Genomic differences between nasal Staphylococcus aureus from hog slaughterhouse workers and their communities

New human pathogens can emerge from the livestock-human interface and spread into human populations through many pathways including livestock products. Occupational contact with livestock is a risk factor for exposure to those pathogens and may cause further spreading of those pathogens in the community. The current study used whole genome sequencing to explore nasal Staphylococcus aureus obtained from hog slaughterhouse workers and their community members, all of whom resided in a livestock-dense region in rural North Carolina. Sequence data were analyzed for lineage distribution, pathogenicity-related genomic features, and mobile genetic elements. We observed evidence of nasal S. aureus differences between hog workers and non-workers. Nasal S. aureus from hog workers showed a greater lineage diversity than nasal S. aureus from community residents. Hog worker isolates were less likely to carry the φSa3 prophage and human-specific immune evasion cluster genes than community resident isolates (φSa3 prophage: 54.5% vs. 91.7%, Benjamini-Hochberg (BH) corrected p = 0.035; immune evasion cluster genes: 66.7% vs. 100%, BH p = 0.021). Hog worker isolates had a lower prevalence and diversity of enterotoxins than community resident isolates, particularly lacking the enterotoxin gene cluster (39.4% vs. 70.8%, BH p = 0.125). Moreover, hog worker isolates harbored more diverse antibiotic resistance genes, with a higher prevalence of carriage of multiple resistance genes, than community resident isolates (75.8% vs. 29.2%, BH p = 0.021). Phylogenetic analysis of all ST5 isolates, the most abundant lineage in the collection, further supported separation of isolates from hog workers and non-workers. Together, our observations suggest impact of occupational contact with livestock on nasal S. aureus colonization and highlight the need for further research on the complex epidemiology of S. aureus at the livestock-human interface.


Introduction
Staphylococcus aureus is an important pathogen causing significant clinical and public health burdens.This situation is more serious now because of the global emergence of antibiotic resistance, including methicillin resistance, in S. aureus [1,2].In the United States, the Centers for Disease Control and Prevention reported that methicillin resistant S. aureus (MRSA) was responsible for 11,000 deaths from invasive skin and soft tissue infections in 2011 [3].Community-associated MRSA (CA-MRSA) now accounts for a significant portion of all MRSA infections, in addition to health care-associated MRSA (HA-MRSA) [3,4].Although certain risk factors for infections by CA-MRSA have been characterized [3], reservoirs of resistant and/or virulent S. aureus outside of health care settings remain poorly understood, and the origins and transmission routes of S. aureus in the community are not elucidated [5].
The widespread use of antibiotics in industrial food animal production has been a major driver of the development and dissemination of resistance among microbes in agricultural settings and results in human exposure to resistant pathogens via food and the environment [6].For instance, Rinsky et al. [7] reported that industrial livestock operation workers carried nasal S. aureus with characteristics that were putatively associated with livestock (e.g., tetracyclineresistant and scn-negative), which were not seen in workers from antibiotic-free livestock operations.Recent studies further show that livestock-associated occupational groups are at increased risk of nasal carriage of antibiotic resistant S. aureus, including MRSA and multidrug resistant S. aureus (MDRSA), compared to the general population [8][9][10].While findings to date indicate that occupational contact with livestock is a risk factor for exposure to livestockassociated S. aureus, including resistant strains, few studies have examined influences of such exposure on the S. aureus population in human nostrils, although this information could have important implications for the epidemiology of S. aureus.
Whole genome sequencing (WGS) and comparative genomics has provided broad insights into the success of S. aureus as a commensal and pathogen of both humans and animals, revealing genomic features associated with antibiotic resistance, adhesion to and invasion of host cells and tissues, evasion of immune responses, and biofilm formation [2,11].Many of these determinants are located on mobile genetic elements (MGEs), such as S. aureus pathogenicity islands (SaPIs), prophages, and plasmids.Indeed, MGEs account for 15-20% of the S. aureus genome, accelerating genotypic/phenotypic variation in S. aureus populations and facilitating the rapid spread of antibiotic resistance and/or virulence through horizontal gene transfer [2,12].Comparisons of strains from various host species further suggest a correlation between MGEs acquisition/loss and host adaptation [13][14][15].
In our previous cross-sectional study conducted at a hog slaughter/processing plant in a livestock-dense region in North Carolina, we found that although the prevalence of nasal carriage of S. aureus was similar in hog workers and their household and community members, S. aureus isolates from hog workers were resistant to a greater number of antibiotic classes [9].Whether that reflected distinct nasal S. aureus populations in hog workers and non-workers was unclear.The present study sought to use WGS to explore genomic features of nasal S. aureus collected in our previous study, including isolates colonizing hog workers, their household members, and non-worker residents in the broader community.Our analysis provides new insights into the epidemiology of S. aureus at the livestock-human interface.

Bacterial isolates
This study included 79 nasal S. aureus isolates obtained in our previous study [9].Study design, informed consent, participant enrollment, and biological sampling are detailed therein.Briefly, we enrolled 162 hog slaughterhouse workers, 63 household members of those workers from 50 different households, and 111 community residents, and collected specimens from both nares of each participant using a swab.S. aureus was isolated and identified from nasal swabs by the Johns Hopkins Hospital Clinical Microbiology Laboratory.One isolate per S. aureus-positive individual was analyzed in this study (a flowchart in S1 Fig).
We also included 22 previously published sequences of human and livestock strains from international studies for comparison (S1 Table ).They represent the same multilocus sequence types (MLSTs) and/or clonal complexes (CCs) as the nasal isolates obtained by us.

DNA preparation and genome sequencing
Isolates were grown on trypticase soy agar with 5% sheep blood (BBL, BD Diagnostic Systems) at 37˚C overnight.DNA was extracted from bacterial cells using a boiling and freeze/thaw method [16].DNA quantity and quality was assessed using a NanoDrop 2000C spectrophotometer (NanoDrop Technologies).
Genomic libraries were constructed as previously described [14].Multiplexed paired-end sequencing was conducted at the Institute of Genomic Sciences, University of Maryland Baltimore.Individual libraries were quantified by quantitative PCR on an ABI 7900HT fast real-time PCR system (Life Technologies) before pools of indexed libraries were loaded onto a flow cell for sequencing.Thirty-five isolates were sequenced on the Illumina MiSeq platform (Illumina, Inc.) to a read length of 251 bp and forty-four isolates on the HiSeq 2500 platform (Illumina, Inc.) to a read length of 101 bp.The median approximate coverage was 102 for all the sequenced isolates.

Quality control and de novo assembly
Raw reads were assessed for quality using FASTQC (http://www.bioinformatics.bbsrc.ac.uk/ projects/fastqc/) and trimmed at both 5' and 3' ends using Trimmomatic [17] to filter out reads of low quality (Phred quality score <30, length <36 bp).Potential contaminating sequences were checked using Kraken [18]; sequencing errors were corrected using Lighter [19].Corrected reads were de novo assembled with SPAdes 3.5.0[20]; assembly quality was evaluated by QUAST [21].Contigs of >200 bp and with !5× average coverage depth were subject to further analysis.

MLST and spa typing
MLST of most of the isolates has been reported [9].Several previously undetermined isolates were characterized here using srst2 [22], which employs bowtie2 [23] to call loci directly from Illumina reads based on the S. aureus MLST database (http://saureus.mlst.net).spa types of all the isolates were determined by identifying spa repeats (http://spa.ridom.de) in the assemblies using BLAST [24], followed by manual verification.

Analysis of virulence and antimicrobial resistance genes
For all the isolates, we screened their genome assemblies against the VirulenceFinder database (https://cge.cbs.dtu.dk/services/data.php)[25] and searched for known staphylococcal enterotoxins (SEs) (sea-see, seg-seq and yent2) as described in [26].The presence of immune evasion cluster (IEC) genes, including chp for chemotaxis inhibitory protein, scn for staphylococcal complement inhibitor, sea or sep for staphylococcal enterotoxin A or P, and sak for staphylokinase, was examined using a method similar to Price et al. [14].S2 Table lists all virulence factors analyzed in this study.
All genes were identified with a threshold of !90% nucleotide identity, !90% length coverage, and !5× sequence depth.

Phylogenetic analysis and genome annotation for the ST5 lineage
Sequence type (ST) 5 was the most abundant lineage in this study (n = 19) and was found in all population groups, providing an opportunity for a deeper analysis.Consequently, a whole genome phylogeny was reconstructed for this lineage using REALPHY [39].Briefly, the 19 ST5 genome assemblies from this study, along with 6 published ST5 genomes, were mapped to 2 ST105 reference genomes by bowtie2 [23].ST105 was chosen as reference because of its relatedness to ST5 within CC5 [26].The resulting alignments were merged, and single nucleotide polymorphisms (SNPs) and nonpolymorphic positions were used for generating a maximum likelihood phylogeny using PhyML with the generalized time-reversible substitution model, the gamma-distributed rate heterogeneity, and midpoint rooting [39,40].
Genome assemblies of the 19 ST5 isolates were further annotated in the CloVR-Microbe pipeline [41].Identified SaPIs and plasmids were compared to SaPI and S. aureus plasmid sequences in the GenBank database using Mauve [42].In addition, the 19 annotated ST5 genome assemblies were input to the CloVR-Comparative pipeline, which uses reference-free whole genome alignment to call SNPs for phylogeny reconstruction [43].

Statistical analysis
All statistical analyses were performed using R (version 3.2.4).Simpson diversity and 95% confidence interval were calculated as described previously [44].The presence/absence of individual genes was compared between isolates from different population groups, using community resident isolates as reference.Differences between proportions of gene-carriage were tested by two-sided Fisher-Boschloo exact test implemented in the "Exact" package [45], and p-Value correction for multiple testing was conducted using the Benjamini-Hochberg (BH) method [46] implemented in the "stats" package.Difference between mean numbers of SEs or antibiotic resistance genes per genome was tested by two-sided ANOVA or Welch t test after Levene's test for variance homogeneity.

Nucleotide accession numbers
The Illumina sequences generated and used in this study are deposited in the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA412599.

Lineage diversity of nasal S. aureus in a community of hog slaughterhouse workers and residents in a region with intensive swine production
In this study, we successfully sequenced 77 of the 79 nasal isolates that were obtained from 336 residents in rural North Carolina (WGS statistics in S4 Table ).The MLST of one isolate was untypable and that isolate was excluded from further analysis, leaving a total of 76 sequenced isolates for genomic analysis.These isolates belonged to 26 STs, including two different singlelocus variants (SLVs) of ST8, one SLV of ST15, one SLV of ST30, one SLV of ST45, one SLV of ST630, and one novel ST.All the SLVs had one SNP difference from the closest STs (S2 Fig).

Virulence genes in nasal S. aureus
A variety of virulence genes were identified in the 76 isolates (Fig 1A and S5 Table).They encode adhesins, exoenzymes, toxins and other virulence factors responsible for adhesion to and invasion of host cells and tissues, evasion of immune responses, and biofilm formation (S2 Table ).
Among the genes for heavy metal and antiseptic resistance that were examined in this study, the arsenic resistance gene arsB was the most prevalent (75/76; 99%), followed by the  ).

Nasal S. aureus of the ST5 lineage
A variety of MGEs were present in these ST5 isolates (Fig 2).Two classes of SaPIs were present in 12 of the 19 nasal isolates.The SaPIm4 (i.e., νSa3 type I) class was predominant, present in most community resident isolates (7/10) and household member isolates (3/5), as well as in one hog worker isolate (1/4).These SaPIm4 elements fell into three subtypes (SaPIm4a, SaPIm4b, SaPIm4c), which shared 98-99% nucleotide identity over a large region but had various genes adjacent to their attR sites (S4A Fig) .In detail, SaPIm4a carried a fhuD gene encoding ferrichrome-binding protein and a lcm gene encoding leucine carboxyl methyltransferase.SaPIm4b carried an ear gene encoding extracellular β-lactamase homolog, in addition to fhuD and lcm.SaPIm4c carried ear and two enterotoxin genes, sec and sel.The second SaPI class, an element akin to SaPI3 (i.e., νSa1), was present in a household member isolate (1/5).It had ear and the enterotoxin gene seb adjacent to its attR (S4A Fig) .Overall, SaPIs in these nasal isolates seemed to have evolved from recombination between SaPIs of various S. aureus strains reported around the world [47][48][49].
Furthermore, there was evidence suggesting distinct genomic features of nasal S. aureus from different population groups.In general, nasal S. aureus from hog workers was less likely to contain any human-specific IEC gene than isolates from community residents (66.7% vs. 100%, BH corrected p = 0.021) (Table 1).The prevalence of the staphylokinase-encoding gene (sak) and the staphylococcal complement inhibitor gene (scn) was significantly lower among hog worker isolates than community resident isolates (51.5% vs. 95.8%,BH p = 0.012 for sak; 66.7% vs. 100%, BH p = 0.021 for scn).The prevalence of the chemotaxis inhibitory proteinencoding gene (chp) was also lower in hog worker isolates than in community resident isolates (48.5% vs. 66.7%),although not statistically significant.Nasal S. aureus from household members of hog workers and community residents did not show statistical differences in carriage of IEC genes (Table 1).
Nasal S. aureus from hog workers was also less likely to contain SEs than isolates from community residents (66.7% vs. 91.7%,BH p = 0.125) (Table 1).Most hog worker isolates had 2 enterotoxins in their genomes, whereas the majority of community resident isolates had !5 enterotoxins in their genomes (Fig 3A).Such a discrepancy was largely due to carriage of the egc (i.e., seg, sei, sem, sen, seo, yent2), which showed lower prevalence in hog worker isolates as compared to community resident isolates (39.4% vs. 70.8%,BH p = 0.125).Nasal S. aureus from household members of hog workers and community residents were similar in SE carriage (Table 1 and Fig 3A).
Distinct genomic features of nasal S. aureus from different population groups were also illustrated by antibiotic resistance genes (Table 1).Overall, a total of 13, 10, and 7 classes of resistance genes were observed in isolates from hog workers, their household members, and community residents, respectively.Isolates from hog workers and their household members tended to harbor a greater number of antibiotic resistance genes in their genomes than isolates from community residents (mean number being 2.8, 2.6 and 1.6; p = 0.025 for hog worker isolates vs. community resident isolates; p = 0.036 for household member isolates vs. community resident isolates) (Fig 3B).The prevalence of carrying !2 resistance genes was significantly higher among isolates from hog workers than those from community residents (75.8% vs. 29.2%,BH p = 0.021).Specifically, isolates from hog workers were more likely to contain the penicillin resistance gene blaZ (84.8% vs. 54.2%,BH p = 0.125).Isolates from household members also showed higher prevalence of carrying !2 resistance genes than community resident isolates (84.2% vs. 54.2%),although not statistically significant.Additionally, the aminoglycoside phosphorylase gene aph(3')-III, two macrolide resistance genes (mphC and msrA), and the streptothricin acetyltransferase gene sat4 were present in several hog worker isolates and one household member isolate but were not seen in community resident isolates.
Nasal S. aureus from the three population groups further showed differences in carriage of prophages and plasmids (Table 1).The prevalence of φSa3 was much lower among hog worker isolates than community resident isolates (54.5% vs. 91.7%,BH p = 0.035), while household member isolates and community resident isolates were similar in φSa3 carriage.The prevalence of plasmids was somewhat higher in hog isolates and household member isolates than in community resident isolates (87.9%, 94.7% and 70.8% for isolates from workers,  household members and community residents, respectively), although not statistically significant.

Discussion
The growing recognition of S. aureus as a community-based pathogen urges a comprehensive understanding of reservoirs of S. aureus outside of health care settings and the origins and transmission routes of S. aureus in the community.The epidemiology of S. aureus is complex at the livestock-human interface, where farm animals provide additional reservoirs of S. aureus.Extensive contact with those animals could lead to human exposure to animal-associated microbiota, which might eventually spread into the community.Bacterial WGS analysis has been used to successfully resolve the evolutionary trajectories of several CA-MRSA strains and in some cases revealed their emergence from the livestock-human interface [15,50,51].
Here we applied WGS to analyze nasal isolates that were collected in our previous cross-sectional study conducted at a hog slaughter/processing plant in rural North Carolina.We observed differences in nasal S. aureus, including lineage diversity and pathogenicity-related genomic features, between hog workers and their community members.
We previously reported greater MLST diversity of nasal isolates from hog workers as compared to those from their household and community members [9].The current study confirms and extends that observation.Analysis based on both MLST and spa typing revealed highest lineage diversity of nasal S. aureus from hog workers and lowest diversity of nasal S. aureus from community residents.The exact sources of S. aureus colonization in the study population are unknown.However, given that all the three population groups were from the same broader community, occupational exposure most likely contributed to the S. aureus diversity in hog workers.Further clues might come from sampling the animals entering the plant and the working environment inside the plant.
Several lines of evidence suggest distinct genomic features of nasal S. aureus of livestockassociated workers and non-workers.Compared to community resident isolates, hog worker isolates were less likely to contain φSa3 prophages and φSa3-associated human-specific IEC genes, particularly the sak and scn genes.φSa3 prophages are present in diverse S. aureus lineages and are more common in colonizing strains [52].The associated IEC genes are suggested to facilitate S. aureus nasal colonization, playing crucial roles in its adaptation to human [34,53].Consequently, the relatively high prevalence of φSa3 and human-specific IEC genes in nasal S. aureus from non-workers could be normal.However, the significantly lower prevalence of φSa3 and human-specific IEC genes in nasal S. aureus from hog workers was unexpected.It is reported that regardless of S. aureus strain lineage and geographic location, φSa3 and IEC genes are present in 83%−100% of human isolates but only in 2%−34% of animal isolates, a phenomenon likely resulting from host adaptation [14,15,36,53,54].More recently, a study focusing on ST5 MRSA reported the complete absence of φSa3 and IEC genes in all isolates sampled from swine, swine facility environments, and humans with short-or long-term exposure to swine.In contrast, the authors found that 90.4% of the examined clinical isolates from persons with no exposure to swine carried φSa3 prophages with IEC genes [55].Therefore, the lack of φSa3 and IEC genes in hog worker isolates in this study could suggest a contribution of zoonotic sources to S. aureus colonization in this occupational group.
Compared to nasal S. aureus from community residents, hog worker isolates also showed a lower prevalence and less diversity of enterotoxins, particularly lacking the egc operon [56].egc was found common in nasal isolates [57] and is suggested to primarily function as a colonization factor, increasing commensal fitness [58].This may explain its relatively high prevalence in nasal S. aureus from non-workers.But the lower prevalence of egc, as well as all SEs in general, among hog worker isolates was unexpected.Previous analysis of global S. aureus strains showed a lower carriage of SEs and/or egc in animal-associated strains than in human strains [59,60].A recent study sampling swine herds from 11 states of the United States, including North Carolina, also found a uniform absence of sea-see enterotoxins in representative swine isolates, regardless of their MLST backgrounds [61].Therefore, the observation of less SEs in hog worker isolates in this study supports our hypothesis that zoonotic sources contributed to S. aureus colonization in this occupational group.
Moreover, nasal S. aureus from hog workers showed greater diversity of antibiotic resistance genes and significantly higher prevalence of carriage of multiple resistance genes than community resident isolates.This finding is consistent with and extends our previous phenotypic observation [9].Genes conferring resistance to aminoglycosides/aminocyclitols and MLS B antibiotics were particularly abundant in hog worker isolates.For instance, one hog worker isolate simultaneously carried the aadE-sat4-aph(3')-III cluster encoding aminoglycoside and streptothricin modifying enzymes as well as the mphC-msrA cluster for resistance to macrolides.Another three hog worker isolates simultaneously had the sat4-aph(3')-III cluster and the mphC-msrA cluster.The accumulation of multiple resistance genes for the same class of drugs in a bacterial genome could result from horizontal gene transfer followed by recombination, which probably reflects certain selective pressure during the strain's evolution [62].Drugs including MLS B antibiotics are currently used in swine production and might have direct or indirect influence on the accumulation of resistance genes in hog worker isolates [6].This is supported by previous reports showing enrichment of clusters of multiple antibiotic resistance genes on MGEs in swine and swine farm environmental microbiomes [63,64].Those studies found enrichment of aminoglycoside resistance genes clustered with macrolide resistance genes on MGEs although aminoglycosides were not fed to swine, and suggested coselection as a potential mechanism.Surprisingly, nasal S. aureus from household members of hog workers also showed greater diversity of antibiotic resistance genes and higher prevalence of carriage of multiple resistance genes than community resident isolates.This raises concerns about potential flow of resistance genes within households of hog workers.Notably, tetracycline resistance was uncommon among our isolates, in contrast to findings of other studies of populations involved in swine farming [7,8].This is largely due to the fact that most tetracycline resistant isolates in those studies were ST398 strains, a lineage uncommon in our collection.
Analysis of all ST5 isolates in our collection further indicated differences between nasal S. aureus of hog workers and non-workers.In the whole-genome inferred phylogeny, isolates from hog workers and non-workers are largely separated.Moreover, hog worker isolates all contained plasmids encoding virulence and penicillin resistance but mostly lacked SaPIs, and only half contained φSa3 prophages.In comparison, the majority of non-worker isolates contained SaPIs with virulence genes and φSa3 prophages with human-specific IEC genes, but many of them lacked plasmids.The ST5 lineage is comprised of both animal and human strains, and MGEs have contributed to the success of globally distributed ST5 human strains and the pathogenicity of frequently emerged ST5 CA-MRSA [13,31,65].Based on swine herds from major swine producing regions in 11 states, ST5 also represents one dominant lineage in swine in the United States [61], and the selected swine-associated ST5 MRSA isolates showed in vitro adherence to human keratinocytes equivalent to clinical ST5 MRSA isolates, suggesting humans in contact with swine have the potential to be colonized with those isolates [66].Our observations, based on WGS analysis, could suggest distinct origins of this lineage in hog workers and non-workers in North Carolina and warrants further research on its dynamics at the livestock-human interface.
Several limitations need to be considered, which might affect this study's robustness.Only one isolate from each S. aureus-positive participant was analyzed and for 18 STs/SLVs, our collection only contained 1 to 2 isolates.WGS of multiple isolates per individual has unveiled considerable within-host diversity of S. aureus, emphasizing the need for analyzing several isolates per person to capture an accurate picture of S. aureus colonization [67].Analysis of multiple isolates from each individual could also help explain the sporadic occurrence of certain lineages and genetic variations across lineages [68].
We were not provided access to hogs or the environment of the slaughter/processing plant.This prevented us from confirming that the nasal isolates from hog workers reflected the S. aureus populations in swine.Further, the hog slaughter/processing plant was located in a livestock-dense area.Hence, the study population may have been exposed to livestock-associated microbiota through environmental pathways.
Overall, however, our data support a growing concern about impacts of occupational contact with livestock on S. aureus populations in human.Findings from this study also add to our understanding of the epidemiology of S. aureus in the community, particularly situations in intensive livestock production areas.Further research is needed to confirm findings in this study and to fully understand the origins and transmission routes of S. aureus in the community.

Fig 1 .
Fig 1. Profiles of (A) virulence factors and (B) antimicrobial resistance genes for the 76 sequenced nasal S. aureus isolates.Each row represents one isolate from an individual, with the color bar on the left side indicating host groups (red, community residents; green, household members; blue, hog workers) and the texts on the right side indicating lineages.https://doi.org/10.1371/journal.pone.0193820.g001

A
whole-genome phylogeny was constructed for ST5, the most abundant lineage in the collection (Fig 2 and S3 Fig).The 19 nasal isolates clustered into two major clades.

Fig 2 .
Fig 2. Maximum likelihood phylogeny of the 19 ST5 S. aureus isolates sequenced in this study.Published genome sequences of 6 ST5 strains (502A, ECT-R2, ED98, Mu3, Mu50, N315) and 2 ST105 strains (JH1 and JH9) were included for comparison, with the country of report and host (H, human; C, chicken) for each reference strain shown in parenthesis (details in S1 Table).Color dots represent host groups in this study (red, community residents; green, household members; blue, hog workers).spa types and MGE types are listed on the right of the phylogeny.Subgroups a and b include isolates of certain relatedness, with subgroup b coming from the same household.Asterisk denotes a ST5 MRSA (SCCmec IV) isolate.https://doi.org/10.1371/journal.pone.0193820.g002

Table 1 . Characteristics of nasal S. aureus from hog slaughterhouse workers, their household members, and community residents.
Percentages indicate prevalence among isolates analyzed from participants of each population group.p 1 : Comparisons between hog worker isolates and community resident isolates.p 2 : Comparisons between household member isolates and community resident isolates.
a n, number of S. aureus isolates.One isolate per S. aureus-positive participant was sequenced.See S1 Fig for number of persons enrolled and tested for nasal S. aureus in the parent cross-sectional study b p-Values were calculated in Fisher-Boschloo exact test, not available for some characteristics due to data type or small sample size; p-Values were corrected for multiple testing with the Benjamini-Hochberg method c Including five SLVs that each had one SNP from the closest ST.SLVs were considered as individual types d The spa type of one ST45 isolate was untypable.Three novel spa types were 04-522-12-21-17-34-22-25 for a ST630-SLV isolate, 04-34-24-34-22-24-34-22-25 for a ST8 isolate, and 07-23-12-34-12-10-23-02-12-23 for a ST15 e g h i