ABSTRACT
We sequenced the genomes of 10 Salmonella enterica serovar Infantis isolates containing blaCTX-M-65 obtained from chicken, cattle, and human sources collected between 2012 and 2015 in the United States through routine National Antimicrobial Resistance Monitoring System (NARMS) surveillance and product sampling programs. We also completely assembled the plasmids from four of the isolates. All isolates had a D87Y mutation in the gyrA gene and harbored between 7 and 10 resistance genes [aph(4)-Ia, aac(3)-IVa, aph(3′)-Ic, blaCTX-M-65, fosA3, floR, dfrA14, sul1, tetA, aadA1] located in two distinct sites of a megaplasmid (∼316 to 323 kb) similar to that described in a blaCTX-M-65-positive S. Infantis isolate from a patient in Italy. High-quality single nucleotide polymorphism (hqSNP) analysis revealed that all U.S. isolates were closely related, separated by only 1 to 38 pairwise high-quality SNPs, indicating a high likelihood that strains from humans, chickens, and cattle recently evolved from a common ancestor. The U.S. isolates were genetically similar to the blaCTX-M-65-positive S. Infantis isolate from Italy, with a separation of 34 to 47 SNPs. This is the first report of the blaCTX-M-65 gene and the pESI (plasmid for emerging S. Infantis)-like megaplasmid from S. Infantis in the United States, and it illustrates the importance of applying a global One Health human and animal perspective to combat antimicrobial resistance.
INTRODUCTION
Salmonella enterica serovar Infantis (S. Infantis) is a serovar found in multiple animal hosts and animal food products. Many countries have reported increasing incidence of S. Infantis infections, and the World Health Organization (WHO) states that S. Infantis is among the top 15 Salmonella serovars reported from all regions (1). Cases of S. Infantis have been on the rise in the United States: the incidence of S. Infantis infections reported through the Foodborne Disease Active Surveillance Network in 2014 was significantly higher than in 2006 to 2008 (2). Information on sources of S. Infantis is limited. There have been a small number of case-control studies to investigate risk factors for S. Infantis infections (3), and none have been conducted in the United States. Based on available information, the reservoir for S. Infantis in other countries is thought to be food animals, particularly poultry (4, 5). In the United States, S. Infantis is commonly isolated from regulatory samples collected by the United States Department of Agriculture's Food Safety Inspection Service (USDA-FSIS) during chicken and cattle production and has been found in the cecal content of pigs (6).
The increasing incidence of S. Infantis infections may be complicated by the development of resistance to medically important antimicrobials, including penicillins and extended-spectrum cephalosporins. Organisms harboring extended-spectrum β-lactamases (ESBLs) exhibit resistance to most β-lactam antimicrobials, including penicillins, expanded-spectrum cephalosporins, and monobactams, but not cephamycins or carbapenems. Although antimicrobial treatment is not generally recommended for S. Infantis infections, it is recommended for certain vulnerable hosts or for invasive infections (7). Infections with ESBL-producing organisms are particularly concerning because they are not only resistant to most of the β-lactam antimicrobials but also are commonly resistant to additional classes of antimicrobials (8), leaving few treatment options and the potential for worse clinical outcomes. Of the ESBL enzymes, the CTX-M family is the most widely reported in the world, particularly in areas of Europe and Asia (9).
The emergence of ESBL-producing S. Infantis has been reported in other areas of the world, including Italy and Israel, and this has been connected with the presence of a unique pESI (plasmid for emerging S. Infantis) or pESI-like megaplasmid that enhances the fitness of the bacterium (8, 10). Until recently, very few Salmonella isolates with ESBL enzymes or genes had been found in the United States.
Here we investigate the recent emergence of the ESBL gene blaCTX-M-65 in S. Infantis isolates from food animals, retail chickens, and humans in the United States (A. C. Brown, J. C. Chen, D. Campbell, J. P. Folster, H. Tate, C. V. Tubbergen, and C. R. Friedman, submitted for publication). Until recently, the blaCTX-M-65 gene had only been described in a single Escherichia coli isolate from a patient in the United States (11). We used whole-genome sequencing (WGS) to characterize the plasmids from select strains used in this study and to determine the similarity, if any, to the S. Infantis pESI-like megaplasmid previously described by Aviv et al. and Franco et al. (8, 10). We also examined the relatedness of food and human isolates from the United States.
RESULTS
Antimicrobial susceptibility.Using CLSI interpretive criteria, we found that 9 of the 10 blaCTX-M-65-positive isolates were resistant to all of the following antimicrobials: ampicillin (Amp), chloramphenicol (Chl), sulfisoxazole (Fis), tetracycline (Tet), ceftriaxone (Cro), ceftiofur (Tio), nalidixic acid (Nal), and trimethoprim-sulfamethoxazole (Sxt) (Table 1). Resistance to other antimicrobials varied; only 4 of the 10 isolates expressed resistance to an aminoglycoside (gentamicin [Gen] or streptomycin [Str]). Nine of the isolates showed intermediate susceptibility to Gen, and all isolates had decreased susceptibility to ciprofloxacin (Cip) (data not shown). All 10 isolates were further tested for the presence of an ESBL using CLSI guidelines. All isolates expressed a ≥3 2-fold concentration decrease in the cefotaxime (Ctx) and ceftazidime (Caz) MICs when tested in combination with clavulanate versus the MICs of cefotaxime and ceftazidime when tested alone (data not shown). These isolates were also resistant to aztreonam and cefotaxime. None of the isolates were resistant to imipenem (Ipm).
Resistance phenotypes and genotypes of selected blaCTX-M-65-positive S. Infantis isolates from humans, retail chicken, and food animals
For the most part, resistance genotypes corresponded with antimicrobial susceptibility test results (Table 1). All Amp-, Chl-, Fis-, Tet-, Cro-, Tio-, Nal-, and Sxt-resistant isolates contained blaCTX-M-65 (which confers resistance to Amp, Cro, and Tio), floR (which confers resistance to Chl), sul1 (which confers resistance to Fis), dfrA14 (which confers resistance to Sxt), and tetA (which confers resistance to Tet). Isolates resistant to gentamicin harbored aac(3)-IVa genes; resistance to streptomycin was associated with the presence of aadA1. The retail chicken isolate contained sul1, aac(3)-IVa, and aph(4)-la genes but was susceptible to sulfisoxazole and displayed intermediate resistance to gentamicin using traditional susceptibility testing methods. Nalidixic acid-resistant strains exhibited a D87Y mutation in the gyrA gene; no parC mutations were found. Sequencing results also revealed that 7 out of the 10 blaCTX-M-65-positive isolates contained the fosA3 gene, which likely confers resistance to fosfomycin, and eight of the 10 isolates contained aph(3′)-lc, which likely confers resistance to kanamycin. MICs were not determined for these two antimicrobials.
Description of plasmids.Conjugation experiments demonstrated that all resistances except for quinolones could be transferred to the E. coli recipient cells (data not shown). We also sequenced the plasmids from four of the S. Infantis isolates and found that they had an average coverage of between 149× and 438×. BLAST analysis of the complete sequences confirmed the presence of an IncFIB-like incompatibility group replicon (Table 2). The plasmids were large, ranging from 316 to 323 kb (Table 2). All four plasmids shared 99% sequence identity (containing between 429 and 446 genes) with the plasmid backbone of the strain from the patient in Italy. The shared backbone regions consisted of 309 syntenic genes with similar codon usage that encoded functions such as plasmid replication/maintenance and membrane transport function (tra genes). The high degree of similarity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor.
Comparison of blaCTX-M-65-positive plasmids from U.S. and Italian isolates
Notably on the backbone of the Italian strain is a gene (mer) conferring resistance to mercury, a hallmark of the transposon Tn21 family and common on plasmids throughout the Enterobacteriaceae family. Our isolates also carried the mer genes on the plasmid. All other antimicrobial resistance determinants were integrated at two sites (site 1 and site 2) harboring laterally acquired DNA, as shown by the deviating nucleotide compositions of these regions (Fig. 1). Site 1, located approximately 160 kb from the origin of replication, contained seven antimicrobial resistance genes, including blaCTX-M-65, fosA3, floR, aph(4)-la, aac(3)-IV, dfrA14, and aph(3′)-lc. Site 2 contained three antimicrobial resistance genes (aadA1, sul1, and tetA), but aadA1 was absent from the retail isolate. All seven antimicrobial resistance genes in site 1 were present in the plasmids from animal isolates collected at slaughter (both the chicken cecal content sample and comminuted chicken product), but fosA3 was not in the plasmids in retail chicken or human isolates. The aph(3′)-lc gene was also absent in the plasmid from the human isolate (Table 2). All resistance genes were surrounded by transposases and/or integrases (data not shown). Since the isolate from the patient in Italy was sequenced with short read technology, we could not determine if all of the resistance genes were clustered in one site or two sites.
Comparisons of plasmids in CTX-M-65 strains. Graphic comparison of plasmid pESI in the Italian strain harboring blaCTX-M-65 (strain 14026835) compared to U.S. isolates. Genetic elements and their orientations are shown in the two integration sites. Colors indicate the source of the plasmid, and resistance genes are highlighted in red. *, chicken ceca and raw comminuted chicken had identical plasmids.
hqSNP analysis.We constructed a high-quality single nucleotide polymorphism (hqSNP) tree to examine the genomic relatedness of animal, food, and human isolates, using the retail meat isolate as a reference for hqSNP calling (Fig. 2). We also added the blaCTX-M-65-containing Italian S. Infantis strain characterized by Franco et al. (8). For comparison, we included an outgroup consisting of an Italian S. Infantis isolate containing blaCTX-M-1 (strain no. 14035093) (8), and we included six additional U.S. S. Infantis isolates that did not have pulsed-field gel electrophoresis (PFGE) pattern JFXX01.0787 and did not contain blaCTX-M-65, but were isolated from humans and retail chickens in 2013 and 2014 (strains N53842, N53846, 2014K-0421, 2014K-0374, N54719, and 2013K-1322). The blaCTX-M-65-positive retail chicken isolate, the two animal isolates from dairy cattle and chicken cecal content, and the three comminuted chicken isolates all clustered closely together, separated by only 1 to 6 hqSNPs. All 10 blaCTX-M-65-positive animal, food, and human isolates from the United States were closely related, separated by an average of 16 SNPs. The Italian strain carrying blaCTX-M-65 was also closely related to the 10 U.S. isolates, differing by 34 to 47 pairwise hqSNPs. The outgroup contained between 115 and 471 pairwise hqSNP differences from the blaCTX-M-65-positive U.S. isolates. The Italian strain carrying blaCTX-M-1 differed from the Italian strain carrying blaCTX-M-65 by 128 pairwise hqSNPs.
hqSNP tree of S. Infantis strains. Shown is a maximum likelihood hqSNP phylogenetic tree examining relatedness of U.S. isolates and Italian strains (blaCTX-M-65 positive, 14026835; blaCTX-M-1 positive, 14035093). blaCTX-M-65-positive isolate labels are color coded by source. The outgroup consists of isolates that do not have PFGE pattern JFXX01.0787 and do not contain blaCTX-M-65. The geographic location and year of isolation are shown. Bootstrap values of >80, hqSNP differences, and isolate source information are displayed using the R package ggtree (32).
DISCUSSION
This study is the first description of an S. Infantis isolate harboring a pESI-like megaplasmid in the United States. We examined select blaCTX-M-65-positive isolates with PFGE pattern JFXX01.0787. The blaCTX-M-65 gene has been well described in E. coli, Klebsiella pneumoniae, Salmonella enterica serovar Indiana, and Salmonella enterica serovar Typhimurium isolated from humans and healthy animals in China and Korea (12–16). It has also been found in commensal bacteria in pediatric populations in Bolivia (17) and produce imported to Switzerland from the Dominican Republic and Vietnam (18). blaCTX-M-65 had been described in E. coli from humans in North America (11, 19), but it may have only recently emerged in Salmonella in the United States (Brown et al., submitted).
The blaCTX-M-65 gene conferred resistance to most of the β-lactam antimicrobials on the National Antimicrobial Resistance Monitoring System (NARMS) panel (except Ipm, amoxicillin-clavulanic acid [Amc], and cefoxitin [Fox]). Of public health concern is the fact that isolates were also resistant to trimethoprim-sulfamethoxazole and had decreased susceptibility to ciprofloxacin, two drugs recommended for therapy of Salmonella infections when therapy is indicated. Additionally, 7 out of the 10 isolates also contained resistance determinants for fosfomycin, an agent used to treat urinary tract infections (20). Taken together, these results demonstrate a highly resistant organism that is perhaps more difficult to treat.
The four plasmids we characterized were very similar to the plasmid recently identified in Italy by Franco et al. in S. Infantis isolates from broilers and humans (8). Of the eight extended-spectrum cephalosporin-resistant Italian isolates analyzed by whole-genome sequencing (WGS) in that study, seven grouped into one phylogenetic cluster that included the ESBL gene blaCTX-M-1, and one human isolate containing the blaCTX-M-65 gene was an outlier. The blaCTX-M-65-positive Italian strain and the 10 U.S. isolates shared many resistance genes [aph(4)-la, aac(3)-IV, blaCTX-M-65, sul1, tetA, and dfrA14]. The total resistance gene composition of the Italian strain perfectly matched 5 of the 10 U.S. isolates (2014-2863, FSIS1502967, FSIS1502973, FSIS1502169, and FSIS1502916). The variability in resistance gene compositions would be expected since all resistance genes were surrounded by transposases and/or integrases, suggesting that they would be transferrable from one genetic locus to another.
Despite being collected from different sources in different states over a period of 2 and a half years, the small SNP difference between the blaCTX-M-65-positive U.S. animal, food, and human isolates indicates a highly stable strain that likely spread by clonal expansion in the recent past and suggests that blaCTX-M-65-positive S. Infantis in humans may be linked to food products from chickens and cattle, although additional epidemiological evidence is needed to confirm this association. How this strain entered the food supply is not known. It is not known whether any ill persons acquired blaCTX-M-65-positive Salmonella via chicken or cattle production facilities or if the blaCTX-M-65-positive transfer into chickens or cattle occurred from Salmonella-infected humans. With today's global agricultural economy, imported feed or feed components as sources of Salmonella isolates that are blaCTX-M-65 positive cannot be ruled out.
There were some important items to note in the study. First, we selected blaCTX-M-65-positive isolates based on initial matches with PFGE pattern JFXX01.0787. Since our initial investigation, there have been additional blaCTX-M-65-positive S. Infantis isolates found in the United States with other PFGE patterns. Further study is needed to determine the location of the blaCTX-M-65 or the type of plasmid in these S. Infantis isolates. Second, while this is one of the first reports of this gene in Salmonella in the United States, this does not mean that this is the first isolate in the United States. Antimicrobial-resistant isolates collected through NARMS were routinely sequenced beginning in 2014. S. Infantis isolates collected prior to 2014 were selected for sequencing based on matches with PFGE pattern JFXX01.0787. It is possible that this gene may have been present in other Salmonella strains from humans, animals, or retail meats collected before 2011. While the Italian blaCTX-M-65-positive strain was collected in 2014, the first Italian isolates with the pESI-like plasmid were collected in 2011, suggesting that this plasmid (and possibly the clone) may have been in circulation for some time. Notably, all of the blaCTX-M-65 isolates tested were resistant to ceftriaxone and ceftiofur but susceptible to cefoxitin. Because ESBLs mediate resistance to extended-spectrum cephalosporins but do not affect cephamycins, cefoxitin susceptibility among ceftriaxone/ceftiofur-resistant isolates has been indicated as a screening tool for potential ESBL production (21). When we used these criteria to review historical NARMS data from isolates collected prior to 2014, we identified 642 Salmonella isolates that could possibly harbor an ESBL gene. Whole-genome sequencing is necessary to confirm the presence and type of ESBL in those historical isolates.
The discovery of pESI-like S. Infantis in the United States is another example of how easily resistant bacteria can spread internationally. This investigation does not resolve whether the plasmid was introduced to the United States through humans or food animals. Regardless, the findings of this investigation highlight the international nature of antimicrobial resistance and the importance of applying a global One Health human and animal perspective to combating this challenge. The findings also emphasize the importance of an “integrated antimicrobial resistance surveillance system” to compare animal, human, and food isolates and the critical role that DNA sequencing technology can play to significantly enhance integrated surveillance. As more bacterial strains are sequenced and examined at the genomic level, our understanding of antimicrobial resistance will improve significantly. As a result, we will not only be equipped to identify different sources and mechanisms of resistance but also will learn how to design more effective interventions.
MATERIALS AND METHODS
Sample selections and susceptibility testing.In 2015, we identified one S. Infantis isolate containing the blaCTX-M-65 gene. The isolate was recovered from chicken samples collected during routine retail meat surveillance for the National Antimicrobial Resistance Monitoring System (NARMS). NARMS is a collaborative program of state and local health departments and universities, the Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and USDA that tracks antimicrobial-resistant enteric bacteria. This isolate also had pulsed-field gel electrophoresis (PFGE) XbaI pattern JFXX01.0787. Subsequently, a total of 67 isolates with pattern JFXX01.0787 occurring between 2012 and 2015 were identified in the National Molecular Subtyping Network for Foodborne Disease Surveillance (PulseNet), a national network of state and local public health laboratories and food regulatory agencies. We characterized 10 of these isolates with pattern JFXX01.0787, including four from humans, one from retail chickens, and five from animals sampled at slaughter facilities (Table 1). All contained the blaCTX-M-65 gene. Isolates were chosen to encompass a variety of factors, including geographic location, animal source, and date of isolation. All NARMS sampling schemes have been described previously (22). The four human isolates were from patients in California, Florida, Massachusetts, and Virginia and were collected by CDC through routine NARMS surveillance during 2013 and 2014. The retail chicken isolate came from a retail chicken sample collected at a grocery store in Tennessee and was submitted to the FDA in 2014 through routine NARMS retail meat surveillance. The five animal isolates from the slaughter facilities were obtained by USDA-FSIS from two samples collected in North Carolina and one each from California, Maine, and New Jersey. Among the animal isolates, two were from animal cecal content—one from young chickens at a slaughter facility in North Carolina and the other from a dairy cow at a slaughter facility in California—and were collected during routine NARMS sampling in 2015. The three remaining isolates were from comminuted poultry products and were acquired through other FSIS sampling programs (both exploratory and investigative) during 2014 and 2015.
Salmonella isolates were cultured in Trypticase soy agar (TSA [Becton Dickinson, NJ]) and in Trypticase soy broth (TSB [Becton]) overnight at 37°C. Isolates were serotyped using agglutination methods and then tested for antimicrobial susceptibility using broth microdilution (Sensititre; Trek Diagnostic Systems, Cleveland, OH, USA) on custom NARMS panels containing 14 antimicrobials: azithromycin (Azi), ampicillin (Amp), amoxicillin-clavulanic acid (Amc), cefoxitin (Fox), ceftiofur (Tio), ceftriaxone (Cro), chloramphenicol (Chl), ciprofloxacin (Cip), gentamicin (Gen), nalidixic acid (Nal), streptomycin (Str), sulfisoxazole (Fis), tetracycline (Tet), and trimethoprim-sulfamethoxazole (Sxt). Results were interpreted using CLSI criteria and NARMS consensus breakpoints as described in the NARMS 2012-2013 annual integrated report (6). Isolates that were resistant to ceftriaxone (MIC, ≥4 μg/ml) were also tested for susceptibility to additional β-lactams, including aztreonam (Atm), cefepime (Fep), cefotaxime (Ctx), cefotaxime-clavulanic acid, ceftazidime (Caz), ceftazidime-clavulanic acid, imipenem (Ipm), and piperacillin-tazobactam (Tzp).
Whole-genome sequencing and assembly.Genomic DNA was purified using the Qiagen DNeasy kit (Qiagen, Valencia, CA, USA), and DNA concentrations were measured using a Qubit fluorometer (Life Technologies, MD, USA). WGS was performed using v3 chemistry with paired-end 2- by 300-bp reads on the MiSeq platform (Illumina, San Diego, CA, USA). Sequencing libraries were prepared according to the Illumina Nextera XT sample preparation guide. Raw reads were assembled de novo using CLC Genomics Workbench 8 (Qiagen, Inc.). Assembly contig (coverage) information was as follows: 2013AM-0055-71, 164×; 2013AM-1918-67, 104×; 2014AM-2863-60, 106×; 2014AM-3028-61, 231×; N55391-117, 55×; FSIS1502967-107, 48×; FSIS1502973-122, 55×; FSIS1502169-102, 49×; FSIS1504606-91, 74×; and FSIS1502916-108, 34×. The whole-genome sequence was analyzed using Resfinder 2.1 (2 June 2016), an acquired resistance determinant database, and Plasmidfinder 1.3 (16 March 2016), a tool for in silico detection and typing of plasmids (23).
Four isolates from retail chickens (CVM N55391), food animals (FSIS1502169 and FSIS1502916), and humans (2014AM-3028) were selected for sequencing on the Pacific Biosciences (PacBio) RS II sequencer, as previously described (24, 25). Analysis of the sequence reads was implemented using SMRT Analysis 2.3.0. The best de novo assembly was established with the PacBio Hierarchical Genome Assembly Process (HGAP3.0) program using the continuous long reads from the four SMRT cells. The assembly outputs from HGAP contain overlapping regions at the end, which can be identified using dot plots in Gepard (26). Genomes were checked manually for even sequencing coverage. Afterwards, the improved consensus sequence was uploaded in SMRT Analysis 2.3.0 to determine the final consensus and accuracy scores using the Quiver consensus algorithm (27). Coverage for the four plasmids was as follows: CVM N55391, 449×; FSIS1502169, 216×; FSIS1502916, 405×; and 2014AM3028, 275×.
hqSNP analysis.High-quality single nucleotide polymorphisms (hqSNPs) were identified using lyve-SET v1.1.4f (https://github.com/lskatz/lyve-SET) and default presets for Salmonella enterica (28). The closed chromosomal sequence of the retail meat isolate N55391 was used as a reference. The resulting maximum likelihood phylogeny (see Data Set S1 in the supplemental material) was generated with RAxML v8.1.16 with 100 bootstrap replicates (29).
Plasmid analysis.Whole-genome shotgun sequences (accession no. ERR1014119) of the Italian human isolate carrying the blaCTX-M-65 gene (14026835) (8) were retrieved from the European Nucleotide Archive (http://www.ebi.ac.uk/ena). The raw reads of the sequences for this isolate were assembled using the Assembler pipeline (version 1.0), which is available in the Center for Genomic Epidemiology (CGE [http://cge.cbs.dtu.dk/services/all.php]). The assembled sequences were annotated with the RAST annotation server (30). The contigs of the isolate from the patient in Italy were aligned against the assembled sequences of the U.S. isolates using BLASTN (https://blast.ncbi.nlm.nih.gov/) to determine the order of these contigs and to identify the homologs between the isolate from the patient in Italy and the U.S. isolates.
Conjugation experiment.We selected six S. Infantis isolates that carried blaCTX-M-65 as donor cells for conjugation experiments: these included four isolates from food animals (FSIS1502973, FSIS1502916, FSIS1502967, and FSIS1502169) and one each from retail meat (N55391) and a human patient (2014AM-3028). E coli Top10 cells (Life Technologies Corporation, Invitrogen, Grand Island, NY), were used as the recipients. Conjugation was performed as described elsewhere (31). Briefly, one loopful of bacteria grown overnight on a blood agar plate was resuspended in 200 μl LB broth; 10 μl of each donor strain was spotted separately on top of seven 10-μl spots of recipient strain on a fresh blood agar plate. Plates were then incubated overnight at 35°C. Each coculture was scraped from the plate and resuspended in 500 μl LB broth. One hundred microliters each of 1:10 and 1:100 dilutions of the cell suspensions was plated on agar plates supplemented with ampicillin (50 μg/ml) and streptomycin (128 μg/ml) to select transconjugants. Transconjugants were then confirmed as E. coli using the Gram-negative card on Vitek2 Compact (bioMérieux, Durham, NC) following the method recommended by the company and determined antimicrobial susceptibility testing (AST) profiles using the Gram-negative panel (CMV3AGNF) on the Sensititre system (Thermo Fisher Scientific, Trek Diagnostics, Cleveland, OH) as described previously (22).
Accession number(s).WGS data were submitted to the National Center for Biotechnology Information (NCBI). Accession numbers for blaCTX-M-65-positive isolates from the U.S. are listed in Tables 1 and 2. Accession numbers for the Italian strains and the blaCTX-M-65-negative S. Infantis isolates are as follows: strain N53842, SRR2407674; N53846, SRR2407678; 2014K-0421, SRR1616805; 2014K-0374, SRR1654337; N54719, SRR2407735; 2013K-1322, SRR1616757; 14035093, ERR1014115; and 14026835, ERR1014119.
ACKNOWLEDGMENTS
We thank the participating state and local health departments that collect samples and submit isolates for NARMS studies. We would also like to acknowledge Claudia Lam for her role in submitting the sequences to NCBI.
This study was conducted as part of routine NARMS work.
The views expressed in this article are those of the authors and do not necessarily reflect the official policy of the Department of Health and Human Services, the U.S. Food and Drug Administration, Centers for Disease Control and Prevention, the U.S. Department of Agriculture, or the U.S. Government. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture or Food and Drug Administration.
FOOTNOTES
- Received 6 March 2017.
- Returned for modification 3 April 2017.
- Accepted 3 May 2017.
- Accepted manuscript posted online 8 May 2017.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00488-17.
- Copyright © 2017 American Society for Microbiology.
