Genome analysis of Salmonella enterica serovar enteritis strains isolated from poultry and humans in Burkina Faso.

Whole-genome sequencing (WGS) was used to characterize four Salmonella enterica Enteritidis isolates from poultry (n=2) and human (n=2) from Ouagadougou, Burkina Faso. Antimicrobial resistance genes, chromosomal mutations, and mobile genetic elements were identified by analysis of WGS data using sequence homology.

An analysis of culture-based methods used for the detection and isolation of Salmonella spp., Escherichia coli, and Enterococcus spp. from surface water: A systematic review.

Identification of methods for the standardized assessment of bacterial pathogens and antimicrobial resistance (AMR) in environmental water can improve the quality of monitoring and data collected, support global surveillance efforts, and enhance the understanding of environmental water sources. We conducted a systematic review to assemble and synthesize available literature that identified methods for assessment of prevalence and abundance of bacterial fecal indicators and pathogens in water for the purposes of monitoring bacterial pathogens and AMR. After screening for quality, 175 unique publications were identified from 15 databases, and data were extracted for analysis. This review identifies the most common and robust methods, and media used to isolate target organisms from surface water sources, summarizes methodological trends, and recognizes knowledge gaps. The information presented in this review will be useful when establishing standardized methods for monitoring bacterial pathogens and AMR in water in the United States and globally.

Infection biology of Salmonella enterica.

Salmonella enterica is the leading cause of bacterial foodborne illness in the USA, with an estimated 95% of salmonellosis cases due to the consumption of contaminated food products. Salmonella can cause several different disease syndromes, with the most common being gastroenteritis, followed by bacteremia and typhoid fever. Among the over 2,600 currently identified serotypes/serovars, some are mostly host-restricted and host-adapted, while the majority of serotypes can infect a broader range of host species and are associated with causing both livestock and human disease. Salmonella serotypes and strains within serovars can vary considerably in the severity of disease that may result from infection, with some serovars that are more highly associated with invasive disease in humans, while others predominantly cause mild gastroenteritis. These observed clinical differences may be caused by the genetic make-up and diversity of the serovars. Salmonella virulence systems are very complex containing several virulence-associated genes with different functions that contribute to its pathogenicity. The different clinical syndromes are associated with unique groups of virulence genes, and strains often differ in the array of virulence traits they display. On the chromosome, virulence genes are often clustered in regions known as Salmonella pathogenicity islands (SPIs), which are scattered throughout different Salmonella genomes and encode factors essential for adhesion, invasion, survival, and replication within the host. Plasmids can also carry various genes that contribute to Salmonella pathogenicity. For example, strains from several serovars associated with significant human disease, including Choleraesuis, Dublin, Enteritidis, Newport, and Typhimurium, can carry virulence plasmids with genes contributing to attachment, immune system evasion, and other roles. The goal of this comprehensive review is to provide key information on the Salmonella virulence, including the contributions of genes encoded in SPIs and plasmids during Salmonella pathogenesis.

Tulathromycin metaphylaxis increases nasopharyngeal isolation of multidrug resistant Mannheimia haemolytica in stocker heifers.

Bovine respiratory disease (BRD) is a leading cause of disease in feedlot and stocker calves with Mannheimia haemolytica (MH) as one of the most common etiologies. One of the most effective means of controlling BRD is through metaphylaxis, which involves administering antimicrobials to all animals at high risk of developing BRD. However, increasing prevalence of multidrug resistant (MDR) MH may reduce efficacy of metaphylaxis due to decreased susceptibility to drugs used for metaphylaxis. Primarily, this study aimed to determine the effect of tulathromycin metaphylaxis and subsequent BRD treatment on antimicrobial resistance (AMR) in MH isolated from stocker calves. Secondary objectives included evaluating the effect of metaphylaxis and treatment for BRD on animal health and comparing the genetic relationship of MH isolated. Crossbred beef heifers (n = 331, mean weight = 232, SD = 17.8 kg) at high risk for BRD were randomly assigned to receive tulathromycin metaphylaxis (META, n = 167) or not (NO META, n = 164). Nasopharyngeal swabs were collected for MH isolation, antimicrobial susceptibility testing and whole genome sequencing at arrival and 3 (WK3) and 10 (WK10) weeks later. Mixed-effects logistic regression was used to identify risk factors for isolation of MH and MDR MH (resistant to ≥3 antimicrobial drug classes) at 3 and 10 weeks, BRD morbidity, and crude mortality. Animals in the META group had higher odds of isolation of MDR MH at 3 weeks [OR (95% CI) = 13.08 (5-30.9), p < 0.0001] and 10 weeks [OR (95% CI) = 5.92 (1.34-26.14), p = 0.019] after arrival. There was no difference in risk of isolation of any MH (resistant or susceptible) between META and NO META groups at all timepoints. Animals in the NO META group had 3 times higher odds of being treated for BRD [WK3: OR (95% CI) = 3.07 (1.70-5.52), p = 0.0002; WK10: OR (95% CI) = 2.76 (1.59-4.80), p = 0.0002]. Antimicrobial resistance genes found within isolates were associated with integrative conjugative element (ICE) genes. Tulathromycin metaphylaxis increased risk of isolation of MDR MH and in this population, the increase in MDR MH appeared to be associated with ICE containing antimicrobial resistance genes for multiple antimicrobial classes. This may have important implications for future efficacy of antimicrobials for control and treatment of BRD.

Distribution of Antibiotic Resistance in a Mixed-Use Watershed and the Impact of Wastewater Treatment Plants on Antibiotic Resistance in Surface Water.

The aquatic environment has been recognized as a source of antibiotic resistance (AR) that factors into the One Health approach to combat AR. To provide much needed data on AR in the environment, a comprehensive survey of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues was conducted in a mixed-use watershed and wastewater treatment plants (WWTPs) within the watershed to evaluate these contaminants in surface water. A culture-based approach was used to determine prevalence and diversity of ARB in surface water. Low levels of AR Salmonella (9.6%) and Escherichia coli (6.5%) were detected, while all Enterococcus were resistant to at least one tested antibiotic. Fewer than 20% of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (17.3%) and carbapenem-resistant Enterobacteriaceae (CRE) (7.7%) were recovered. Six ARGs were detected using qPCR, primarily the erythromycin-resistance gene, ermB. Of the 26 antibiotics measured, almost all water samples (98.7%) had detectable levels of antibiotics. Analysis of wastewater samples from three WWTPs showed that WWTPs did not completely remove AR contaminants. ARGs and antibiotics were detected in all the WWTP effluent discharges, indicating that WWTPs are the source of AR contaminants in receiving water. However, no significant difference in ARGs and antibiotics between the upstream and downstream water suggests that there are other sources of AR contamination. The widespread occurrence and abundance of medically important antibiotics, bacteria resistant to antibiotics used for human and veterinary purposes, and the genes associated with resistance to these antibiotics, may potentially pose risks to the local populations exposed to these water sources.

A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

Polymerase chain reaction for the in vitro detection of the pESI plasmid associated with the globally circulating Salmonella Infantis outbreak strain.

A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.

Use of automated capillary immunoassays for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins.

The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In the present study, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations was also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. The chicken immunoglobin G (IgG) antibody response to the FliD protein from the immunized group was 1110- and 51,400-fold higher than that from the un-immunized chickens both two- and three-weeks post-vaccination, respectively. It was also observed that IgM antibody against the FliD protein from the immunized chickens was 1030-fold higher than that from the un-immunized chickens two weeks post-vaccination, but the IgM response declined to 120-fold between two groups from two weeks to three weeks after immunization. The IgM antibody response to the FimA protein from the immunized group was 1.84- and 1.12-fold higher than that from the un-immunized group, respectively, both two- and three-weeks post-vaccination, while the IgG antibody response from the immunized group was 8.07- and 27.6-fold higher than that from the un-immunized group, respectively, during the same period. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

Whole-genome sequencing of Listeria innocua recovered from retail milk and dairy products in Egypt.

The similarity of the Listeria innocua genome with Listeria monocytogenes and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an in-depth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five L. innocua isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, fosX, in the L. innocua isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Listeria Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of L. innocua. The five isolates possessed an ATP-dependent protease (clpL) gene, which mediates heat resistance, on a rep25 type plasmids. Blast analysis of clpL-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of L. monocytogenes strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to L. monocytogenes that was responsible for a serious outbreak, this is the first report of L. innocua containing clpL-carrying plasmids. Various genetic mechanisms of virulence transfer among Listeria species and other genera could raise the possibility of the evolution of virulent strains of L. innocua. Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures.

Buffered Peptone Water Formulation Does Not Influence Growth of pESI-positive Salmonella enterica Serovar Infantis.

Salmonella enterica is a major cause of human foodborne illness and is often attributed to poultry food sources. S. enterica serovar Infantis, specifically those carrying the pESI plasmid, has become a frequently isolated serotype from poultry meat samples at processing and has caused numerous recent human infections. In 2016, the USDA-Food Safety and Inspection Service changed the official sampling method for raw poultry products from BPW to using neutralizing BPW (nBPW) as the rinsing agent in order to prevent residual antimicrobial effects from acidifying and oxidizing processing aids. This change was contemporaneous to the emergence of pESI-positive ser. Infantis as a prevalent serovar in poultry, prompting some to question if nBPW could be selecting for this prevalent serovar. We performed two experiments: a comparison of ser. Infantis growth in BPW versus nBPW, and a simulation of regulatory sampling methods. We found that when inoculated into both broths, ser. Infantis initially grows slightly slower in nBPW than in BPW but little difference was seen in abundance after 6 h of growth. Additionally, the use of nBPW to simulate poultry rinse sample and overnight cold shipping to a regulatory lab did not affect the survival or subsequent growth of ser. Infantis in BPW. We concluded that the change in USDA-FSIS methodology to include nBPW in sampling procedures has likely not affected the emergence of S. ser. Infantis as a prevalent serovar in chicken and turkey meat product samples.

Freshwater environment as a reservoir of extended-spectrum ß-lactamase-producing Enterobacteriaceae.

Surface water receives large quantities of wastes from human and animal sources, thus providing an ideal setting for the accumulation, development, and dissemination of antibiotic resistant bacteria, including extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. The rapid spread of ESBL-producing Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, is a growing threat to public health, and there have been increasing reports on the prevalence and abundance of ESBL-producing Enterobacteriaceae in aquatic environments all over the globe. The objective of this review is to understand the extent of ESBL-producing Enterobacteriaceae contamination in aquatic environments and to enhance our knowledge on the role of the freshwater environment as a reservoir and transmission routes for these bacteria. In this review, we present the prevalence and distribution of ESBL-producing Enterobacteriaceae and their ESBL genes in the freshwater environment, potential sources of these bacteria in the aquatic environment, as well as their potential drivers in the environment, including anthropogenic and environmental factors.

Increased Prevalence of Salmonella Infantis Isolated from Raw Chicken and Turkey Products in the United States Is Due to a Single Clonal Lineage Carrying the pESI Plasmid.

Infantis has recently become one of the most common serotypes of Salmonella isolated in the U.S. from raw meat samples collected in processing facilities and in retail stores. Investigations have determined that the majority of these isolates contain the pESI plasmid, but there has not been a large-scale investigation of the chromosome of these isolates. Here, we investigated 3276 whole-genome sequences of Salmonella Infantis with and without the pESI plasmid to understand chromosomal differences between plasmid carriage groups. S. Infantis genomes arranged into multiple clades with a single clade containing the isolates carrying the plasmid. Fifty-eight SNPs were identified in complete linkage disequilibrium between isolates that did and did not carry the plasmid. However, there were no unique genes present only in the genomes of isolates containing the plasmid. On average, isolates with the plasmid did contain more insertion sequences than those without (p < 0.05). Given that S. Infantis isolates carrying pESI form a single clade, it can be inferred that the increase in carriage of this plasmid in the U.S. is due to rapid clonal expansion of a single strain rather than as a result of multiple transfer events. As this S. Infantis clone does not contain any unique chromosomal genes, its proliferation appears to be due to pESI plasmid-encoded genes that may be advantageous in the chickens and turkeys or in their environment.

Non-point source fecal contamination from aging wastewater infrastructure is a primary driver of antibiotic resistance in surface waters.

Antibiotic resistance is a global threat to human health. Many surface water resources are environmental hotspots of antibiotic resistant gene (ARG) transfer, with agricultural runoff and human waste highlighted as common sources of ARGs to aquatic systems. Here we quantified fecal marker genes and ARGs in 992 stream water samples collected seasonally during a 5-year period from 115 sites across the Upper Oconee watershed (Georgia, USA), an area characterized by gradients of agricultural and urban development. Widespread fecal contamination was found from humans (48% of samples), ruminants (55%), and poultry (19%), and 73% of samples tested positive for at least one of the six targeted ARGs (ermB, tet(B), blaCTX-M-1, blaKPC, blaSHV, and qnrS). While ARGs were strongly correlated with human fecal markers, many highly contaminated samples were not associated with sewage outfalls, an expected source of fecal and ARG pollution. To determine sources of contamination, we synthesized ARG and fecal marker data with geospatial data on land use/land cover and wastewater infrastructure across the watershed. This novel analysis found strong correlations between ARGs and measures of sewer density, sewer length, and septic system age within sample watersheds, indicating non-point sources of fecal contamination from aging wastewater infrastructure can be critical disseminators of anthropogenic ARGs in the environment.

Distribution and Transfer of Plasmid Replicon Families among Multidrug-Resistant Enterococcus faecalis and Enterococcus faecium from Poultry.

The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to the dissemination of antimicrobial resistance. However, the prevalence of plasmids from commensal bacteria, such as the enterococci, in food animals remains largely unknown. In this study, the diversity and prevalence of plasmid families from multidrug-resistant (MDR; resistance to three or more antimicrobials) enterococci from poultry carcasses were determined. Plasmid-positive MDR enterococci were also tested for the ability to transfer plasmids to other enterococci using conjugation. MDR Enterococcus faecalis (n = 98) and Enterococcus faecium (n = 696) that were isolated from poultry carcass rinsates between 2004 and 2011 were tested for the presence of 21 plasmid replicon (rep) families using multiplex PCR. Approximately 48% of E. faecalis (47/98) and 16% of E. faecium (110/696) were positive for at least one rep-family. Fourteen rep-families were detected overall, and ten rep-families were shared between E. faecalis and E. faecium. The rep7 and rep17 families were unique to E. faecalis, while the rep5 and rep8 families were unique to E. faecium. The rep9 family was predominant in both E. faecalis and E. faecium for all the years tested. The greatest number of rep-families detected was in 2005 (n = 10), and the least was in 2009 (n = 1). Eight rep-families were transferred from E. faecalis donors to the E. faecalis JH2-2 recipient using conjugation. Results from this study showed that E. faecalis and E. faecium from poultry carcasses contain numerous and diverse rep-families that are capable of conjugal transfer.

Resistance Genes, Plasmids, Multilocus Sequence Typing (MLST), and Phenotypic Resistance of Non-Typhoidal Salmonella (NTS) Isolated from Slaughtered Chickens in Burkina Faso.

The emergence of antimicrobial-resistant bacteria in developing countries increases risks to the health of both such countries' residents and the global community due to international travel. It is consequently necessary to investigate antimicrobial-resistant pathogens in countries such as Burkina Faso, where surveillance data are not available. To study the epidemiology of antibiotic resistance in Salmonella, 102 Salmonella strains isolated from slaughtered chickens were subjected to whole-genome sequencing (WGS) to obtain information on antimicrobial resistance (AMR) genes and other genetic factors. Twenty-two different serotypes were identified using WGS, the most prevalent of which were Hato (28/102, 27.5%) and Derby (23/102, 22.5%). All strains analyzed possessed at least one and up to nine AMR genes, with the most prevalent being the non-functional aac(6')-Iaa gene, followed by aph(6)-Id. Multi-drug resistance was found genotypically in 36.2% of the isolates for different classes of antibiotics, such as fosfomycin and ß-lactams, among others. Plasmids were identified in 43.1% of isolates (44/102), and 25 plasmids were confirmed to carry AMR genes. The results show that chicken can be considered as a reservoir of antibiotic-resistant Salmonella strains. Due to the prevalence of these drug-resistant pathogens and the potential for foodborne illnesses, poultry processing and cooking should be performed with attention to prescribed safe handling methods to avoid cross-contamination with chicken products.

Analysis of Salmonella enterica Isolated from a Mixed-Use Watershed in Georgia, USA: Antimicrobial Resistance, Serotype Diversity, and Genetic Relatedness to Human Isolates.

As the cases of Salmonella enterica infections associated with contaminated water are increasing, this study was conducted to address the role of surface water as a reservoir of S. enterica serotypes. We sampled rivers and streams (n = 688) over a 3-year period (2015 to 2017) in a mixed-use watershed in Georgia, USA, and 70.2% of the total stream samples tested positive for Salmonella. A total of 1,190 isolates were recovered and characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). A wide range of serotypes was identified, including those commonly associated with humans and animals, with S. enterica serotype Muenchen being predominant (22.7%) and each serotype exhibiting a high degree of strain diversity by PFGE. About half (46.1%) of the isolates had PFGE patterns indistinguishable from those of human clinical isolates in the CDC PulseNet database. A total of 52 isolates (4.4%) were resistant to antimicrobials, out of which 43 isolates were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). These 52 resistant Salmonella isolates were screened for the presence of antimicrobial resistance genes, plasmid replicons, and class 1 integrons, out of which four representative MDR isolates were selected for whole-genome sequencing analysis. The results showed that 28 MDR isolates resistant to 10 antimicrobials had blacmy-2 on an A/C plasmid. Persistent contamination of surface water with a high diversity of Salmonella strains, some of which are drug resistant and genetically indistinguishable from human isolates, supports a role of environmental surface water as a reservoir for and transmission route of this pathogen. IMPORTANCE Salmonella has been traditionally considered a foodborne pathogen, as it is one of the most common etiologies of foodborne illnesses worldwide; however, recent Salmonella outbreaks attributed to fresh produce and water suggest a potential environmental source of Salmonella that causes some human illnesses. Here, we investigated the prevalence, diversity, and antimicrobial resistance of Salmonella isolated from a mixed-use watershed in Georgia, USA, in order to enhance the overall understanding of waterborne Salmonella. The persistence and widespread distribution of Salmonella in surface water confirm environmental sources of the pathogen. A high proportion of waterborne Salmonella with clinically significant serotypes and genetic similarity to strains of human origin supports the role of environmental water as a significant reservoir of Salmonella and indicates a potential waterborne transmission of Salmonella to humans. The presence of antimicrobial-resistant and MDR Salmonella demonstrates additional risks associated with exposure to contaminated environmental water.

Genomic Comparison of Conjugative Plasmids from Salmonella enterica and Escherichia coli Encoding Beta-Lactamases and Capable of Mobilizing Kanamycin Resistance Col-like Plasmids.

Salmonella enterica and Escherichia coli are important human pathogens that frequently contain plasmids, both large and small, carrying antibiotic resistance genes. Large conjugative plasmids are known to mobilize small Col plasmids, but less is known about the specificity of mobilization. In the current study, six S. enterica and four E. coli strains containing large plasmids were tested for their ability to mobilize three different kanamycin resistance Col plasmids (KanR plasmids). Large conjugative plasmids from five isolates, four S. enterica and one E. coli, were able to mobilize KanR plasmids of various types. Plasmids capable of mobilizing the KanR plasmids were either IncI1 or IncX, while IncI1 and IncX plasmids with no evidence of conjugation had disrupted transfer regions. Conjugative plasmids of similar types mobilized similar KanR plasmids, but not all conjugative plasmid types were capable of mobilizing all of the KanR plasmids. These data describe some of the complexities and specificities of individual small plasmid mobilization.

Emergence of Multidrug-Resistant Escherichia coli Producing CTX-M, MCR-1, and FosA in Retail Food From Egypt.

In this study, multidrug-resistant (MDR) Escherichia coli isolates from retail food and humans assigned into similar Multilocus Sequence Types (MLST) were analyzed using whole genome sequencing (WGS). In silico analysis of assembled sequences revealed the existence of multiple resistance genes among the examined E. coli isolates. Of the six CTX-M-producing isolates from retail food, blaCTX-M-14 was the prevalent variant identified (83.3%, 5/6). Two plasmid-mediated fosfomycin resistance genes, fosA3, and fosA4, were detected from retail food isolates (one each from chicken and beef), where fosA4 was identified in the chicken isolate 82CH that also carried the colistin resistance gene mcr-1. The blaCTX-M-14 and fosA genes in retail food isolates were located adjacent to insertion sequences ISEcp1 and IS26, respectively. Sequence analysis of the reconstructed mcr-1 plasmid (p82CH) showed 96-97% identity to mcr-1-carrying IncI2 plasmids previously identified in human and food E. coli isolates from Egypt. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of chicken isolate 82CH, co-harboring mcr-1 and fosA4 genes, with a chicken E. coli isolate from China at the HC200 level (≤200 core genome allelic differences). As E. coli co-harboring mcr-1 and fosA4 genes has only been recently reported, this study shows rapid spread of this genotype that shares similar genetic structures with regional and international E. coli lineages originating from both humans and food animals. Adopting WGS-based surveillance system is warranted to facilitate monitoring the international spread of MDR pathogens.

Diversity of Plasmids and Genes Encoding Resistance to Extended-Spectrum ß-Lactamase in Escherichia coli from Different Animal Sources.

Antimicrobial resistance associated with the spread of plasmid-encoded extended-spectrum ß-lactamase (ESBL) genes conferring resistance to third generation cephalosporins is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. The purpose of this study was to investigate potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing (WGS). Three hundred isolates were typed into different phylogroups, characterized by BOX AIR-1 PCR and tested for resistance to antimicrobials. Of the 300 isolates, 59.7% were resistant to sulfisoxazole, 49.3% to tetracycline, 32.3% to cephalothin, 22.3% to ampicillin, 20% to streptomycin, 16% to ticarcillin; resistance to the remaining 12 antimicrobials was less than 10%. Phylogroups A and B1 were most prevalent with A (n = 92, 30%) and B1 (87 = 29%). A total of nine E. coli isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant (MDR) to at least three antimicrobial drug classes. Using WGS, significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli. These isolates carry plasmids, such as IncFIA(B), IncFII, IncX1, IncX4, IncQ1, CollRNAI, Col440I, and acquired ARGs aph(6)-Id, aph(3″)-Ib, aadA5, aph(3')-Ia, blaCTX-M-15, blaTEM-1B, mphA, ermB, catA1, sul1, sul2, tetB, dfrA17. One of the E. coli isolates from swine with ST 410 was resistant to nine antibiotics and carried more than 28 virulence factors, and this ST has been shown to belong to an international high-risk clone. Our data suggests that ESBL producing E. coli are widely distributed in different animal sources, but swine and dairy cattle may be their main reservoir.

AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence.

Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.