J Antimicrob Chemother 2014; 69: 2573 – 2590 doi:10.1093/jac/dku137 Advance Access publication 3 May 2014
Enterococcal multiresistance gene cluster in methicillin-resistant Staphylococcus aureus from various origins and geographical locations
1
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany; 2Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, P. R. China; 3The Hong Kong Polytechnic University, Hong Kong, China; 4Institute for Medical Microbiology and Hygiene, Carl-ThiemKlinikum Cottbus, Cottbus, Germany; 5Alere Technologies GmbH, Jena, Germany; 6Institute for Medical Microbiology and Hygiene, Technische Universita¨t Dresden, Dresden, Germany *Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-143; E-mail: [email protected]
Keywords: MRSA, resistance gene transfer, Enterococcus
Sir, The recently conducted analysis of methicillin-resistant Staphylococcus aureus (MRSA) ST398 and methicillin-susceptible S. aureus (MSSA) ST9 isolates of human origin from Spain, and MRSA ST9 isolates from swine in China, revealed the presence of multiresistance gene clusters, which are most likely of enterococcal origin.1 – 4 To date, two types of enterococcal-like multiresistance gene clusters have been identified in S. aureus.1 – 3 Both clusters harbour the novel resistance genes lsa(E) (pleuromutilin/lincosamide/streptogramin A resistance) 5 and spw (spectinomycin resistance), 6 and the resistance genes aadE (streptomycin resistance) and lnu(B) (lincosamide resistance).1 – 3 In addition, the cluster identified on plasmid pV7037 from porcine MRSA ST9 carries an aacA-aphD gene (gentamicin/kanamycin/tobramycin resistance) and an erm(B) gene (macrolide/lincosamide/streptogramin B resistance).2,3 Several spectinomycin-resistant and pleuromutilin-resistant MRSA isolates from various origins and geographical locations have subsequently been found to harbour lsa(E) and spw.5,6 The aims of the present study were: (i) to investigate the genotypic relationships of these spectinomycin-resistant and pleuromutilinresistant MRSA isolates; (ii) to gain insight into the genetic environment of the lsa(E) and spw genes; and (iii) to determine
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
2573
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Francisco on March 10, 2015
Sarah Wendlandt1, Jun Li2, Jeff Ho3, Marc Armengol Porta4, Andrea T. Feßler1, Yang Wang2, Kristina Kadlec1, Stefan Monecke5,6, Ralf Ehricht5, Maureen Boost3 and Stefan Schwarz1*
whether these two genes form part of a multiresistance gene cluster. Eleven clonal complex (CC) 9 MRSA isolated from frozen or chilled chicken carcasses (n¼ 8) and pig carcasses (n¼ 2) obtained from five different wet markets and a supermarket in Hong Kong, and from a butcher working at a sixth wet market, were included in this study. An MRSA CC398 isolate of a nasally colonized human patient in a German hospital was also included in the study (Table 1). SCCmec, spa and dru typing was performed on all isolates as previously described.7 The isolates were also analysed with an S. aureus-specific DNA microarray (StaphyType, Alere, Jena, Germany).8 The 12 isolates were screened for the lsa(E), lnu(B), spw, aadE, erm(B) and aacA-aphD resistance genes using specific PCR assays.5 – 7 To observe more subtle variations between the isolates that could not be detected with the sequence-based typing methods, PFGE using the ApaI enzyme was conducted on the CC9 isolates as previously described.7 All CC9 isolates had the same atypical/truncated SCCmec type V and apart from one isolate were spa type t899 (Table 1). Eight of the 11 CC9 isolates were dru type dt12w, with the remaining three isolates dt12y, dt11by or dt10ca. In contrast, the CC398 isolate harboured an SCCmec type V and was spa type t011 and dru type dt11a. Five resistance phenotypes and four resistance genotypes were detected among the CC9 isolates, all of which were considered to be multiresistant due to their resistance to three or more classes of antimicrobial agents.9 The four chicken CC9 isolates from wet market 2 exhibited indistinguishable or very similar PFGE patterns (A, A1 and A2). The porcine CC9 isolate from wet market 4 and the chicken CC9-t899 isolate from wet market 3 had closely related PFGE patterns (B and B1). The remaining five CC9 isolates had individual PFGE patterns (C, D, E, F and G). To analyse the 12 isolates for the presence of the previously described multiresistance gene clusters, 10 overlapping PCR assays were used as previously described.2 Four PCRs covered about 5 kb downstream of the lsa(E) gene and included the resistance gene lnu(B). The remaining six PCRs covered 10 kb upstream of lsa(E) and included the resistance genes spw, aadE, erm(B) and aacA-aphD. The PCR mapping showed that all CC9 isolates were positive in PCR4–PCR8 including the resistance genes aadE, spw, lsa(E) and lnu(B). The CC398 isolate, although positive in PCR4 – PCR7, was negative in PCR8. Whole-genome sequencing was performed on all isolates. First, the whole-genome shotgun library of each isolate was constructed and sequenced using the Illumina HiSeq 2000 platform (Berry Genomics Company, Beijing, China). Second, a de novo assembly for the multiresistance gene cluster-carrying contig was conducted using CLC Genomics Workbench 5.5 (CLC Bio, Aarhus, Denmark). The sequence was then compared with the nucleotide sequences deposited in the National Center for Biotechnology Information (NCBI) database using the BLASTn
Isolate
Market
District
Host
CC
spa type
SCCmec type
dru type PFGE
C24W
wet market 1 Kowloon East
chicken (chilled)
CC9
t899
V-atyp./truncateda
dt10ca
D
C60
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt11by
A2
C59
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
A1
C58Y
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
A
C55
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
A
C208
wet market 3 Kowloon West
chicken (chilled)
CC9
t1234
V-atyp./truncateda
dt12w
E
C227
supermarket
New Territories East
chicken (chilled)
CC9
t899
V-atyp./truncateda
dt12w
C
C219
wet market 3 Kowloon West
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
B
370
wet market 4 Kowloon East
pig (carcasses nares) CC9
t899
V-atyp./truncateda
dt12w
B1
310– 1
wet market 5 Kowloon Central
pig (carcasses nares) CC9
t899
V-atyp./truncateda
dt12y
G
CW01
wet market 6 Hong Kong Island East
human (butcher)
CC9
t899
V-atyp./truncateda
dt12w
F
13941
NA
human (nasal colonization)
CC398
t011
V
dt11a
NT
Brandenburg, Germany
Resistance phenotype
Resistance genotype
BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN KAN, STR, SPT, CHL, FFN, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, FFN, ENR BLA, TET, CLI, TIA, TMP, GEN, KAN, STR, SPT, ENR BLA, TET, CLI, TIA, TMP, GEN, KAN, STR, SPT, ENR BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, FFN, ENR BLA, TET, CLI, TIA, KAN, STR, SPT
mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, fexA mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, fexA mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, fexA mecA, blaZ/I/R, tet(K), tet(M), lnu(B), lsa(E), aphA3, aadE, sat, spw
BLA, b-lactam antibiotics; CHL, chloramphenicol; CLI, clindamycin; ENR, enrofloxacin; FFN, florfenicol; GEN, gentamicin; KAN, kanamycin; MLSB, macrolides/lincosamides/streptogramin B; SPT, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole/trimethoprim; TET, tetracyclines; TIA, tiamulin; TMP, trimethoprim; NA, not applicable; NT, not tested. a Isolates with the atypical/truncated SCCmec type V element were mecA-positive and ugpQ-positive, but negative for recombinase genes.
Research letters
2574 Table 1. Characteristics of the 12 MRSA isolates included in this study
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Francisco on March 10, 2015
JAC
Research letters
Acknowledgements We thank Vivian Hensel and Ute Beermann for excellent technical assistance.
Funding This study was funded by the German Federal Ministry of Education and Research (BMBF) through the German Aerospace Center (DLR), grant number 01KI1301D (MedVet-Staph 2) and the Major State Basic Research Development Program of China (973 Program, No. 2013CB127200). S. W. is supported by scholarships of the Akademie fu¨r Tiergesundheit (AfT) e.V. and the University of Veterinary Medicine Hannover, Foundation.
Transparency declarations S. M. and R. E. are employees of Alere Technologies GmbH, the company that manufactures the microarrays used for this study. S. M. and R. E. do not own stock or options in the company. All other authors: none to declare.
References 1 Lozano C, Aspiroz C, Saenz Y et al. Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus
and other staphylococci of animal and human origin. J Antimicrob Chemother 2012; 67: 2804– 8. 2 Li B, Wendlandt S, Yao J et al. Detection and new genetic environment of the pleuromutilin– lincosamide– streptogramin A resistance gene lsa(E) in methicillin-resistant Staphylococcus aureus of swine origin. J Antimicrob Chemother 2013; 68: 1251– 5. 3 Wendlandt S, Li B, Ma Z et al. Complete sequence of the multi-resistance plasmid pV7037 from a porcine methicillin-resistant Staphylococcus aureus. Vet Microbiol 2013; 166: 650– 4. 4 Li X-S, Dong W-C, Wang X-M et al. Presence and genetic environment of pleuromutilin – lincosamide – streptogramin A resistance gene lsa(E) in enterococci of human and swine origin. J Antimicrob Chemother 2014; 69: 1424 –6. 5 Wendlandt S, Lozano C, Kadlec K et al. The enterococcal ABC transporter gene lsa(E) confers combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-susceptible and methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 2013; 68: 473–5. 6 Wendlandt S, Li B, Lozano C et al. Identification of the novel spectinomycin resistance gene spw in methicillin-resistant and methicillin-susceptible Staphylococcus aureus of human and animal origin. J Antimicrob Chemother 2013; 68: 1679–80. 7 Wendlandt S, Kadlec K, Feßler AT et al. Resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus isolates from broiler chickens at slaughter and abattoir workers. J Antimicrob Chemother 2013; 68: 2458– 63. 8 Monecke S, Coombs G, Shore AC et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One 2011; 6: e17936. 9 Schwarz S, Silley P, Simjee S et al. Assessing the antimicrobial susceptibility of bacteria obtained from animals. J Antimicrob Chemother 2010; 65: 601–4.
J Antimicrob Chemother 2014 doi:10.1093/jac/dku139 Advance Access publication 3 May 2014
Emergence of genetically unrelated NDM-1-producing Acinetobacter pittii strains in Paraguay Fernando Pasteran1, Mario Martinez Mora2, Ezequiel Albornoz1, Diego Faccone1, Rossana Franco2, Juana Ortellado3, Nancy Melgarejo2, Sonia Gomez1, Irma Riquelme3, Jorge Matheu4, Pilar Ramon-Pardo4 and Alejandra Corso1* 1
Servicio Antimicrobianos, Instituto Nacional de Enfermedades Infecciosas (INEI), ANLIS ‘Dr Carlos G. Malbra´n’, Ciudad Auto´noma de Buenos Aires, Argentina; 2Servicio Antimicrobianos, Laboratorio Central de Salud Pu´blica, Asuncio´n, Paraguay; 3Centro Materno Infantil-Hospital de Clı´nicas, Universidad Nacional de Asuncio´n, San Lorenzo, Departamento Central, Paraguay; 4Antimicrobial Resistance and Infection Control Program, International Regulations, Alert and Response and Epidemic Diseases and Water
2575
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Francisco on March 10, 2015
function. Sequence analysis was conducted using the ORF Finder and the BLASTp functions at NCBI. For each isolate, multiresistance gene cluster-carrying contigs ranging in size between 9613 bp and 9690 bp were obtained. They comprised the region from the aadE gene to downstream of lnu(B). A comparison with the 17577 bp sequence of the cluster located on the multiresistance plasmid pV7037 (GenBank accession number JX560992) revealed a single nucleotide exchange (AC) in the MRSA CC398 isolate at position 9474 in the non-coding region between spw and orf4. An additional nucleotide exchange (AG), which, however, did not change the amino acid sequence, was seen in all 12 isolates at position 10071 within orf4. All CC9 isolates carried a transposase gene downstream of the gene lnu(B). The CC398 isolate exhibited at this position two genes coding for a resolvase and a nucleotidyltransferase with greatest amino acid identities of 99.5% and 100%, respectively, with the corresponding proteins from Enterococcus faecium Aus0085 plasmid p3 (GenBank accession number NC_021988.1). In all cases, the repeatedly negative results of conjugation, protoplast transformation and electrotransformation experiments suggested that the cluster was located in the chromosomal DNA, as previously seen for the majority of the Chinese porcine MRSA CC9 isolates and the Spanish MRSA ST398 and MSSA ST9 isolates of human origin.1,2 In conclusion, a large part of the pV7037-associated multiresistance gene cluster, including the novel resistance genes lsa(E) and spw, was identified in MRSA CC9 isolated from different food samples and a butcher in Hong Kong as well as from a single MRSA CC398 of human origin in Germany. These and previous observations suggest that a multiresistance gene cluster, closely related to the one that has recently been detected in enterococci of human and porcine origin,4 has been acquired by MRSA/MSSA CC9 and CC398 several times on different occasions in Europe and Asia.
Enterococcal multiresistance gene cluster in methicillin-resistant Staphylococcus aureus from various origins and geographical locations
1
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany; 2Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, P. R. China; 3The Hong Kong Polytechnic University, Hong Kong, China; 4Institute for Medical Microbiology and Hygiene, Carl-ThiemKlinikum Cottbus, Cottbus, Germany; 5Alere Technologies GmbH, Jena, Germany; 6Institute for Medical Microbiology and Hygiene, Technische Universita¨t Dresden, Dresden, Germany *Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-143; E-mail: [email protected]
Keywords: MRSA, resistance gene transfer, Enterococcus
Sir, The recently conducted analysis of methicillin-resistant Staphylococcus aureus (MRSA) ST398 and methicillin-susceptible S. aureus (MSSA) ST9 isolates of human origin from Spain, and MRSA ST9 isolates from swine in China, revealed the presence of multiresistance gene clusters, which are most likely of enterococcal origin.1 – 4 To date, two types of enterococcal-like multiresistance gene clusters have been identified in S. aureus.1 – 3 Both clusters harbour the novel resistance genes lsa(E) (pleuromutilin/lincosamide/streptogramin A resistance) 5 and spw (spectinomycin resistance), 6 and the resistance genes aadE (streptomycin resistance) and lnu(B) (lincosamide resistance).1 – 3 In addition, the cluster identified on plasmid pV7037 from porcine MRSA ST9 carries an aacA-aphD gene (gentamicin/kanamycin/tobramycin resistance) and an erm(B) gene (macrolide/lincosamide/streptogramin B resistance).2,3 Several spectinomycin-resistant and pleuromutilin-resistant MRSA isolates from various origins and geographical locations have subsequently been found to harbour lsa(E) and spw.5,6 The aims of the present study were: (i) to investigate the genotypic relationships of these spectinomycin-resistant and pleuromutilinresistant MRSA isolates; (ii) to gain insight into the genetic environment of the lsa(E) and spw genes; and (iii) to determine
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
2573
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Francisco on March 10, 2015
Sarah Wendlandt1, Jun Li2, Jeff Ho3, Marc Armengol Porta4, Andrea T. Feßler1, Yang Wang2, Kristina Kadlec1, Stefan Monecke5,6, Ralf Ehricht5, Maureen Boost3 and Stefan Schwarz1*
whether these two genes form part of a multiresistance gene cluster. Eleven clonal complex (CC) 9 MRSA isolated from frozen or chilled chicken carcasses (n¼ 8) and pig carcasses (n¼ 2) obtained from five different wet markets and a supermarket in Hong Kong, and from a butcher working at a sixth wet market, were included in this study. An MRSA CC398 isolate of a nasally colonized human patient in a German hospital was also included in the study (Table 1). SCCmec, spa and dru typing was performed on all isolates as previously described.7 The isolates were also analysed with an S. aureus-specific DNA microarray (StaphyType, Alere, Jena, Germany).8 The 12 isolates were screened for the lsa(E), lnu(B), spw, aadE, erm(B) and aacA-aphD resistance genes using specific PCR assays.5 – 7 To observe more subtle variations between the isolates that could not be detected with the sequence-based typing methods, PFGE using the ApaI enzyme was conducted on the CC9 isolates as previously described.7 All CC9 isolates had the same atypical/truncated SCCmec type V and apart from one isolate were spa type t899 (Table 1). Eight of the 11 CC9 isolates were dru type dt12w, with the remaining three isolates dt12y, dt11by or dt10ca. In contrast, the CC398 isolate harboured an SCCmec type V and was spa type t011 and dru type dt11a. Five resistance phenotypes and four resistance genotypes were detected among the CC9 isolates, all of which were considered to be multiresistant due to their resistance to three or more classes of antimicrobial agents.9 The four chicken CC9 isolates from wet market 2 exhibited indistinguishable or very similar PFGE patterns (A, A1 and A2). The porcine CC9 isolate from wet market 4 and the chicken CC9-t899 isolate from wet market 3 had closely related PFGE patterns (B and B1). The remaining five CC9 isolates had individual PFGE patterns (C, D, E, F and G). To analyse the 12 isolates for the presence of the previously described multiresistance gene clusters, 10 overlapping PCR assays were used as previously described.2 Four PCRs covered about 5 kb downstream of the lsa(E) gene and included the resistance gene lnu(B). The remaining six PCRs covered 10 kb upstream of lsa(E) and included the resistance genes spw, aadE, erm(B) and aacA-aphD. The PCR mapping showed that all CC9 isolates were positive in PCR4–PCR8 including the resistance genes aadE, spw, lsa(E) and lnu(B). The CC398 isolate, although positive in PCR4 – PCR7, was negative in PCR8. Whole-genome sequencing was performed on all isolates. First, the whole-genome shotgun library of each isolate was constructed and sequenced using the Illumina HiSeq 2000 platform (Berry Genomics Company, Beijing, China). Second, a de novo assembly for the multiresistance gene cluster-carrying contig was conducted using CLC Genomics Workbench 5.5 (CLC Bio, Aarhus, Denmark). The sequence was then compared with the nucleotide sequences deposited in the National Center for Biotechnology Information (NCBI) database using the BLASTn
Isolate
Market
District
Host
CC
spa type
SCCmec type
dru type PFGE
C24W
wet market 1 Kowloon East
chicken (chilled)
CC9
t899
V-atyp./truncateda
dt10ca
D
C60
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt11by
A2
C59
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
A1
C58Y
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
A
C55
wet market 2 Kowloon East
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
A
C208
wet market 3 Kowloon West
chicken (chilled)
CC9
t1234
V-atyp./truncateda
dt12w
E
C227
supermarket
New Territories East
chicken (chilled)
CC9
t899
V-atyp./truncateda
dt12w
C
C219
wet market 3 Kowloon West
chicken (frozen)
CC9
t899
V-atyp./truncateda
dt12w
B
370
wet market 4 Kowloon East
pig (carcasses nares) CC9
t899
V-atyp./truncateda
dt12w
B1
310– 1
wet market 5 Kowloon Central
pig (carcasses nares) CC9
t899
V-atyp./truncateda
dt12y
G
CW01
wet market 6 Hong Kong Island East
human (butcher)
CC9
t899
V-atyp./truncateda
dt12w
F
13941
NA
human (nasal colonization)
CC398
t011
V
dt11a
NT
Brandenburg, Germany
Resistance phenotype
Resistance genotype
BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN KAN, STR, SPT, CHL, FFN, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, ENR, SXT BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, FFN, ENR BLA, TET, CLI, TIA, TMP, GEN, KAN, STR, SPT, ENR BLA, TET, CLI, TIA, TMP, GEN, KAN, STR, SPT, ENR BLA, TET, MLSB, TIA, TMP, GEN, KAN, STR, SPT, CHL, FFN, ENR BLA, TET, CLI, TIA, KAN, STR, SPT
mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, cat mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, fexA mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, fexA mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadE, spw mecA, blaZ/I/R, tet(L), erm(C), lnu(B), lsa(E), dfrK, aacA-aphD, aadD, aadE, spw, fexA mecA, blaZ/I/R, tet(K), tet(M), lnu(B), lsa(E), aphA3, aadE, sat, spw
BLA, b-lactam antibiotics; CHL, chloramphenicol; CLI, clindamycin; ENR, enrofloxacin; FFN, florfenicol; GEN, gentamicin; KAN, kanamycin; MLSB, macrolides/lincosamides/streptogramin B; SPT, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole/trimethoprim; TET, tetracyclines; TIA, tiamulin; TMP, trimethoprim; NA, not applicable; NT, not tested. a Isolates with the atypical/truncated SCCmec type V element were mecA-positive and ugpQ-positive, but negative for recombinase genes.
Research letters
2574 Table 1. Characteristics of the 12 MRSA isolates included in this study
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Francisco on March 10, 2015
JAC
Research letters
Acknowledgements We thank Vivian Hensel and Ute Beermann for excellent technical assistance.
Funding This study was funded by the German Federal Ministry of Education and Research (BMBF) through the German Aerospace Center (DLR), grant number 01KI1301D (MedVet-Staph 2) and the Major State Basic Research Development Program of China (973 Program, No. 2013CB127200). S. W. is supported by scholarships of the Akademie fu¨r Tiergesundheit (AfT) e.V. and the University of Veterinary Medicine Hannover, Foundation.
Transparency declarations S. M. and R. E. are employees of Alere Technologies GmbH, the company that manufactures the microarrays used for this study. S. M. and R. E. do not own stock or options in the company. All other authors: none to declare.
References 1 Lozano C, Aspiroz C, Saenz Y et al. Genetic environment and location of the lnu(A) and lnu(B) genes in methicillin-resistant Staphylococcus aureus
and other staphylococci of animal and human origin. J Antimicrob Chemother 2012; 67: 2804– 8. 2 Li B, Wendlandt S, Yao J et al. Detection and new genetic environment of the pleuromutilin– lincosamide– streptogramin A resistance gene lsa(E) in methicillin-resistant Staphylococcus aureus of swine origin. J Antimicrob Chemother 2013; 68: 1251– 5. 3 Wendlandt S, Li B, Ma Z et al. Complete sequence of the multi-resistance plasmid pV7037 from a porcine methicillin-resistant Staphylococcus aureus. Vet Microbiol 2013; 166: 650– 4. 4 Li X-S, Dong W-C, Wang X-M et al. Presence and genetic environment of pleuromutilin – lincosamide – streptogramin A resistance gene lsa(E) in enterococci of human and swine origin. J Antimicrob Chemother 2014; 69: 1424 –6. 5 Wendlandt S, Lozano C, Kadlec K et al. The enterococcal ABC transporter gene lsa(E) confers combined resistance to lincosamides, pleuromutilins and streptogramin A antibiotics in methicillin-susceptible and methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 2013; 68: 473–5. 6 Wendlandt S, Li B, Lozano C et al. Identification of the novel spectinomycin resistance gene spw in methicillin-resistant and methicillin-susceptible Staphylococcus aureus of human and animal origin. J Antimicrob Chemother 2013; 68: 1679–80. 7 Wendlandt S, Kadlec K, Feßler AT et al. Resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus isolates from broiler chickens at slaughter and abattoir workers. J Antimicrob Chemother 2013; 68: 2458– 63. 8 Monecke S, Coombs G, Shore AC et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One 2011; 6: e17936. 9 Schwarz S, Silley P, Simjee S et al. Assessing the antimicrobial susceptibility of bacteria obtained from animals. J Antimicrob Chemother 2010; 65: 601–4.
J Antimicrob Chemother 2014 doi:10.1093/jac/dku139 Advance Access publication 3 May 2014
Emergence of genetically unrelated NDM-1-producing Acinetobacter pittii strains in Paraguay Fernando Pasteran1, Mario Martinez Mora2, Ezequiel Albornoz1, Diego Faccone1, Rossana Franco2, Juana Ortellado3, Nancy Melgarejo2, Sonia Gomez1, Irma Riquelme3, Jorge Matheu4, Pilar Ramon-Pardo4 and Alejandra Corso1* 1
Servicio Antimicrobianos, Instituto Nacional de Enfermedades Infecciosas (INEI), ANLIS ‘Dr Carlos G. Malbra´n’, Ciudad Auto´noma de Buenos Aires, Argentina; 2Servicio Antimicrobianos, Laboratorio Central de Salud Pu´blica, Asuncio´n, Paraguay; 3Centro Materno Infantil-Hospital de Clı´nicas, Universidad Nacional de Asuncio´n, San Lorenzo, Departamento Central, Paraguay; 4Antimicrobial Resistance and Infection Control Program, International Regulations, Alert and Response and Epidemic Diseases and Water
2575
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Francisco on March 10, 2015
function. Sequence analysis was conducted using the ORF Finder and the BLASTp functions at NCBI. For each isolate, multiresistance gene cluster-carrying contigs ranging in size between 9613 bp and 9690 bp were obtained. They comprised the region from the aadE gene to downstream of lnu(B). A comparison with the 17577 bp sequence of the cluster located on the multiresistance plasmid pV7037 (GenBank accession number JX560992) revealed a single nucleotide exchange (AC) in the MRSA CC398 isolate at position 9474 in the non-coding region between spw and orf4. An additional nucleotide exchange (AG), which, however, did not change the amino acid sequence, was seen in all 12 isolates at position 10071 within orf4. All CC9 isolates carried a transposase gene downstream of the gene lnu(B). The CC398 isolate exhibited at this position two genes coding for a resolvase and a nucleotidyltransferase with greatest amino acid identities of 99.5% and 100%, respectively, with the corresponding proteins from Enterococcus faecium Aus0085 plasmid p3 (GenBank accession number NC_021988.1). In all cases, the repeatedly negative results of conjugation, protoplast transformation and electrotransformation experiments suggested that the cluster was located in the chromosomal DNA, as previously seen for the majority of the Chinese porcine MRSA CC9 isolates and the Spanish MRSA ST398 and MSSA ST9 isolates of human origin.1,2 In conclusion, a large part of the pV7037-associated multiresistance gene cluster, including the novel resistance genes lsa(E) and spw, was identified in MRSA CC9 isolated from different food samples and a butcher in Hong Kong as well as from a single MRSA CC398 of human origin in Germany. These and previous observations suggest that a multiresistance gene cluster, closely related to the one that has recently been detected in enterococci of human and porcine origin,4 has been acquired by MRSA/MSSA CC9 and CC398 several times on different occasions in Europe and Asia.
