BackgroundSalmonella enterica is a significant foodborne pathogen, which can be transmitted via several distinct routes, and reports on acquisition of antimicrobial resistance (AMR) are increasing. To better understand the association between human Salmonella clinical isolates and the potential environmental/animal reservoirs, whole genome sequencing (WGS) was used to investigate the epidemiology and AMR patterns within Salmonella isolates from two adjacent US states.ResultsWGS data of 200 S. enterica isolates recovered from human (n = 44), swine (n = 32), poultry (n = 22), and farm environment (n = 102) were used for in silico prediction of serovar, distribution of virulence genes, and phylogenetically clustered using core genome single nucleotide polymorphism (SNP) and feature frequency profiling (FFP). Furthermore, AMR was studied both by genotypic prediction using five curated AMR databases, and compared to phenotypic AMR using broth microdilution. Core genome SNP-based and FFP-based phylogenetic trees showed consistent clustering of isolates into the respective serovars, and suggested clustering of isolates based on the source of isolation. The overall correlation of phenotypic and genotypic AMR was 87.61% and 97.13% for sensitivity and specificity, respectively. AMR and virulence genes clustered with the Salmonella serovars, while there were also associations between the presence of virulence genes in both animal/environmental isolates and human clinical samples.ConclusionsWGS is a helpful tool for Salmonella phylogenetic analysis, AMR and virulence gene predictions. The clinical isolates clustered closely with animal and environmental isolates, suggesting that animals and environment are potential sources for dissemination of AMR and virulence genes between Salmonella serovars.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5137-4) contains supplementary material, which is available to authorized users.

The aim of this study was to characterize the plasmids carrying antimicrobial resistance (AMR) determinants in multiple Salmonella serotypes recovered from the commercial swine farm environment after manure application on land. Manure and soil samples were collected on day 0 before and after manure application on six farms in North Carolina, and sequential soil samples were recollected on days 7, 14, and 21 from the same plots. All environmental samples were processed for Salmonella, and their plasmid contents were further characterized. A total of 14 isolates including Salmonella enterica serotypes Johannesburg (n ϭ 2), Ohio (n ϭ 2), Rissen (n ϭ 1), Typhimurium var5Ϫ (n ϭ 5), Worthington (n ϭ 3), and 4,12:i:Ϫ (n ϭ 1), representing different farms, were selected for plasmid analysis. Antimicrobial susceptibility testing was done by broth microdilution against a panel of 14 antimicrobials on the 14 confirmed transconjugants after conjugation assays. The plasmids were isolated by modified alkaline lysis, and PCRs were performed on purified plasmid DNA to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis and to compare profiles and create phylogenetic trees. A class 1 integron with an ANT(2Љ)-Ia-aadA2 cassette was detected in the 50-kb IncN plasmids identified in S. Worthington isolates. We identified 100-kb and 90-kb IncI1 plasmids in S. Johannesburg and S. Rissen isolates carrying the bla CMY-2 and tet(A) genes, respectively. An identical 95-kb IncF plasmid was widely disseminated among the different serotypes and across different farms. Our study provides evidence on the importance of horizontal dissemination of resistance determinants through plasmids of multiple Salmonella serotypes distributed across commercial swine farms after manure application. IMPORTANCEThe horizontal gene transfer of antimicrobial resistance (AMR) determinants located on plasmids is considered to be the main reason for the rapid proliferation and spread of drug resistance. The deposition of manure generated in swine production systems into the environment is identified as a potential source of AMR dissemination. In this study, AMR gene-carrying plasmids were detected in multiple Salmonella serotypes across different commercial swine farms in North Carolina. The plasmid profiles were characterized based on Salmonella serotype donors and incompatibility (Inc) groups. We found that different Inc plasmids showed evidence of AMR gene transfer in multiple Salmonella serotypes. We detected an identical 95-kb plasmid that was widely distributed across swine farms in North Carolina. These conjugable resistance plasmids were able to persist on land after swine ma-

Land application of swine manure in commercial hog farms is an integral part of their waste management system which recycles the nutrients back to the soil. However, manure application can lead to the dissemination of bacterial pathogens in the environment and pose a serious public health threat. The aim of this study was to determine the dissemination of antimicrobial resistant Salmonella in the environment due to manure application in commercial swine farms in North Carolina (n = 6) and Iowa (n = 7), two leading pork producing states in the US. We collected manure and soil samples twice on day 0 (before and after manure application) from four distinct plots of lands (5 soil samples/plot) located at 20 feet away from each other in the field. Subsequent soil samples were collected again on days 7, 14, 21 from the same plots. A total of 1,300 soil samples (NC = 600; IA = 700) and 130 manure samples (NC = 60; IA = 70) were collected and analyzed in this study. The overall Salmonella prevalence was 13.22% (189/1,430), represented by 10.69% and 38.46% prevalence in soil and manure, respectively. The prevalence in NC (25.45%) was significantly higher than in IA (2.73%) (P<0.001) and a consistent decrease in Salmonella prevalence was detected from Day 0-Day 21 in all the farms that tested positive. Salmonella serotypes detected in NC were not detected in IA, thereby highlighting serotype association based on manure storage and soil application method used in the two regions. Antimicrobial susceptibility testing was done by the broth microdilution method to a panel of 15 antimicrobial drugs. A high frequency of isolates (58.73%) were multidrug resistant (resistance to three or more class of antimicrobials) and the most frequent resistance was detected against streptomycin (88.36%), sulfisoxazole (67.2%), and tetracycline (57.67%). Genotypic characterization by pulse field gel electrophoresis revealed clonally related Salmonella in both manure and soil at multiple time points in the positive farms. Our study highlights the potential role of swine manure application in the dissemination and persistence of antimicrobial resistant Salmonella in the environment.

Salmonella enterica serovar Rissen has been recognized as one of the most common serovar among humans and pork production systems in different parts of the world, especially Asia. In the United States, this serovar caused outbreaks but its epidemiologic significance remains unknown. The objectives of this study were to compare the phenotypic (antimicrobial susceptibility) and genotypic attributes of Salmonella Rissen isolated in Thailand (Thai) and the United States (US). All the Thai isolates (n = 30) were recovered from swine faecal samples. The US isolates (n = 35) were recovered from swine faecal samples (n = 29), cattle (n = 2), chicken (n = 2), dog (n = 1) and a ready-to-eat product (n = 1). The antimicrobial susceptibility of isolates was determined using the Kirby-Bauer disk diffusion method with a panel of 12 antimicrobials. Pulse-field gel electrophoresis (PFGE) was used to determine the genotypic diversity of isolates. All Thai isolates showed multidrug resistance (MDR) with the most frequent antibiotic resistance shown against ampicillin (100%), sulfisoxazole (96.7%), tetracycline (93.3%), streptomycin (90%) and chloramphenicol (30%). About half of the isolates of USA origin were pan-susceptible and roughly 30% were resistant to only tetracycline (R-type: Te). Salmonella Rissen isolated from Thailand and the USA in this study were found to be clonally unrelated. Genotypic analyses indicated that isolates were clustered primarily based on the geographic origin implying the limited clonality among the strains. Clonal relatedness among different host species within the same geography (USA) was found. We found genotypic similarity in Thai and US isolates in few instances but with no epidemiological link. Further studies to assess propensity for increased inter-regional transmission and dissemination is warranted.

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This study was conducted to analyze the prevalence and quantitative loads of Salmonella spp. on pig farms in Chiang Mai, Lamphun, Thailand to assess loading levels before slaughtering. The serotype diversity, antimicrobial-resistance pattern and pulse-field type of Salmonella spp. were also characterized to assess the dynamic propagation of the pathogen. The Salmonella-positive prevalence was 246/805 (30.56%), and the quantitative loads varied from 1.48~4.04 Log10MPN/g, with a mean ± standard deviation of 2.11 ± 0.57. AMP/S/TE (ampicillin/streptomycin/tetracycline) was the highest frequency antimicrobial resistance pattern found in this study. In addition, Salmonella Rissen was the primary serotype in this region. PFGE results indicated the occurrence of infection by cross contamination among pig farms. Our study showed that pork is easily contaminated with this pathogen. Farm control programs must be based on strict biosecurity and hygienic measures, which could further reduce the contamination pressure at slaughterhouses or retail shops.

The deposition of manure originating from food animal farms in the environment can lead to the dissemination of antimicrobial-resistant (AMR) bacterial foodborne pathogens, thereby potentially impacting human health. The objective of our study was to determine the dissemination of multidrug methicillin-resistant Staphylococcus sciuri (MDR-MRSS) in the environment after land application of manure on commercial swine farms. A total of 400 environmental samples (40 manure and 360 soil) were collected after repeated sampling from four commercial swine farms located in North Carolina (n = 1) and Iowa (n = 3) in the United States. At each farm, we collected 10 manure and 40 soil samples (20 samples before and after 2 h of manure application) from four plots (five soil samples/plot) on day 0. Subsequently, 20 soil samples were collected on day 7, 14, and 21 from the same plots. A total of 67 (16.75%) MRSS were isolated from the 400 samples. The prevalence in soil and manure was 13.33% (48/360) and 47.5% (19/40), respectively. Prevalence was highest in the soil samples collected after 2 h of manure application on day 0 and decreased subsequently on 7, 14, and 21 days. Antimicrobial susceptibility testing was done against a panel of 12 antibiotics. A majority of S. sciuri isolates exhibited resistance against ampicillin (AMP; 95.5%), penicillin (PEN; 95.5%), clindamycin (CLI; 95.5%), cefoxitin (FOX; 92.5%), ceftiofur (XNL; 92.5%), tetracycline (TET; 86.56%), and erythromycin (ERY; 50.74%). The MDR pattern AMP FOX CLI PEN TET XNL (n = 24; 35.8%) was the most commonly observed. We detected multiple AMR genes, including mecA, aac(6'), Ie-aph(2″)Ia, tetM, tetK, mphC, ermA, ermB, and ermC. Pulsed-field gel electrophoresis clustered isolates from different sample collection days from the same farm into one group. Overall, our study identifies swine manure as an important reservoir of MDR-MRSS and highlights its dissemination in the environment upon spreading of manure.

Acinetobacter is a bacteria found in the environment and clinical specimens, causing nosocomial infection and antimicrobial resistance (AMR) threats. This study examined the prevalence, species, and AMR characteristics of Acinetobacter isolated from surgical practice and the laboratory dog husbandry room environments (n = 235) at Rajamangala University of Technology Tawan-ok veterinary hospital during 2018-2019. The prevalence of Acinetobacter in the laboratory dog husbandry room and veterinary belongings were 2.55% and 0.43%, respectively. Species determination was Acinetobacter hemolyticus (2.1%) and Acinetobacter baumannii (0.4%) from environments in the laboratory dog husbandry room, and Acinetobacter junii (0.4%) from the shoes used in the surgical practice room. AMR was observed in both study environments and the specimens sent to the Veterinary Diagnostic Center. These isolates had a high resistant percentage to amoxicillin-clavulanic acid (84.62%), sulfamethoxazole-trimethoprim (61.54%), and cephalexin (53.85%) but were susceptible to imipenem. Compared to the isolates recovered from the clinical specimens, most isolates derived from environments exhibited multidrug resistance and shared correlated resistance patterns. These results highlight the need for sanitization in the dog husbandry room. Furthermore, the AMR results can be used as a preliminary baseline for studying AMR Acinetobacter contamination in animals and their environments.