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Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro hospitals
Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro hospitals Raiane Cardoso Chamon a, Natalia Lopes Pontes Iorio b, Sthefanie da Silva Ribeiro a, Fernanda Sampaio Cavalcante a, Kátia Regina Netto dos Santos a,⁎ a b
Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Departamento de Ciências Básicas, Universidade Federal Fluminense, Nova Friburgo, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 8 July 2015 Received in revised form 22 August 2015 Accepted 6 September 2015 Available online xxxx
a b s t r a c t In a collection of 50 pvl-positive Staphylococcus aureus isolates from 10 Rio de Janeiro hospitals, 18 (36%) were from bloodstream infections, and 31 (62%) carried the SCCmec IV. Among 25 (50%) isolates of the USA1100/ ST30/CC30 lineage present in 8 hospitals, 1 isolate was characterized as vancomycin-intermediate S. aureus. © 2015 Elsevier Inc. All rights reserved.
Keywords: Staphylococcus aureus USA1100/ST30/CC30 Panton-Valentine leukocidin Vancomycin-intermediate S. aureus
Staphylococcus aureus is an important cause of infections worldwide (David and Daum, 2010), and the presence of genes that encode for Panton-Valentine leukocidin (PVL) is associated to a wide range of diseases, from uncomplicated to severe infections (Tristan et al., 2007). The occurrence of PVL is associated with community-acquired S. aureus isolates, both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) (Boan et al., 2015). However, recent reports had described the presence of hospital-acquired isolates carrying these genes (Nichol et al., 2013), indicating a changing in epidemiology of PVL-positive S. aureus isolates. In the current study, we characterized 50 PVL-positive S. aureus isolates, detected among 940 isolates of our collection and isolated from 10 different hospitals in Rio de Janeiro, between 2004 and 2012. They were isolated from bloodstream infections (BSIs) (18 isolates/ 36%), skin and soft tissue infections (SSTIs) (11/22%), nostrils (9/18%), and other clinical sources (12/24%). The antimicrobial susceptibility was determined for 12 agents by the disk diffusion method, according to the CLSI (2012), except for mupirocin (Finlay et al., 1997) and tigecycline (European Committee on Antimicrobial Susceptibility Testing, 2011). The MIC for oxacillin and vancomycin was determined by broth microdilution test (CLSI, 2012). The mecA gene and SCCmec types were detected by PCR (Milheiriço et al., 2007), and the clonal lineages were determined by pulsed field gel electrophoresis (PFGE) (Vivoni et al., 2005) and Multilocus Sequence Typing (MLST) method (Enright et al., 2000). The presence of genes that encode for adhesins, such as bbp (bone sialo⁎ Corresponding author. Tel.: +55-21-2560-8344; fax: +55-21-2560-8028. E-mail address: [email protected] (K.R.N. dos Santos).
binding protein), cna (collagen-binding protein), ebpS (elastin-binding protein), fnbB (fibronectin-binding protein B) (Tristan et al., 2003), and fnbA (fibronectin-binding protein A) (Peacock et al., 2002), as well as enterotoxins sea-see (Johnson et al., 1991; Lovseth et al., 2004; Monday and Bohach, 1999) and seh (Sila et al., 2009), and toxin esfoliative (tst) (Johnson et al., 1991) were investigated by PCR. Among the 50 PVL-positive S. aureus isolates, 31 were MRSA and carried the SCCmec type IV, and 20 of them were resistant only to β-lactams. All 50 isolates were susceptible for linezolid, mupirocin, rifampin, and tigecycline. The higher resistance rate was observed for erythromycin (28%), and 14% were resistant to clindamycin, including 2 MSSA isolates with inducible resistance by erythromycin. MRSA isolates presented oxacillin MIC90 of 16 μg/mL and for vancomycin of 2 μg/mL. For MSSA isolates, both the MIC90 values were 1 μg/mL (data not shown). One isolate, number 1342a, which was obtained from bloodstream infection of a male newborn, with 2405 g admitted at a neonatal intensive care unit, suffering from meconium aspiration syndrome exhibited vancomycin MIC of 4 μg/mL. It was confirmed as vancomycin-intermediate S. aureus (VISA) by populational analysis profile (PAP) (Nunes et al., 2006), and its agr polymorphism was determined (Gilot et al., 2002) as type 3 (Supplementary Material). S. aureus isolates were distributed in 15 lineages, with most of the isolates (35/70%) included in 3 lineages: USA1100/ST30/CC30 (25 isolates), USA400/ST1/CC1 (7 isolates) and USA800/ST/CC5 (3 isolates), and 94% of these isolates were MRSA (Fig. 1). The ST2369 of the CC30 was described for the first time in the present study. The USA1100/ ST30/CC30 isolates were present in 8 hospitals, between 2006 and 2009 and in 2012. Eighteen (72%) of them were detected as MRSA and presented the same PFGE subtype (Table 1). These identical isolates
http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Chamon RC, et al, Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro ho..., Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004
2
R.C. Chamon et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Fig. 1. Dendrogram of the PFGE patterns and characteristics related to the genetic background of 50 PVL-positive S. aureus isolates. Isolates showing a similarity coefficient ≥80% were considered genetically related. ST = sequence type; CC = clonal complex. One isolate, 1155a, was used as control of the USA1100/ST30 A1 pulsotype (Schuenck et al., 2012).
were detected in 7 hospitals in 4 different periods. Over 11 months, 8 isolates presenting similar resistance and virulence gene profiles were recovered at hospital H1. Similarly, 4 isolates were recovered from blood of newborns at hospital H3 in a period of 5 months during 2009. One of these isolates (number 1342) was characterized as VISA. Among the virulence genes analyzed, the bbp was found in all USA1100/ST30/CC30 isolates (P b 0.05), and the seh was related to the USA400/ST1/CC1 isolates (P b 0.05). Overall, the enterotoxins genes were not related to a specific lineage, being the sea gene the most prevalent one (80%). Some virulence genes were rare, such as the fnbB gene found in 2 isolates (USA300/ST8 and ST97 lineages) and the tst gene, related to the ST484/CC30 and ST97/CC1 isolates. S. aureus PVL-positive isolates have been related to severe disease (Tristan et al., 2007; Cavalcante et al., 2015) making its characterization very important. Furthermore, analysis of a collection of bacterial isolates having a common characteristic, such as PVL genes, can help to understand the relationship between them. However, in general, available studies demonstrate only the occurrence of these isolates (Nichol et al., 2013; Cavalcante et al., 2015; Caboclo et al., 2013; Egea et al., 2014; Fernandez et al., 2012; Schuenck et al., 2012). In the present study, we analyzed a collection of 50 PVL-positive S. aureus isolates obtained in a 6-year period in Rio de Janeiro hospitals as their phenotypic and molecular characteristics. Despite most of our isolates present a community-associated profile, we could not inferred that our isolates were community acquired, as we did not had access to most patient's clinical data, such as day of admission and previous risk factors for MRSA infection, being this is a limiting factor of the study.
The USA1100/ST30/CC30 lineage was present in majority of Rio de Janeiro hospitals. This lineage carrier of PVL genes had already been reported in Argentina (Egea et al., 2014; Fernandez et al., 2012) and in Brazil (Cavalcante et al., 2015; Caboclo et al., 2013; Schuenck et al., 2012). Moreover, in the present study, a VISA isolate belonging to USA1100/ST30 was identified, indicating the relevance of this lineage among the PVL-positive MRSA isolates. Although there are studies reporting the occurrence of S. aureus isolates carrying the PVL genes capable to express the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype (Maor et al., 2009), to our knowledge, this is the first study to identify a PVL-positive VISA isolate belonging to USA1100/ST30/CC30 lineage. The bbp virulence gene, whose presence could be associated to osteomyelitis and arthritis (Tristan et al., 2003) had been related to the ST30 S. aureus lineage found in an orthopedic hospital (Schuenck et al., 2012). Here, we also found a correlation between the ST30 and the bbp gene, suggesting that this gene could be a possible marker of this lineage. Similarly, we observed that the seh gene was associated with the USA400/ ST1/CC1 lineage, as previously reported (Tristan et al., 2007). It is interesting to mention that, although the USA400 lineage is becoming prevalent in Brazilian hospitals, it is usually negative for PVL genes (Caboclo et al., 2013; Schuenck et al., 2012), contrary to what is observed at the USA (Tristan et al., 2007). Further studies characterizing such a collection of PVL-positive S. aureus isolates and their relationship to genetic backgrounds are necessary, as these lineages are becoming more common in the hospital environment affecting patients already debilitated. This study showed a
Please cite this article as: Chamon RC, et al, Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro ho..., Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004
R.C. Chamon et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
3
Table 1 Clinical and microbiological aspects of 25 isolates belonging to the PVL-positive Brazilian prevalent lineage USA1100/ST30/CC30. Hospital (number of isolates) H1 (n = 11) 601 596 809 806 826 827 932 943 1086 1079 1195 H2 (n = 3) 1155 1165 1174 H3 (n = 4) 1341 1342 1343 1352 H5 (n = 1) 1012 H6 (n = 1) 1013 H7 (n = 2) 1179 1180 H8 (n = 2) 1463 1462 H10 (n = 1) 1025
Isolation date (dd/mm/yy)
Isolation source
PFGE profile
SCCmec type
09/01/2006 02/02/2006 06/07/2006 16/08/2006 24/10/2006 24/10/2006 14/11/2006 14/11/2006 10/01/2008 03/04/2008 20/05/2008
SSTI SSTI Nostril Nostril BSI SSTI Nostril SSTI BSI BSI BSI
A1 A1 A1 A1 A1 A1 A1 A1 A4 A6 A4
IV IV IV IV IV IV IV IV NA NA NA
03/05/2006 01/08/2006 17/01/2007
Bone secretion SSTI SSTI
A1 A4 A5
24/02/2009 24/03/2009 30/03/2009 10/06/2009
BSI BSI BSI BSI
20/08/2007
Resistance profile
MIC (mg/mL)
Virulence genes profilea
OXA
VAN
fox, eri fox fox fox fox fox fox fox NA sut sut, tet
4 4 4 2 1 1 0.5 8 0.25 0.125 0.125
0.5 2 1 1 1 1 1 1 1 1 1
fnbA, sea, seb fnbA, sea sea, seb, seh sea fnbA, sea, seb fnbA, sea, seh seh fnbA, sea, seh fnbA, sea NA fnbA, sea, seb
IV NA NA
fox sut, tet clo
8 0.5 1
1 1 1
fnbA, sea fnbA, sea, seb fnbA
A1 A1 A1 A1
IV IV IV IV
fox fox fox fox
2 8 2 2
2 4 2 2
sea sea fnbA, sea fnbA, sea, seh
BSI
A1
IV
fox, cli, eri
4
0.5
fnbA, sea
22/08/2007
SSTI
A1
IV
fox, tec
1
1
fnbA, sea, seb
05/02/2009 09/02/2009
SSTI SSTI
A1 A1
IV IV
fox fox, eri, tet
8 16
1 1
fnbA, sea sea
18/04/2012 18/04/2012
SSTI SSTI
A1 A3
IV NA
fox clo
4 2
1 2
sec, sed, see sec, sed, see
18/11/2007
SSTI
A2
IV
fox
4
1
fnbA, sea
SSTI = skin and soft tissue infection; BSI = bloodstream infection; NA = Not applicable (MSSA isolates); fox = cefoxitin; eri = erythromycin; cli = clindamycin; tec = teicoplanin; tet = tetracycline; OXA = oxacillin; VAN = vancomycin. a All isolates also presented the adhesion genes: bbp, ebpS, cna, clfA, and clfB.
USA1100/ST30/CC30 PVL-positive S. aureus lineage present in majority of Rio de Janeiro hospitals and its association with the bbp gene. Moreover, the occurrence of a VISA isolate causing bloodstream infection in a newborn, among this well-adapted PVL-positive MRSA lineage is of great concern, since vancomycin is the antimicrobial of choice to treat invasive MRSA infections. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2015.09.004. Competing interests None declared. Acknowledgments This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento Pessoal de Nível Superior, Fundação Universitária José Bonifácio, and Programa de Núcleos de Excelência. We are indebted to Dr Dennis de Carvalho Ferreira (Programa de Pós Graduação em Odontologia, Universidade Estácio de Sá), Dr Simone Aranha Nouér (Faculdade de Medicina, Universidade Federal do Rio de Janeiro), and Pharmacist Fernanda Maia (Laboratório de Bacteriologia, Hospital Naval Marcilio Dias) for providing some of the staphylococcal isolates and Dr Rosana Barreto Rocha Ferreira (Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro) for providing language help.
References Boan P, Tan HL, Pearson J, Coombs G, Heath CH, Robinson JO. Epidemiological, clinical, outcome and antibiotic susceptibility differences between PVL positive and PVL negative Staphylococcus aureus infections in Western Australia: a case control study. BMC Infect Dis 2015;15:10. Caboclo RM, Cavalcante FS, Iorio NL, Schuenck RP, Olendzki AN, Felix MJ, et al. Methicillinresistant Staphylococcus aureus in Rio de Janeiro hospitals: dissemination of the USA400/ST1 and USA800/ST5 SCCmec type IV and USA100/ST5 SCCmec type II lineages in a public institution and polyclonal presence in a private one. Am J Infect Control 2013;41(3):e21–6. Cavalcante FS, Abad ED, Lyra YC, Saintive SB, Ribeiro M, Ferreira DC, et al. High prevalence of methicillin resistance and PVL genes among Staphylococcus aureus isolates from the nares and skin lesions of pediatric patients with atopic dermatitis. Braz J Med Biol Res 2015;48(7):588–94. Clinical Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing. M02-A11. Wayne, PA: National Committee for Clinical Laboratory Standards; 2012. David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev 2010;23(3):616–87. Egea AL, Gagetti P, Lamberghini R, Faccone D, Lucero C, Vindel A, et al. New patterns of methicillin-resistant Staphylococcus aureus (MRSA) clones, community-associated MRSA genotypes behave like healthcare-associated MRSA genotypes within hospitals, Argentina. Int J Med Microbiol 2014;304(8):1086–99. Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38(3):1008–15. European Committee on Antimicrobial Susceptibility Testing. MIC and inhibition zone diameter distributions of microorganisms without and with resistance mechanisms. http://www.eucast.org/mic_distributions/, 2011. Fernandez S, de Vedia L, Lopez Furst MJ, Gardella N, Di Gregorio S, Ganaha MC, et al. Methicillin-resistant Staphylococcus aureus ST30-SCCmec IVc clone as the major cause of community-acquired invasive infections in Argentina. Infect Genet Evol 2012;14:401–5. Finlay JE, Miller LA, Poupard JA. Interpretive criteria for testing susceptibility of staphylococci to mupirocin. Antimicrob Agents Chemother 1997;41(5):1137–9.
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glycopeptide susceptibility profiles and cell wall thickening. Int J Antimicrob Agents 2006;27(4):307–15. Peacock SJ, Moore CE, Justice A, Kantzanou M, Story L, Mackie K, et al. Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infect Immun 2002;70:4987–96. Schuenck RP, Cavalcante FS, Emery E, Giambiagi-de Marval M, dos Santos KR. Staphylococcus aureus isolates belonging to different multilocus sequence types present specific virulence gene profiles. FEMS Immunol Med Microbiol 2012;65(3):501–4. Sila J, Sauer P, Kolar M. Comparison of the prevalence of genes coding for enterotoxins, exfoliatins, panton-valentine leukocidin and tsst-1 between methicillin-resistant and methicillin-susceptible isolates of Staphylococcus aureus at the university hospital in Olomouc. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009;153(3):215–8. Tristan A, Ying L, Bes M, Etienne J, Vandenesch F, Lina G. Use of multiplex PCR to identify S. aureus adhesions involved in human haematogenous infections. J Clin Microbiol 2003;41:4465–7. Tristan A, Bes M, Meugnier H, Lina G, Bozdogan B, Courvalin P, et al. Global distribution of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus, 2006. Emerg Infect Dis 2007;13(4):594–600. Vivoni AM, Santos KR, de-Oliveira MP, Giambiagi-deMarval M, Ferreira AL, Riley LW, et al. Mupirocin for controlling methicillin-resistant Staphylococcus aureus: lessons from a decade of use at a university hospital. Infect Control Hosp Epidemiol 2005;26(7): 662–7.
Please cite this article as: Chamon RC, et al, Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro ho..., Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Note
Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro hospitals Raiane Cardoso Chamon a, Natalia Lopes Pontes Iorio b, Sthefanie da Silva Ribeiro a, Fernanda Sampaio Cavalcante a, Kátia Regina Netto dos Santos a,⁎ a b
Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Departamento de Ciências Básicas, Universidade Federal Fluminense, Nova Friburgo, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 8 July 2015 Received in revised form 22 August 2015 Accepted 6 September 2015 Available online xxxx
a b s t r a c t In a collection of 50 pvl-positive Staphylococcus aureus isolates from 10 Rio de Janeiro hospitals, 18 (36%) were from bloodstream infections, and 31 (62%) carried the SCCmec IV. Among 25 (50%) isolates of the USA1100/ ST30/CC30 lineage present in 8 hospitals, 1 isolate was characterized as vancomycin-intermediate S. aureus. © 2015 Elsevier Inc. All rights reserved.
Keywords: Staphylococcus aureus USA1100/ST30/CC30 Panton-Valentine leukocidin Vancomycin-intermediate S. aureus
Staphylococcus aureus is an important cause of infections worldwide (David and Daum, 2010), and the presence of genes that encode for Panton-Valentine leukocidin (PVL) is associated to a wide range of diseases, from uncomplicated to severe infections (Tristan et al., 2007). The occurrence of PVL is associated with community-acquired S. aureus isolates, both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) (Boan et al., 2015). However, recent reports had described the presence of hospital-acquired isolates carrying these genes (Nichol et al., 2013), indicating a changing in epidemiology of PVL-positive S. aureus isolates. In the current study, we characterized 50 PVL-positive S. aureus isolates, detected among 940 isolates of our collection and isolated from 10 different hospitals in Rio de Janeiro, between 2004 and 2012. They were isolated from bloodstream infections (BSIs) (18 isolates/ 36%), skin and soft tissue infections (SSTIs) (11/22%), nostrils (9/18%), and other clinical sources (12/24%). The antimicrobial susceptibility was determined for 12 agents by the disk diffusion method, according to the CLSI (2012), except for mupirocin (Finlay et al., 1997) and tigecycline (European Committee on Antimicrobial Susceptibility Testing, 2011). The MIC for oxacillin and vancomycin was determined by broth microdilution test (CLSI, 2012). The mecA gene and SCCmec types were detected by PCR (Milheiriço et al., 2007), and the clonal lineages were determined by pulsed field gel electrophoresis (PFGE) (Vivoni et al., 2005) and Multilocus Sequence Typing (MLST) method (Enright et al., 2000). The presence of genes that encode for adhesins, such as bbp (bone sialo⁎ Corresponding author. Tel.: +55-21-2560-8344; fax: +55-21-2560-8028. E-mail address: [email protected] (K.R.N. dos Santos).
binding protein), cna (collagen-binding protein), ebpS (elastin-binding protein), fnbB (fibronectin-binding protein B) (Tristan et al., 2003), and fnbA (fibronectin-binding protein A) (Peacock et al., 2002), as well as enterotoxins sea-see (Johnson et al., 1991; Lovseth et al., 2004; Monday and Bohach, 1999) and seh (Sila et al., 2009), and toxin esfoliative (tst) (Johnson et al., 1991) were investigated by PCR. Among the 50 PVL-positive S. aureus isolates, 31 were MRSA and carried the SCCmec type IV, and 20 of them were resistant only to β-lactams. All 50 isolates were susceptible for linezolid, mupirocin, rifampin, and tigecycline. The higher resistance rate was observed for erythromycin (28%), and 14% were resistant to clindamycin, including 2 MSSA isolates with inducible resistance by erythromycin. MRSA isolates presented oxacillin MIC90 of 16 μg/mL and for vancomycin of 2 μg/mL. For MSSA isolates, both the MIC90 values were 1 μg/mL (data not shown). One isolate, number 1342a, which was obtained from bloodstream infection of a male newborn, with 2405 g admitted at a neonatal intensive care unit, suffering from meconium aspiration syndrome exhibited vancomycin MIC of 4 μg/mL. It was confirmed as vancomycin-intermediate S. aureus (VISA) by populational analysis profile (PAP) (Nunes et al., 2006), and its agr polymorphism was determined (Gilot et al., 2002) as type 3 (Supplementary Material). S. aureus isolates were distributed in 15 lineages, with most of the isolates (35/70%) included in 3 lineages: USA1100/ST30/CC30 (25 isolates), USA400/ST1/CC1 (7 isolates) and USA800/ST/CC5 (3 isolates), and 94% of these isolates were MRSA (Fig. 1). The ST2369 of the CC30 was described for the first time in the present study. The USA1100/ ST30/CC30 isolates were present in 8 hospitals, between 2006 and 2009 and in 2012. Eighteen (72%) of them were detected as MRSA and presented the same PFGE subtype (Table 1). These identical isolates
http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004 0732-8893/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Chamon RC, et al, Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro ho..., Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004
2
R.C. Chamon et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
Fig. 1. Dendrogram of the PFGE patterns and characteristics related to the genetic background of 50 PVL-positive S. aureus isolates. Isolates showing a similarity coefficient ≥80% were considered genetically related. ST = sequence type; CC = clonal complex. One isolate, 1155a, was used as control of the USA1100/ST30 A1 pulsotype (Schuenck et al., 2012).
were detected in 7 hospitals in 4 different periods. Over 11 months, 8 isolates presenting similar resistance and virulence gene profiles were recovered at hospital H1. Similarly, 4 isolates were recovered from blood of newborns at hospital H3 in a period of 5 months during 2009. One of these isolates (number 1342) was characterized as VISA. Among the virulence genes analyzed, the bbp was found in all USA1100/ST30/CC30 isolates (P b 0.05), and the seh was related to the USA400/ST1/CC1 isolates (P b 0.05). Overall, the enterotoxins genes were not related to a specific lineage, being the sea gene the most prevalent one (80%). Some virulence genes were rare, such as the fnbB gene found in 2 isolates (USA300/ST8 and ST97 lineages) and the tst gene, related to the ST484/CC30 and ST97/CC1 isolates. S. aureus PVL-positive isolates have been related to severe disease (Tristan et al., 2007; Cavalcante et al., 2015) making its characterization very important. Furthermore, analysis of a collection of bacterial isolates having a common characteristic, such as PVL genes, can help to understand the relationship between them. However, in general, available studies demonstrate only the occurrence of these isolates (Nichol et al., 2013; Cavalcante et al., 2015; Caboclo et al., 2013; Egea et al., 2014; Fernandez et al., 2012; Schuenck et al., 2012). In the present study, we analyzed a collection of 50 PVL-positive S. aureus isolates obtained in a 6-year period in Rio de Janeiro hospitals as their phenotypic and molecular characteristics. Despite most of our isolates present a community-associated profile, we could not inferred that our isolates were community acquired, as we did not had access to most patient's clinical data, such as day of admission and previous risk factors for MRSA infection, being this is a limiting factor of the study.
The USA1100/ST30/CC30 lineage was present in majority of Rio de Janeiro hospitals. This lineage carrier of PVL genes had already been reported in Argentina (Egea et al., 2014; Fernandez et al., 2012) and in Brazil (Cavalcante et al., 2015; Caboclo et al., 2013; Schuenck et al., 2012). Moreover, in the present study, a VISA isolate belonging to USA1100/ST30 was identified, indicating the relevance of this lineage among the PVL-positive MRSA isolates. Although there are studies reporting the occurrence of S. aureus isolates carrying the PVL genes capable to express the heterogeneous vancomycin-intermediate S. aureus (hVISA) phenotype (Maor et al., 2009), to our knowledge, this is the first study to identify a PVL-positive VISA isolate belonging to USA1100/ST30/CC30 lineage. The bbp virulence gene, whose presence could be associated to osteomyelitis and arthritis (Tristan et al., 2003) had been related to the ST30 S. aureus lineage found in an orthopedic hospital (Schuenck et al., 2012). Here, we also found a correlation between the ST30 and the bbp gene, suggesting that this gene could be a possible marker of this lineage. Similarly, we observed that the seh gene was associated with the USA400/ ST1/CC1 lineage, as previously reported (Tristan et al., 2007). It is interesting to mention that, although the USA400 lineage is becoming prevalent in Brazilian hospitals, it is usually negative for PVL genes (Caboclo et al., 2013; Schuenck et al., 2012), contrary to what is observed at the USA (Tristan et al., 2007). Further studies characterizing such a collection of PVL-positive S. aureus isolates and their relationship to genetic backgrounds are necessary, as these lineages are becoming more common in the hospital environment affecting patients already debilitated. This study showed a
Please cite this article as: Chamon RC, et al, Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro ho..., Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004
R.C. Chamon et al. / Diagnostic Microbiology and Infectious Disease xxx (2015) xxx–xxx
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Table 1 Clinical and microbiological aspects of 25 isolates belonging to the PVL-positive Brazilian prevalent lineage USA1100/ST30/CC30. Hospital (number of isolates) H1 (n = 11) 601 596 809 806 826 827 932 943 1086 1079 1195 H2 (n = 3) 1155 1165 1174 H3 (n = 4) 1341 1342 1343 1352 H5 (n = 1) 1012 H6 (n = 1) 1013 H7 (n = 2) 1179 1180 H8 (n = 2) 1463 1462 H10 (n = 1) 1025
Isolation date (dd/mm/yy)
Isolation source
PFGE profile
SCCmec type
09/01/2006 02/02/2006 06/07/2006 16/08/2006 24/10/2006 24/10/2006 14/11/2006 14/11/2006 10/01/2008 03/04/2008 20/05/2008
SSTI SSTI Nostril Nostril BSI SSTI Nostril SSTI BSI BSI BSI
A1 A1 A1 A1 A1 A1 A1 A1 A4 A6 A4
IV IV IV IV IV IV IV IV NA NA NA
03/05/2006 01/08/2006 17/01/2007
Bone secretion SSTI SSTI
A1 A4 A5
24/02/2009 24/03/2009 30/03/2009 10/06/2009
BSI BSI BSI BSI
20/08/2007
Resistance profile
MIC (mg/mL)
Virulence genes profilea
OXA
VAN
fox, eri fox fox fox fox fox fox fox NA sut sut, tet
4 4 4 2 1 1 0.5 8 0.25 0.125 0.125
0.5 2 1 1 1 1 1 1 1 1 1
fnbA, sea, seb fnbA, sea sea, seb, seh sea fnbA, sea, seb fnbA, sea, seh seh fnbA, sea, seh fnbA, sea NA fnbA, sea, seb
IV NA NA
fox sut, tet clo
8 0.5 1
1 1 1
fnbA, sea fnbA, sea, seb fnbA
A1 A1 A1 A1
IV IV IV IV
fox fox fox fox
2 8 2 2
2 4 2 2
sea sea fnbA, sea fnbA, sea, seh
BSI
A1
IV
fox, cli, eri
4
0.5
fnbA, sea
22/08/2007
SSTI
A1
IV
fox, tec
1
1
fnbA, sea, seb
05/02/2009 09/02/2009
SSTI SSTI
A1 A1
IV IV
fox fox, eri, tet
8 16
1 1
fnbA, sea sea
18/04/2012 18/04/2012
SSTI SSTI
A1 A3
IV NA
fox clo
4 2
1 2
sec, sed, see sec, sed, see
18/11/2007
SSTI
A2
IV
fox
4
1
fnbA, sea
SSTI = skin and soft tissue infection; BSI = bloodstream infection; NA = Not applicable (MSSA isolates); fox = cefoxitin; eri = erythromycin; cli = clindamycin; tec = teicoplanin; tet = tetracycline; OXA = oxacillin; VAN = vancomycin. a All isolates also presented the adhesion genes: bbp, ebpS, cna, clfA, and clfB.
USA1100/ST30/CC30 PVL-positive S. aureus lineage present in majority of Rio de Janeiro hospitals and its association with the bbp gene. Moreover, the occurrence of a VISA isolate causing bloodstream infection in a newborn, among this well-adapted PVL-positive MRSA lineage is of great concern, since vancomycin is the antimicrobial of choice to treat invasive MRSA infections. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2015.09.004. Competing interests None declared. Acknowledgments This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento Pessoal de Nível Superior, Fundação Universitária José Bonifácio, and Programa de Núcleos de Excelência. We are indebted to Dr Dennis de Carvalho Ferreira (Programa de Pós Graduação em Odontologia, Universidade Estácio de Sá), Dr Simone Aranha Nouér (Faculdade de Medicina, Universidade Federal do Rio de Janeiro), and Pharmacist Fernanda Maia (Laboratório de Bacteriologia, Hospital Naval Marcilio Dias) for providing some of the staphylococcal isolates and Dr Rosana Barreto Rocha Ferreira (Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro) for providing language help.
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Please cite this article as: Chamon RC, et al, Molecular characterization of Staphylococcus aureus isolates carrying the Panton-Valentine leukocidin genes from Rio de Janeiro ho..., Diagn Microbiol Infect Dis (2015), http://dx.doi.org/10.1016/j.diagmicrobio.2015.09.004