbreaks of human infections by S. Dublin have been reported in several countries including high-income countries. A high proportion of S. Dublin cases in humans are associated with invasive disease and systemic illness. The genetic basis of virulence in S. Dublin is not well characterized.MethodsWhole genome sequencing was applied to a set of clinical invasive and non-invasive S. Dublin isolates from different countries in order to characterize the putative genetic determinants involved in the virulence and invasiveness of S. Dublin in humans.ResultsWe identified several virulence factors that form the bacterial invasome and may contribute to increasing bacterial virulence and pathogenicity including mainly Gifsy-2 prophage, two different type 6 secretion systems (T6SSs) harbored by Salmonella pathogenicity islands; SPI-6 and SPI-19 respectively and virulence genes; ggt and PagN. Although Vi antigen and the virulence plasmid have been reported previously to contribute to the virulence of S. Dublin we did not detect them in all invasive isolates indicating that they are not the main virulence determinants in S. Dublin.ConclusionSeveral virulence factors within the genome of S. Dublin might contribute to the ability of S. Dublin to invade humans’ blood but there were no genomic markers that differentiate invasive from non-invasive isolates suggesting that host immune response play a crucial role in the clinical outcome of S. Dublin infection.

Whole-Genome Sequencing of Drug-Resistant Salmonella enterica Isolates from Dairy Cattle and Humans in New York and Washington States Reveals Source and Geographic Associations.

Multidrug-resistant (MDR) Salmonella enterica can be spread from cattle to humans through direct contact with animals shedding Salmonella as well as through the food chain, making MDR Salmonella a serious threat to human health. The objective of this study was to use whole-genome sequencing to compare antimicrobial-resistant (AMR) Salmonella enterica serovars Typhimurium, Newport, and Dublin isolated from dairy cattle and humans in Washington State and New York State at the genotypic and phenotypic levels. A total of 90 isolates were selected for the study (37 S Typhimurium, 32 S Newport, and 21 S Dublin isolates). All isolates were tested for phenotypic antibiotic resistance to 12 drugs using Kirby-Bauer disk diffusion. AMR genes were detected in the assembled genome of each isolate using nucleotide BLAST and ARG-ANNOT. Genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.2 and specificity of 85.2. Sulfamethoxazole-trimethoprim resistance was observed only in human isolates (P < 0.05), while resistance to quinolones and fluoroquinolones was observed only in 6 S Typhimurium isolates from humans in Washington State. S Newport isolates showed a high degree of AMR profile similarity, regardless of source. S Dublin isolates from New York State differed from those from Washington State based on the presence/absence of plasmid replicons, as well as phenotypic AMR susceptibility/nonsusceptibility (P < 0.05). The results of this study suggest that distinct factors may contribute to the emergence and dispersal of AMR S. enterica in humans and farm animals in different regions.IMPORTANCE The use of antibiotics in food-producing animals has been hypothesized to select for AMR Salmonella enterica and associated AMR determinants, which can be transferred to humans through different routes. Previous studies have sought to assess the degree to which AMR livestock- and human-associated Salmonella strains overlap, as well as the spatial distribution of Salmonella's associated AMR determinants, but have often been limited by the degree of resolution at which isolates can be compared. Here, a comparative genomics study of livestock- and human-associated Salmonella strains from different regions of the United States shows that while many AMR genes and phenotypes were confined to human isolates, overlaps between the resistomes of bovine and human-associated Salmonella isolates were observed on numerous occasions, particularly for S Newport. We have also shown that whole-genome sequencing can be used to reliably predict phenotypic resistance across Salmonella isolated from bovine sources.

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations.

Genetic and virulence variation in an environmental population of the opportunistic pathogen Aspergillus fumigatus

Environmental populations of the opportunistic pathogen Aspergillus fumigatus have been shown to be genotypically diverse and to contain a range of isolates with varying pathogenic potential. In this study, we combined two RAPD primers to investigate the genetic diversity of environmental isolates from Manchester collected monthly over 1 year alongside Dublin environmental isolates and clinical isolates from patients. RAPD analysis revealed a diverse genotype, but with three major clinical isolate clusters. When the pathogenicity of clinical and Dublin isolates was compared with a random selection of Manchester isolates in a Galleria mellonella larvae model, as a group, clinical isolates were significantly more pathogenic than environmental isolates. Moreover, when relative pathogenicity of individual isolates was compared, clinical isolates were the most pathogenic, Dublin isolates were the least pathogenic and Manchester isolates showed a range in pathogenicity. Overall, this suggests that the environmental population is genetically diverse, displaying a range in pathogenicity, and that the most pathogenic strains from the environment are selected during patient infection.

Repression of Flagella Is a Common Trait in Field Isolates of Salmonella enterica Serovar Dublin and Is Associated with Invasive Human Infections

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:-:-, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.

One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB, gyrB, and ycfQ. Simultaneous gene scanning of fljB, gyrB, and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S. Dublin isolates, an additional single PCR assay was developed for specific identification of S. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.

Characterisation of antibiotic resistance in host-adapted Salmonella enterica

Salmonella enterica serovars Dublin, Choleraesuis and Pullorum are host-adapted serovars that cause disease primarily in cattle, swine and poultry, respectively. In addition, serovars Dublin and Choleraesuis are important human pathogens that are disproportionately associated with severe invasive infections that require antimicrobial therapy. Because of the potential increased emergence and spread of antimicrobial resistance, isolates of 42 S. enterica serovars Dublin, Choleraesuis and Pullorum were characterised to evaluate resistance. Antimicrobial susceptibility testing, detection of resistance genes and integrons, pulsed-field gel electrophoresis and plasmid analysis were carried out to characterise the isolates. Seventy-nine percent of the isolates were resistant to at least one of the antimicrobial agents tested, whilst 38% of the isolates were resistant to six or more antimicrobial agents. Resistance was most commonly detected to tetracycline (64%), streptomycin (57%) and kanamycin (52%). Overall, when resistance was seen, a corresponding resistance gene was detected 86.7% of the time. The results of this study indicate that antimicrobial resistance is a major concern in serovars Dublin and Choleraesuis isolates owing to the presence of multidrug resistance.

Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle.

Salmonella enterica serovar Dublin is a host-restricted serovar associated with typhoidal disease in cattle. In contrast, the fowl-associated serovar S. enterica serovar Gallinarum is avirulent in calves, yet it invades ileal mucosa and induces enteritis at levels comparable to those induced by S. enterica serovar Dublin. Suppression subtractive hybridization was employed to identify S. enterica serovar Dublin strain SD3246 genes absent from S. enterica serovar Gallinarum strain SG9. Forty-one S. enterica serovar Dublin fragments were cloned and sequenced. Among these, 24 mobile-element-associated genes were identified, and 12 clones exhibited similarity with sequences of known or predicted function in other serovars. Three S. enterica serovar Dublin-specific regions were homologous to regions from the genome of Enterobacter sp. strain 638. Sequencing of fragments adjacent to these three sequences revealed the presence of a 21-kb genomic island, designated S. enterica serovar Dublin island 1 (SDI-1). PCR analysis and Southern blotting showed that SDI-1 is highly conserved within S. enterica serovar Dublin isolates but rarely found in other serovars. To probe the role of genes identified by subtractive hybridization in vivo, 24 signature-tagged S. enterica serovar Dublin SD3246 mutants lacking loci not present in Salmonella serovar Gallinarum SG9 were created and screened by oral challenge of cattle. Though attenuation of tagged SG9 and SD3246 Salmonella pathogenicity island-1 (SPI-1) and SPI-2 mutant strains was detected, no obvious defects of these 24 mutants were detected. Subsequently, a DeltaSDI-1 mutant was found to exhibit weak but significant attenuation compared with the parent strain in coinfection of calves. SDI-1 mutation did not impair invasion, intramacrophage survival, or virulence in mice, implying that SDI-1 does not influence fitness per se and may act in a host-specific manner.

Antimicrobial resistance pattern of Salmonella serotypes isolated from food items and personnel in Addis Ababa, Ethiopia

Antimicrobial susceptibility test of 98 isolates of Salmonella was assayed from September 2003 to February 2004 using the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).The result revealed that 32.7% of Salmonella isolates were resistant to one or more of the 24 antimicrobials tested. Generally resistance for 13 different antimicrobial drugs was recognized. The most common resistance was to streptomycin (24/32, 75%), ampicillin (19/32, 59.4%), tetracycline (15/32, 46.9%), spectinomycin (13/32, 40.6%) and sulfisoxazole (13/32, 40.6%). All the three Salmonella Kentucky isolates showed resistance to at least 8 antimicrobials. Out of the 12 Salmonella Braenderup isolates, 10 (83.3%) showed multidrug resistance to ampicillin, spectinomycin, streptomycin, sulfisoxazole, sulfamethoxazole/trimethoprim, amoxicillin/clavulanic acid and trimethoprim. Among the 8 S. Hadar isolates 7 (86.5%) showed antimicrobial resistance. All the 6 S. Dublin isolates were resistant to carbadox (100%). All the 6 S. Haifa isolates were resistant for at least ampicillin, streptomycin and tetracycline. Up to ten different antimicrobial resistances pattern was observed. Multiple antimicrobial drug resistance was observed in 23 Salmonella isolates (23.5%). The level of antimicrobial resistance was significantly higher for isolates from chicken carcass (18/29, 62.1%) and pork isolates (5/22, 22.7%) (p = 0.003). The findings of the present study ascertain that significant proportion Salmonella isolates have developed resistance for routinely prescribed antimicrobial drugs and poses considerable health hazards to the consumers unless prudent control measures are instituted.

Changes in antimicrobial susceptibility in a population of Salmonella enterica serovar Dublin isolated from cattle in Japan from 1976 to 2005

We investigated the antimicrobial susceptibilities and resistance mechanisms of cattle-adapted Salmonella enterica serovar Dublin isolated in Japan in the past 30 years. This study is an example of evaluation of the impact of introduction of antimicrobials in veterinary medical practice on the selection of resistance in S. enterica. The antimicrobial susceptibilities and prevalence of R-plasmids in Salmonella Dublin isolated in Japan from 1976 to 2005 were investigated. To evaluate the importance of gyrA mutation and active efflux, we derived the gyrA revertants and acrAB deletion mutants, and then compared with their parental strains the MICs of quinolone antimicrobials such as nalidixic acid, enrofloxacin, ofloxacin and ciprofloxacin. Salmonella Dublin isolates with R-plasmids and resistance to more than three antimicrobials were predominant between 1981 and 1995. From the latter half of the 1990s to the present, Salmonella Dublin isolates without R-plasmids became dominant. The introduction of nalidixic acid into the veterinary field in the mid-1980s was followed by the emergence of nalidixic acid-resistant isolates, which are now predominant. We found only a single gyrA mutation (Asp-87-->Tyr) among the nalidixic acid-resistant isolates. Although the reduced susceptibilities to the fluoroquinolones were observed among the nalidixic acid-resistant isolates, none of the isolates was resistant to the fluoroquinolones used in this study. The MIC data for the fluoroquinolones differed up to 4-fold. Results of the susceptibility test using gyrA revertants and acrAB mutants suggest that the isolates with the gyrA mutation were selected by the use of nalidixic acid, and the AcrAB-TolC system accounts for the decreased fluoroquinolone susceptibilities. These data suggest that the introduction of nalidixic acid in veterinary medicine seemed to affect the susceptibilities of Salmonella Dublin among the cattle population in Japan, whereas the introduction of enrofloxacin has not caused any additional effect. The prudent use of antimicrobials in the veterinary field should be continuously stressed.