TY - JOUR
AU - Kyes, Randall C
AB - Abstract Swine nasal samples [n = 282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n = 59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates. MRSA, ST22, Nepal, swine, macaques INTRODUCTION Nepal is in South Asia and has ∼29 million people. Various studies on isolation [14.6% to 69%] and characterization of MRSA from humans in hospital settings (patients or healthcare workers) in Nepal have been published (Ansari et al. 2014; Pahadi et al. 2014; Shrestha et al. 2014; Pokhrel et al. 2016; Khatri et al. 2017). In contrast, no data exist on MRSA in livestock or wildlife such as primates in this area. The carriage of MRSA in livestock including swine is of interest because, unlike LA-MRSA ST398 isolates identified in North America and Europe (Sahibzada et al. 2017), in most Asian countries MRSA ST9 predominates among livestock including pigs (Chuang and Huang 2015). Similarly, there are very limited data on the carriage of Staphylococcus aureus or MRSA in captive primates with no data on MRSA carriage in wild primates in their natural habitat (Hanley et al. 2015; Taylor et al. 1998; Weese 2010; Schaumburg et al. 2013; Soge et al. 2016). A few studies have been done on primates residing in research or zoological centers (Schaumburg et al. 2013; Soge et al. 2016; Hsu et al. 2017). The most prevalent in all three studies was MRSA and/or S. aureus ST188 SCCmec type IV, which is almost exclusively identified in humans from Pan-Asia and very rare in North America (Soge et al. 2016). However, MRSA isolated from wild primates have not been identified or characterized previously. MRSA ST22 SCCmec IV (EMRSA-15) is predominantly a pandemic MRSA isolated both in hospital and community settings. It is endemic in Ireland and the United Kingdom hospitals and a common cause of nosocomial infections in Asia, Australia, Europe and North America (Holden et al. 2013; Hsu et al. 2015; Kinnevey et al. 2016). MRSA ST22 SCCmec IV was first identified in Nepal from hospitalized patients in 2012 (Pokhrel et al. 2016). In contrast, a second study that sampled humans in Nepal did not isolate MRSA ST22 (Joshi et al. 2017). In Asia, two other pandemic clones have also previously been identified; ST5 and ST239 (Pokhrel et al. 2016). In the current study, we characterize MRSA isolated from domestic swine and wild rhesus macaques (Macaca mulatta) residing in Nepal. Using culture and molecular methods, we found that 2.1% of the swine and 6.8% of the macaque samples were positive for MRSA, with three of four macaque MRSAs and five of six swine MRSA identified as ST22 SCCmec IV, which were further characterized. MATERIAL AND METHODS Swine collection The samples were collected from both anterior nares of asymptomatic animals using a single sterile cotton swab (HiMedia Laboratories, Mumbai, India) moistened with sterile saline. Collection occurred between June 2015 and June 2016. The swabs were placed into a tube of Stuart's transport medium and transported to the laboratory. The samples were directly plated onto mannitol salt agar (HiMedia Laboratories) and incubated at 37°C for 24–48 h. Yellow colonies from mannitol plates were sub-cultured on 5% blood agar plates (HiMedia Laboratories) and incubated at 37°C for 24 h. Colonies that showed β-hemolysis on blood plates were further characterized (see below). A total of 282 nasal swabs were collected from 12 farms selected randomly from three districts of Nepal (Kathmandu valley) and included both weaners [n = 103] and growers [n = 179]. Primate collection Fifty-nine salvia samples from wild rhesus macaques living in and around temple areas of the Kathmandu valley, where human–macaque interaction is common, were collected in June 2017. The collection technique involved an adaptation of the non-invasive oral sampling method in Evans et al. 2015. Briefly, the SalivaBio Children's Swabs (Salimetrics LLC, State College PA USA) were soaked in a sterile glucose solution (10% w/v) and tossed to the macaques. A new pair of disposable gloves were used before taking the swab out of the tube and tossed to the monkey. After being chewed for several seconds/minutes, the monkey, realized the swab was not food and discarded it. The swab was then collected by a second person using gloves who stored it individually in a new tube that had been labeled. The storage tube contained enrichment broth Bacto® m Staphylococcus Broth (Difco Laboratories, Sparks, MD) supplemented with a final concentration of 75 mg/L of polymyxin B, 0.01% potassium tellurite and either with or without 12.5 mg/L nystatin (Sigma-Aldrich, St Louis, MO). The tubes were returned to the laboratory the same day and incubated at 37°C until turbid (24–96 h) as previously described (Roberts et al. 2011a). The broth was streaked for isolation onto mannitol salt agar plates (HiMedia Laboratories) and yellow colonies sub-cultured onto blood agar plates (HiMedia Laboratories). Colonies that had β-hemolysis were verified as S. aureus as described below. Ethical statement The research protocol for the sampling of free-ranging primates in the Kathmandu area was approved by the Kathmandu District Forest Office, Department of Forest under the Ministry of Forestry, Government of Nepal (Reference Letter Number: 074/074/1010). This research also complied with the animal use protocol for primates (#3143-04) approved by the Institutional Animal Care and Use Committee at the University of Washington, USA, and the American Society of Primatologists Principles for the Ethical Treatment of Nonhuman Primates. Identification of S. aureus and MRSA Colonies that showed β-hemolysis on blood plates were verified as S. aureus by Gram stain and with the Staphaurex test as previously described (Remel, Lenexa, KS; Roberts et al. 2011b). MRSA isolates were initially identified by their ability to grow on the Mueller-Hinton agar (HiMedia Laboratories) supplemented with 4 mg/L of oxacillin (HiMedia Laboratories). These were verified as MRSA using the Thermo Scientific PBP2’ latex agglutination test kit according to the manufacturer's instructions (Thermo Fisher Scientific Remel Products, Lenexa, KS; Roberts et al. 2011b). The MRSA isolates were further characterized, while the S. aureus were stored for future analysis. Genetic characterization of the MRSA isolates SCCmec typing SCCmec types were determined for the MRSA isolates using SCCmec I-V multiplex PCR, as previously described (Roberts et al. 2011b). The SCCmec types were confirmed as previously described (Zhang et al. 2005; Roberts et al. 2011a,b). Positive and negative controls for SCCmec I-V were used as previously described (Zhang et al. 2005; Roberts et al. 2011a,b). Multilocus sequence Multilocus sequence typing (MLST) was performed on all isolates using PCR assays with amplicons sequenced bi-directionally by ID Genomics (Seattle, WA) using Sanger sequencing and sequence analysis—alleles and ST assignment using the database https://pubmlst.org/saureus/. Alleles were assigned by comparison of DNA sequences to the corresponding loci in the S. aureus MLST database and ST assigned. Sterile water was used as negative control and a clinical USA 300 was used as positive control as described elsewhere (Soge et al. 2016). Antibiotic susceptibility Antibiotic resistant profiles were determined using the Kirby–Bauer disc diffusion method on Mueller–Hinton [MH] agar (Difco Laboratories, Division Becton-Dickinson) according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2015). Isolates were streaked onto the MH agar to form a lawn, and antibiotic discs were aseptically placed on the plate for each strain and incubated at 36.5°C for 18–24 h. Methicillin susceptible S. aureus ATCC 25 923 was used as a control. The following antibiotic discs were included in testing: chloramphenicol [CHL]; erythromycin [ERY]; clindamycin [CLN]; linezolid [LZD]; ciprofloxacin [CIP]; gentamicin [GEN]; tetracycline [TET] and trimethoprim-sulfamethoxazole [SXT] (Soge et al. 2009). The discs were supplied by Becton Dickinson Microbiology Systems (Franklin Lakes, NJ). Whole Genome Sequencing Whole genome sequencing (WGS) was performed on eight MRSA isolates. The creation of whole genome libraries was performed as previously described (Greninger et al. 2015, 2016). One ng of bacterial genomic DNA [gDNA] was used as input for dual-indexed Nextera XT library preparation with 14 cycles of amplification and sequenced on a 2 × 300 bp run on an Illumina MiSeq to achieve approximately 1 million reads per isolate. Paired-end reads were trimmed using trimmomatic, de novo assembled using SPAdes v3.11, and annotated using prokka v1.11 (Bankevich et al. 2012; Bolger, Lohse and Usadel 2014; Seemann 2014). Sequence type and species were confirmed by MLST and JSpecies analysis of the de novo assemblies (Richter et al. 2016; Page et al. 2017). Antibiotic resistance genes were profiled using prokka annotations and the Comprehensive Antibiotic Resistance Database (CARD) (Jia, Raphenya and Alcock 2017). Core genome hqSNP phylogenies were created as described previously using bwa-mem, samtools and vcftools using six MRSA ST22 human Singaporean isolates isolated between 2003 and 2010 [SRA accessions ERR029385, ERR029386, ERR030150, ERR030162, ERR030262, ERR030367] and three other ST22 controls [NCBI accessions CP007659, NC_017763 and SRR1050076] as control genomes (Holden et al. 2013; Hsu et al. 2015; Sabirova et al. 2014; https://www.ncbi.nlm.nih.gov/nuccore/JDRN00000000.1/) based on mapping to the S. aureus NCTC 8325 reference genome [NC_007995] using minDP 10, minQ 100, and minGQ 10 parameters in vcftools (Li and Durbin 2010; Kozyreva et al. 2016, 2017; Goncalves da Silva et al. 2017). The Singaporean ST22 control isolates were selected based on the greatest temporal and geographical diversity in the Singaporean MRSA sequencing study (Hsu et al. 2015). This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accessions PJLP-PJLW00000000. Accession # are SAMN08146085 [isolate MR3] to SAMN08146093 [isolate MR56]. Data analysis Survey data was input into REDCap and descriptive univariate analysis was performed on all variables using SAS v 9.4 (SAS Institute, Cary, NC). Genes identified from CARD (https://omictools.com/comprehensive-antibiotic-resistance-database-tool visited Nov. 19, 2017). Specific antibiotic resistance genes often associated with MRSA ST22 were screened (Holden et al. 2013; Shore et al. 2016). These included aminoglycoside, aacC-aphD; erythromycin and streptogramin B ATP-binding transporter genes, msr(A), msr(D); rRNA methylase genes, erm(A), erm(B), erm(C), erm(F), erm(Q), erm(33), erm(43), erm(44), erm(46), erm(48); lincosamide resistance, lnu(A), lnu(B); multidrug resistant genes including lincosamides, oxazolidinones, streptogramin A, phenicols and pleuromutilins cfr, cfr(B); lincosamide and streptogramin A, lsa(B), lsa(E); ≥1 of the following antibiotics, lincosamides, streptogramin A, pleuromutilins, vga(A), vga(C), vga(C); phenicol, fexA; oxazolidinones and phenicols, optrA; tetracycline, tet(K), tet(M); ampicillin resistance, blaZ; and fosfomycin, fosB. We examined the portions of genes for domains II and V of 23S rRNA and the genes for ribosomal proteins L3 [rplC], L4 [rplD] and L22 [rplV] for mutations associated with linezolid resistance in Streptococcus pneumoniae (Wolter et al. 2005) and S. aureus (Locke, Hilgers and Shaw 2009). Mutations in gyrA associated with ciprofloxacin resistance were also examined (McCurdy et al. 2017). In addition, 17 staphylococcal enterotoxin genes [sak, scn, sea, seb, sec1, sec2, sec3, sed, see, she, sej, sel, sem, sek, sei, seg, tst; nine adhesins [epbS, fnbA, fib, sdrD, sdrE, cna, clfA, fbpA, map], seven leukocidins [lukE, lukD, lukM, lukX, lukY, lukF-PV, lukS-PV] six haemolysins [hlgA, hlgB, hlgC, hla, hld, hig2] and chemotaxis inhibitory protein [chp] genes were screened using the WGS generated for the eight Nepal ST22 isolates. RESULTS Swine MRSA Two hundred eighty-two swine nasal samples were processed, 58 [20.5%] were S. aureus positive and additional six (2.1%) isolates were MRSA. The samples came from animals in five [41.6%] of the 12 different swine herds tested. Five isolates were ST22 SCCmec type IV and one ST88 SCCmec type V (Table 1). All isolates were resistant to ciprofloxacin, six intermediate and one resistant to clindamycin and either resistant [n = 6] or intermediate resistant [n = 3] to erythromycin and all susceptible to tetracycline (Table 1). Four isolates, MR3, MR9, MR11 and MR15 were resistant to gentamicin. M6 was intermediate to gentamicin (Table 1). Two isolates MR10 [ST22 SCCmec type IV], MR11 [ST88 SCCmec type V] were resistant to linezolid, resistant to ciprofloxacin and intermediately resistant to erythromycin and clindamycin (Table 1). The presence of acquired antibiotic resistance and mutational resistance associated with linezolid resistance in MR10 was determined by WGS. The other ST22 isolates were examined for both acquired and mutational antibiotic resistance as well as for the presence of various virulence factors using their whole genome sequence data (Shore et al. 2016) [See below]. Table 1. Characterization of the swine and primate MRSA from Nepal.           Antibiotic susceptibility patternsa        Strains ID Swine  Herd  MLST  SCCmec type  ERY  CLN  LZD  CIP  CHL  GEN  SXT  TET  AR Genesb  Chromosomal mutationsc  Other virulence genesd  MR3  A  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu  sak, scn, tst, sem, seg, ebpS, fib, sdrD, sdrE, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR6  A  ST22  IV  R  I  S  R  S  I  I  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, sem, seg, ebpS, fib, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR9  B  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR10  C  ST22  IV  I  I  R  R  S  S  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu; L22 [A29V] and an insertion in L4 [68KG69]  sak, scn, tst, sec3, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR15  E  ST22  IV  I  I  S  R  S  R  S  S  aacC-aphD  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR11  D  ST88  V  I  I  R  R  S  R  S  S  ND  ND     Primates  Location                                         MR12  T  ST22  IV  S  S  S  R  S  I  S  S  aacC-aphD, blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR48e  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, seg, ebpS, fnbA, fib, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC  MR56  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C)  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, sei, seg, ebpS, fnbA, sdrD, sdrE, map, lukS-PV, lukF-PV, hlgA, hlgB  MR19  T  ST239  III  R  R  S  R  S  R  R  R  ND  ND               Antibiotic susceptibility patternsa        Strains ID Swine  Herd  MLST  SCCmec type  ERY  CLN  LZD  CIP  CHL  GEN  SXT  TET  AR Genesb  Chromosomal mutationsc  Other virulence genesd  MR3  A  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu  sak, scn, tst, sem, seg, ebpS, fib, sdrD, sdrE, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR6  A  ST22  IV  R  I  S  R  S  I  I  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, sem, seg, ebpS, fib, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR9  B  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR10  C  ST22  IV  I  I  R  R  S  S  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu; L22 [A29V] and an insertion in L4 [68KG69]  sak, scn, tst, sec3, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR15  E  ST22  IV  I  I  S  R  S  R  S  S  aacC-aphD  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR11  D  ST88  V  I  I  R  R  S  R  S  S  ND  ND     Primates  Location                                         MR12  T  ST22  IV  S  S  S  R  S  I  S  S  aacC-aphD, blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR48e  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, seg, ebpS, fnbA, fib, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC  MR56  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C)  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, sei, seg, ebpS, fnbA, sdrD, sdrE, map, lukS-PV, lukF-PV, hlgA, hlgB  MR19  T  ST239  III  R  R  S  R  S  R  R  R  ND  ND     a ERY = erythromycin; CLN = clindamycin; LZD = linezolid; CIP = ciprofloxacin; CHL = chloramphenicol; GEN = gentamicin; SXT = trimethoprim-sulfamethoxalzole; TET = tetracycline. bAcquired antibiotic resistance genes from screened genes; aminoglycoside,aacC-aphD; erythromycin and streptogramin B ATP-binding transporter genes, msr(A), msr(D); rRNA methylase genes, erm(A), erm(B), erm(C), erm(F), erm(Q), erm(33), erm(43), erm(44), erm(46), erm(48); lincosamide resistance, lnu(A), lnu(B);multidrug resistant genes including lincosamides, oxazolidinones, streptogramin A, phenicols and pleuromutilins cfr, cfr(B); lincosamide and streptogramin A, lsa(B), lsa(E); ≥1 of the following antibiotics, lincosamides, streptogramin A, pleuromutilins, vga(A), vga(C), vga(C); phenicol, fexA; oxazolidinones and phenicols, optrA; tetracycline, tet(K), tet(M); ampicillin resistance, blaZ; and fosfomycin, fosB. cChromosomal mutations conferring ciprofloxacin resistance [gyrA Ser84Leu] and linezolid resistant ribosomal protein L22 [A29V] and an insertion in L4 [68KG69]. dOther virulence genes included 17 staphylococcal enterotoxin genes [sak, scn, sea, seb, sec1, sec2, sec3, sed, see, she, sej, sel, sem, sek, sei, seg, tst; nine adhesins [epbS, fnbA, fib, sdrD, sdrE, cna, clfA, fbpA, map], seven leukocidins [lukE, lukD, lukM, lukX, lukY, lukF-PV, lukS-PV] six haemolysins [hlgA, hlgB, hlgC, hla, hld, hig2] and chemotaxis inhibitory protein [chp] genes were screened. All isolates carry scn, sak, ebpS, map, hlgA, hlgB, tst genes. eMR48 carried an incomplete gene for aacC-aphD, while MR10 appear to carry a complete aacC-aphD gene even though all are phenotypically susceptible to gentamicin. This gene is listed in the table. View Large Table 1. Characterization of the swine and primate MRSA from Nepal.           Antibiotic susceptibility patternsa        Strains ID Swine  Herd  MLST  SCCmec type  ERY  CLN  LZD  CIP  CHL  GEN  SXT  TET  AR Genesb  Chromosomal mutationsc  Other virulence genesd  MR3  A  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu  sak, scn, tst, sem, seg, ebpS, fib, sdrD, sdrE, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR6  A  ST22  IV  R  I  S  R  S  I  I  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, sem, seg, ebpS, fib, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR9  B  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR10  C  ST22  IV  I  I  R  R  S  S  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu; L22 [A29V] and an insertion in L4 [68KG69]  sak, scn, tst, sec3, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR15  E  ST22  IV  I  I  S  R  S  R  S  S  aacC-aphD  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR11  D  ST88  V  I  I  R  R  S  R  S  S  ND  ND     Primates  Location                                         MR12  T  ST22  IV  S  S  S  R  S  I  S  S  aacC-aphD, blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR48e  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, seg, ebpS, fnbA, fib, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC  MR56  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C)  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, sei, seg, ebpS, fnbA, sdrD, sdrE, map, lukS-PV, lukF-PV, hlgA, hlgB  MR19  T  ST239  III  R  R  S  R  S  R  R  R  ND  ND               Antibiotic susceptibility patternsa        Strains ID Swine  Herd  MLST  SCCmec type  ERY  CLN  LZD  CIP  CHL  GEN  SXT  TET  AR Genesb  Chromosomal mutationsc  Other virulence genesd  MR3  A  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu  sak, scn, tst, sem, seg, ebpS, fib, sdrD, sdrE, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR6  A  ST22  IV  R  I  S  R  S  I  I  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, sem, seg, ebpS, fib, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR9  B  ST22  IV  R  I  S  R  S  R  S  S  aacC-aphD, erm(C), blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC, hld  MR10  C  ST22  IV  I  I  R  R  S  S  S  S  aacC-aphD, erm(C)  gyrA Ser84Leu; L22 [A29V] and an insertion in L4 [68KG69]  sak, scn, tst, sec3, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR15  E  ST22  IV  I  I  S  R  S  R  S  S  aacC-aphD  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrD, sdrE, cna, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR11  D  ST88  V  I  I  R  R  S  R  S  S  ND  ND     Primates  Location                                         MR12  T  ST22  IV  S  S  S  R  S  I  S  S  aacC-aphD, blaZ  gyrA Ser84Leu  sak, scn, tst, sec3, seo, sem, sei, sen, seg, ebpS, sdrE, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hld  MR48e  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C), blaZ  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, seg, ebpS, fnbA, fib, cna, clfA, map, chp, lukS-PV, lukF-PV, hlgA, hlgB, hlgC  MR56  G  ST22  IV  R  S  S  R  S  S  S  S  erm(C)  gyrA Ser84Leu  sak, scn, sec3, tst, seo, sem, sei, seg, ebpS, fnbA, sdrD, sdrE, map, lukS-PV, lukF-PV, hlgA, hlgB  MR19  T  ST239  III  R  R  S  R  S  R  R  R  ND  ND     a ERY = erythromycin; CLN = clindamycin; LZD = linezolid; CIP = ciprofloxacin; CHL = chloramphenicol; GEN = gentamicin; SXT = trimethoprim-sulfamethoxalzole; TET = tetracycline. bAcquired antibiotic resistance genes from screened genes; aminoglycoside,aacC-aphD; erythromycin and streptogramin B ATP-binding transporter genes, msr(A), msr(D); rRNA methylase genes, erm(A), erm(B), erm(C), erm(F), erm(Q), erm(33), erm(43), erm(44), erm(46), erm(48); lincosamide resistance, lnu(A), lnu(B);multidrug resistant genes including lincosamides, oxazolidinones, streptogramin A, phenicols and pleuromutilins cfr, cfr(B); lincosamide and streptogramin A, lsa(B), lsa(E); ≥1 of the following antibiotics, lincosamides, streptogramin A, pleuromutilins, vga(A), vga(C), vga(C); phenicol, fexA; oxazolidinones and phenicols, optrA; tetracycline, tet(K), tet(M); ampicillin resistance, blaZ; and fosfomycin, fosB. cChromosomal mutations conferring ciprofloxacin resistance [gyrA Ser84Leu] and linezolid resistant ribosomal protein L22 [A29V] and an insertion in L4 [68KG69]. dOther virulence genes included 17 staphylococcal enterotoxin genes [sak, scn, sea, seb, sec1, sec2, sec3, sed, see, she, sej, sel, sem, sek, sei, seg, tst; nine adhesins [epbS, fnbA, fib, sdrD, sdrE, cna, clfA, fbpA, map], seven leukocidins [lukE, lukD, lukM, lukX, lukY, lukF-PV, lukS-PV] six haemolysins [hlgA, hlgB, hlgC, hla, hld, hig2] and chemotaxis inhibitory protein [chp] genes were screened. All isolates carry scn, sak, ebpS, map, hlgA, hlgB, tst genes. eMR48 carried an incomplete gene for aacC-aphD, while MR10 appear to carry a complete aacC-aphD gene even though all are phenotypically susceptible to gentamicin. This gene is listed in the table. View Large Primate MRSA Fifty-nine macaque samples were processed and 25 (42.4%) were S. aureus positive. This included four (6.8%) samples that were MRSA positive and 21 samples were S. aureus positive. All four MRSA isolates were from samples collected in two of the temple areas sampled in the study. Like the swine MRSA, all macaque MRSA isolates were resistant to ciprofloxacin and variable resistant to other antibiotics (Table 1). The MR19 [ST239 SCCmec type III] had a unique resistance profile with resistance to erythromycin, clindamycin, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxalzole and tetracycline in addition to carrying the mecA gene. While the two ST22 SCCmec type IV [MR48, MR56] were resistant to erythromycin and MR12 was intermediate resistant to gentamicin (Table 1). The ST22 isolates were examined for both acquired and mutational antibiotic resistance as well as for the presence of various virulence factors using their whole genome sequence data [See below]. WGS of eight MRSA ST22 SCCmec type IV WGS was performed on the eight ST22 SCCmec type IV isolates and compared to multiple previously sequenced human ST22 SCCmec type IV isolates (Fig. 1 and Table 2). All eight isolates were confirmed as ST22 by MLST analysis on de novo assembled whole genome sequences. The swine isolates MR3, MR6 (Farm A), MR9 (Farm B), MR10 (Farm C) clustered together, while MR15 (Farm E) clustered with the three primate isolates (Fig. 1). However, all the Nepal ST22 isolates were much more closely related [17–39 SNP] to each other than to any of the other ST22 isolates examined (Table 2 and Fig. 1). The three control ST22 are closely related to each other. We did not have human ST22 WGS from Nepal for comparison. However, we did use six different ST22 WGS isolated from humans in Singapore for comparison. These six isolates were closely related to each other even though we picked those that were most diverse and not closely related to the three controls. The Singapore ST22 had 269–285 SNP differences with Nepal isolates. In contrast, the most closely related ST22 control to the Nepal isolates was SRR1050076 [170–176 SNP] isolated in California USA. The most distant controls were ST22 CP007659 and NC_017763 with >500 SNP differences with the Nepal isolates (Table 2 and Fig. 1). Figure 1. View largeDownload slide Core genome dendrogram analysis of MRSA ST22 SCCmec IV isolates from swine and rhesus macaque. Swine isolates are MR3, MR6, MR9, MR10 and MR15; rhesus macaque isolates are MR12, MR48 and MR56. Figure 1. View largeDownload slide Core genome dendrogram analysis of MRSA ST22 SCCmec IV isolates from swine and rhesus macaque. Swine isolates are MR3, MR6, MR9, MR10 and MR15; rhesus macaque isolates are MR12, MR48 and MR56. Table 2. SNP pairwise comparison.   CP007659  ERR029385  ERR029386  ERR030150  ERR030162  ERR030262  ERR030267  MR 10  MR 12  MR15  MR3  MR48  MR56  MR6  MR9  NC_017763  SRR1050076  CP007659                                    ERR029385 Singapore  314                                  ERR029386 Singapore  320  14                                ERR030150 Singapore  316  6  16                              ERR030162 Singapore  320  10  20  12                            ERR030262 Singapore  324  14  24  16  20                          ERR030267 Singapore  314  4  14  6  10  12                        MR10 Swine  524  275  281  277  281  285  275                      MR12 Primate  526  277  283  279  283  287  277  32                    MR15 Swine  518  269  275  271  275  279  269  24  22                  MR3 Swine  521  272  278  274  278  282  272  25  29  21                MR48 Primate  523  274  280  276  280  284  274  29  27  15  26              MR56 Primate  521  272  278  274  278  282  272  27  25  17  24  22            MR6 Swine  524  275  281  277  281  285  275  28  32  24  23  29  27          MR9 Swine  531  282  288  284  288  292  282  35  39  31  30  36  34  33        NC_017763  613  341  347  343  347  351  341  575  577  569  572  574  572  575  582      SRR1050076  459  209  215  211  215  219  209  176  178  170  173  175  173  176  183  509      CP007659  ERR029385  ERR029386  ERR030150  ERR030162  ERR030262  ERR030267  MR 10  MR 12  MR15  MR3  MR48  MR56  MR6  MR9  NC_017763  SRR1050076  CP007659                                    ERR029385 Singapore  314                                  ERR029386 Singapore  320  14                                ERR030150 Singapore  316  6  16                              ERR030162 Singapore  320  10  20  12                            ERR030262 Singapore  324  14  24  16  20                          ERR030267 Singapore  314  4  14  6  10  12                        MR10 Swine  524  275  281  277  281  285  275                      MR12 Primate  526  277  283  279  283  287  277  32                    MR15 Swine  518  269  275  271  275  279  269  24  22                  MR3 Swine  521  272  278  274  278  282  272  25  29  21                MR48 Primate  523  274  280  276  280  284  274  29  27  15  26              MR56 Primate  521  272  278  274  278  282  272  27  25  17  24  22            MR6 Swine  524  275  281  277  281  285  275  28  32  24  23  29  27          MR9 Swine  531  282  288  284  288  292  282  35  39  31  30  36  34  33        NC_017763  613  341  347  343  347  351  341  575  577  569  572  574  572  575  582      SRR1050076  459  209  215  211  215  219  209  176  178  170  173  175  173  176  183  509    View Large Table 2. SNP pairwise comparison.   CP007659  ERR029385  ERR029386  ERR030150  ERR030162  ERR030262  ERR030267  MR 10  MR 12  MR15  MR3  MR48  MR56  MR6  MR9  NC_017763  SRR1050076  CP007659                                    ERR029385 Singapore  314                                  ERR029386 Singapore  320  14                                ERR030150 Singapore  316  6  16                              ERR030162 Singapore  320  10  20  12                            ERR030262 Singapore  324  14  24  16  20                          ERR030267 Singapore  314  4  14  6  10  12                        MR10 Swine  524  275  281  277  281  285  275                      MR12 Primate  526  277  283  279  283  287  277  32                    MR15 Swine  518  269  275  271  275  279  269  24  22                  MR3 Swine  521  272  278  274  278  282  272  25  29  21                MR48 Primate  523  274  280  276  280  284  274  29  27  15  26              MR56 Primate  521  272  278  274  278  282  272  27  25  17  24  22            MR6 Swine  524  275  281  277  281  285  275  28  32  24  23  29  27          MR9 Swine  531  282  288  284  288  292  282  35  39  31  30  36  34  33        NC_017763  613  341  347  343  347  351  341  575  577  569  572  574  572  575  582      SRR1050076  459  209  215  211  215  219  209  176  178  170  173  175  173  176  183  509      CP007659  ERR029385  ERR029386  ERR030150  ERR030162  ERR030262  ERR030267  MR 10  MR 12  MR15  MR3  MR48  MR56  MR6  MR9  NC_017763  SRR1050076  CP007659                                    ERR029385 Singapore  314                                  ERR029386 Singapore  320  14                                ERR030150 Singapore  316  6  16                              ERR030162 Singapore  320  10  20  12                            ERR030262 Singapore  324  14  24  16  20                          ERR030267 Singapore  314  4  14  6  10  12                        MR10 Swine  524  275  281  277  281  285  275                      MR12 Primate  526  277  283  279  283  287  277  32                    MR15 Swine  518  269  275  271  275  279  269  24  22                  MR3 Swine  521  272  278  274  278  282  272  25  29  21                MR48 Primate  523  274  280  276  280  284  274  29  27  15  26              MR56 Primate  521  272  278  274  278  282  272  27  25  17  24  22            MR6 Swine  524  275  281  277  281  285  275  28  32  24  23  29  27          MR9 Swine  531  282  288  284  288  292  282  35  39  31  30  36  34  33        NC_017763  613  341  347  343  347  351  341  575  577  569  572  574  572  575  582      SRR1050076  459  209  215  211  215  219  209  176  178  170  173  175  173  176  183  509    View Large All eight isolates were ciprofloxacin resistant and carried the gyrA Ser84Leu mutation (Table 1). We had 87.5% correlation between phenotypic expression of clindamycin and erythromycin, intermediate or resistance as measured by disk diffusion assay and the presence of a resistance gene erm(C). The one exception was swine MR15 that was phenotypically intermediate to both antibiotics but not carrying any of the 12 acquired genes know to confer macrolide resistance in S. aureus [msr(A), msr(D); rRNA methylase genes, erm(A), erm(B), erm(C), erm(F), erm(Q), erm(33), erm(43), erm(44), erm(46), erm(48)] that were examined. In contrast, the other swine isolates [MR3, MR6, MR9, MR10] and primate isolates [MR48, MR56] were resistant or intermediate to erythromycin and clindamycin and carried the erm(C) gene (Table 1). Swine MR6, MR9 MR15 carried the blaZ gene, while MR3, MR6, MR9, MR11, MR15, and primate MR19 were intermediate or resistant to gentamicin and 5 carried the aacC-aphD gene. The swine MR10 isolate was phenotypically resistant to linezolid (Table 1) but did not carry the cfr, cfr(B), optrA, fexA or other known genes associated with linezolid resistance [lnu(A), lnu(B), lsa(B), lsa(E), vga(A), vga(C), vga(C)] when its WGS was examined. Similarly, we did not find any mutations in the 23S rRNA alleles (Shore et al. 2016). However, a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69] were identified (Shore et al. 2016; Table 1). Using WGS data, we looked for the presence of sequences associated with 39 virulence genes. All eight ST22 isolates carry these 11 genes: scn, sak, sem, tst, seg, map, ebpS, hlgA, hlgB, lukF-PV and lukS-PV genes. No isolates carried the following 16 genes: sea, seb, sec1, sec2, sed, see, seh, sej, fbpA, lukE, lukD, lukM, lukX, lukY, hla or hig2 genes. Among the remaining 12 genes carriage was variable (Table 1) DISCUSSION Five of six swine MRSA isolates and three of four macaque MRSA were ST22 SCCmec IV. The other swine isolate was ST88, and the other macaque isolate was MRSA ST239. The ST88 is a pandemic strain primarily of community origin found Asia, Africa, the Middle East and Europe (Mediavilla et al. 2012), while the ST239 is an Asian pandemic clone (Pokhrel et al. 2016). The MRSA ST22 SCCmec IV is an epidemic clone reported around the world but has not previously been found in primates (Soge et al. 2016) or livestock [M. David personal communication] (Monecke et al. 2011; Holden et al. 2013). Approximately ∼98% of ST22 isolates reported in the literature have been isolated from humans [M. David personal communication]. Both S. aureus [MSSA] ST22 and MRSA ST22 have been isolated from hospitalized patients in Nepal at two general hospitals in Kathmandu, Nepal (Pokhrel et al. 2016). Of these, 32% were MRSA and four were MRSA ST22. Unfortunately, we did not have access to the Nepalese human MRSA ST22 isolates for WGS comparisons but did compare our isolates to six previously characterized human isolates from Singapore (Table 2 and Fig. 1). The six human isolates from Singapore were not closely related to the Nepal ST22 isolates, while SRR1050076 from California was the most closely related of the three control ST22 isolates used in the study. This is the first report of linezolid-resistant MRSA in Nepal from either livestock or humans. The two swine isolates, MR10 ST22 SCCmec IV and MR11 ST88 SCCmec V were isolated from separate farms. Based on available information from all the pig farms included in the study, linezolid has not been used as a chemotherapeutic. Based on the WGS analysis of MR10, this isolate had no acquired linezolid resistance genes. However, a mutation in ribosomal protein L22 [A29V], previously reported in linezolid resistant S. aureus (Shore et al. 2016), and an insertion in L4 [68KG69] previously reported in linezolid resistant Streptococcus pneumoniae (Wolter et al. 2005) were identified (Table 1). The insertion in L4 has not previously been identified in Staphylococcus spp., but spontaneous mutations with changes in amino acids have been identified at Lys68 in S. aureus have been associated with linezolid resistance (Locke, Hilgers and Shaw 2009) under laboratory conditions. The fluoroquinolone resistance ST22 clone is thought to have emerged from a hospital-adapted background by the 1990’s in England (Holden et al. 2013); thus, it was not surprising that all of our MRSA isolates are resistance to ciprofloxacin and carried the gyrA Ser84Leu mutation, previously associated with ciprofloxacin resistance (Shore et al. 2016; Soge et al. 2016; Table 1). The other swine MRSA isolate was ST88 SCCmec type V [MR11]. ST88 has predominately been reported in Asia from animals, humans and the environment [M. David personal communication]. A ST88-SCCmec IV has been reported in pigs from Senegal and has been labeled the ‘African clone’ (Fall et al. 2012). No Nepalese swine workers were screened during the current study period. No workers reported a history of hospitalization in the past six months prior to the isolation of the swine during the study, recent infections and/or the use of antibiotics [S. Paudel MS thesis]. One macaque MRSA was ST239 SCCmec III and from the literature 154 separate isolates of ST239 have been identified carrying varying SCCmec elements. These have been isolated from humans in Asia, Australia, Americas and Europe [M. David personal communication]. A limited number of studies on S. aureus/MRSA colonization or disease in primates have been published. Most publications have been from primates in captivity for research, breeding, and/or in zoological facilities (Taylor and Grady 1998; Hanley et al. 2015; Soge et al. 2016). One study examined wild chimpanzees [Pan troglodytes schweinfurthii] and lemurs [Eulemur rufifrons and Propithecus verreauxi] in an African sanctuary (Schaumburg et al. 2013) but found no MRSA. In other facilities, MRSA in macaques and chimpanzees have been either commonly found clone [ST8] (Hanley et al. 2012), or ST188 and the novel ST2368 not previously found (Soge et al. 2016). A few primate-to-human transfer of ST188 or ST3268 have been described in either the USA (Soge et al. 2016) or in the Singapore (Hsu et al. 2017) primate facilities. The current hypothesis is that the source of the MRSA ST22 and ST239 isolates in the wild Nepalese macaques was from humans. 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TI - The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: is man the common source?
JF - FEMS Microbiology Ecology
DO - 10.1093/femsec/fiy052
DA - 2018-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-human-clone-st22-sccmec-iv-methicillin-resistant-staphylococcus-4hheASM2uc
SP - fiy052
VL - 94
IS - 5
DP - DeepDyve
ER -