Molecular Characterization of Salmonella spp. from Cattle and Chicken Farms in Uganda

ObjectiveDetermine the AMR phenotypes and genotypes of Salmonella isolates recovered from cattle and poultry farms in the Wakiso District of Uganda.IntroductionAntimicrobial resistance (AMR) is a major concern in developing countries. Uganda is one of many developing countries that are beginning to implement a surveillance program countrywide to monitor AMR within the animal, environmental, and human sectors. Not only is there a concern for AMR, but the emergence of multidrug resistance (MDR) of Salmonella is also becoming a major One Health problem. Few new drugs are being produced. When current treatments fail, new antimicrobials for treatment of these microorganisms are limited (5). In Salmonella, AMR genes are usually found on plasmids that are transferable. Most plasmids that carry resistance are conjugative plasmids, promoting the transfer of DNA from cell to cell (1). Class I Integrons are located on transposable plasmids and are known to transfer AMR through an assortment of gene cassettes (3). Extended-spectrum β-lactamases (ESBLs) are also known to encode genes located on integrons and transposons (2). ESBLs confer resistance to third generation cephalosporins, a drug of choice for treatment of Salmonella infections. ESBLs are now reported in Enterobacteriaceae all over the world. Examples of common ESBLs include blaCTX-M, blaOXA, blaTEM, blaCMY, and blaSHV (2). It has been reported that ESBLs evolved from the Kluverya species chromosome by mutation and gene transposition (4).In our previous study, we phenotypically characterized Salmonella from cattle and poultry farms within the Wakiso District of Uganda. Based on the high prevalence of MDR in the isolates collected we continued investigating at the molecular level. For the Salmonella isolates, we wanted to characterize genotypes by first analyzing the relatedness of the isolates with pulse field gel electrophoresis (PFGE). Next, we wanted to look to see which DNA plasmids were present. We looked at 28 replicon plasmids and the Class 1 Integron, Int1. The Salmonella isolates were also screened for ESBL genes based on their resistant profiles.MethodsFecal and environmental samples from cattle and poultry farms were cultured using standard laboratory methods. AMR profiles were identified among all poultry and cattle Salmonella using the SensitireTM system per manufacturer’s directions. Fifty-six Salmonella isolates were screened for 28 replicon type plasmids, ESBL genes, and Class I integrons by PCR. The 56 isolates were subjected to PFGE to determine relatedness.ResultsSalmonella was recovered from 51/379 (13.5%) and 5/400 (1.3%) of poultry and cattle samples, respectively. Salmonella Enteritidis 16/51 (31.7%) and Kentucky 11/51 (21.6%) were most often recovered on poultry farms. Salmonella was most often resistant to Tetracycline and Sulfisoxazole. All Salmonella Kentucky isolates were resistant to Ciprofloxacin. Five replicon plasmids were identified among all poultry and cattle Salmonella: IncFIIS 18/56 (32.1%), IncI1α 12/56 (21.4%), IncP 8/56 (14.3%), IncX1 8/56 (14.3%), and IncX2 1/56 (1.8%). The Class I integron, Int1, was positive in one poultry isolate presenting MDR. PFGE cluster analysis of the 56 isolates showed 17 distinctive cluster types and displayed distinct clusters by replicon types IncP, IncX, IncFIIS, and IncI1α. No isolates displayed the ESBL genes that were screened.ConclusionsIn conclusion, we observed some degree of association between the AMR and plasmids. These plasmids also have an association with the PFGE cluster types and the Salmonella serotypes presented in this study. These Salmonella serotypes may be harboring these particular plasmids which confer resistance to select antimicrobials. Future work with these isolates will include whole genome sequence screening to detect differences between AMR phenotypes and genotypes.References1. Bennett, P. M. (2008). Plasmid-encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol, 153 Suppl 1, S347-357. doi:10.1038/sj.bjp.07076072. Bradford, P. A. (2001). Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev, 14(4), 933-951, table of contents. doi:10.1128/cmr.14.4.933-951.20013. Fluit, A. C., & Schmitz, F. J. (2004). Resistance integrons and super-integrons. Clin Microbiol Infect, 10(4), 272-288. doi:10.1111/j.1198-743X.2004.00858.x4. Humeniuk, C., Arlet, G., Gautier, V., Grimont, P., Labia, R., & Philippon, A. (2002). Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types. Antimicrob Agents Chemother, 46(9), 3045-3049.5. Ling, L. L., Schneider, T., Peoples, A. J., Spoering, A. L., Engels, I., Conlon, B. P., Lewis, K. (2015). A new antibiotic kills pathogens without detectable resistance. Nature, 517(7535), 455-459. doi:10.1038/nature14098

Persistence of fluoroquinolone-resistant Salmonella enterica serovar Kentucky from poultry and poultry sources in Nigeria

Introduction: This study investigated the antimicrobial resistance and clonality of Salmonella enterica serotype Kentucky in poultry and poultry sources in Nigeria, and compared the isolates with the clone of S. Kentucky STI98-X1 CIPR using (PFGE) and (MIC). Methodology: Fecal samples from chickens and poultry sources (litter, water, rodent and lizard fecal samples) were collected from fourteen (14) poultry farms in 2007, 2010 and 2011 and were analyzed for S. Kentucky. Results and conclusions: Six percent of the samples were positive for S. Kentucky – all resistant to nalidixic acid and ciprofloxacin. The isolates are grouped within the PFGE cluster X1 of S. Kentucky STI98 CIPR, indicating the association to the emerging and widely spread CIPR S. Kentucky clone with poultry and poultry sources.

Pork Contaminated with Salmonellaenterica Serovar 4,[5],12:i:−, an Emerging Health Risk for Humans

ABSTRACT Salmonella enterica subsp. enterica serovar 4,[5],12:i:− is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes bla TEM1-like, strA-str B, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:− and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:− strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.

Variable Number of Tandem Repeats and Pulsed-Field Gel Electrophoresis Cluster Analysis of Enterohemorrhagic Escherichia coli Serovar O157 Strains

Ninety-five enterohemorrhagic Escherichia coli serovar O157 strains, including 30 strains isolated from 13 intrafamily outbreaks and 14 strains isolated from 3 mass outbreaks, were studied by pulsed-field gel electrophoresis (PFGE) and variable number of tandem repeats (VNTR) typing, and the resulting data were subjected to cluster analysis. Cluster analysis of the VNTR typing data revealed that 57 (60.0%) of 95 strains, including all epidemiologically linked strains, formed clusters with at least 95% similarity. Cluster analysis of the PFGE patterns revealed that 67 (70.5%) of 95 strains, including all but 1 of the epidemiologically linked strains, formed clusters with 90% similarity. The number of epidemiologically unlinked strains forming clusters was significantly less by VNTR cluster analysis than by PFGE cluster analysis. The congruence value between PFGE and VNTR cluster analysis was low and did not show an obvious correlation. With two-step cluster analysis, the number of clustered epidemiologically unlinked strains by PFGE cluster analysis that were divided by subsequent VNTR cluster analysis was significantly higher than the number by VNTR cluster analysis that were divided by subsequent PFGE cluster analysis. These results indicate that VNTR cluster analysis is more efficient than PFGE cluster analysis as an epidemiological tool to trace the transmission of enterohemorrhagic E. coli O157.