Abstract. Background. Editor: G. Lina

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1 ORIGINAL ARTICLE BACTERIOLOGY Evidence of transmission of a Panton Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus clone: a family affair P. Cocchi 1, G. Taccetti 2, C. Montagnani 1, S. Campana 2, L. Galli 1, C. Braggion 2 and M. de Martino 1 1) Department of Sciences for Woman and Child s Health, University of Florence and 2) Cystic Fibrosis Centre, Anna Meyer Children s University Hospital, Florence, Italy Abstract Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) may represent a serious public health problem, owing to the spread of toxin-producing lineages. The presence of genes encoding for Panton Valentine leukocidin (PVL) is an important virulence marker, as the clinical sequelae of PVL-positive infections are often described as more severe than those of PVL-negative S. aureus infections. To date, the presence of PVL has not appeared to be common in Italy; we describe the intrafamilial transmission of an epidemic PVL-producing CA-MRSA lineage, Southwest Pacific clone (SWP). Our data suggested that the strain circulated from the father, who was recurrently affected by a soft tissue infection, to the mother, who showed nasal colonization, and to their child, who was hospitalized with symptoms of necrotizing pneumonia. In this case, we found that a recurrent skin infection that is not normally taken into account may represent a serious threat if caused by a PVL-producing strain. Our findings may have considerable implications for strategies for infection control and treatment of methicillin-resistant S. aureus infections. Keywords: CA-MRSA, infection, MLST, PVL, transmission Original Submission: 29 October 2012; Revised Submission: 31 December 2012; Accepted: 9 January 2013 Editor: G. Lina Article published online: 17 January 2013 Clin Microbiol Infect 2013; 19: / Corresponding author: P. Cocchi, Cystic Fibrosis Laboratory, Department of Sciences for Woman and Child s Health, University of Florence, Florence, Italy s: p.cocchi@meyer.it, priscilla.cocchi@unifi.it Background Currently, methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequently isolated pathogens, especially in clinical settings. Since its discovery, MRSA has been found to develop antimicrobial resistance extremely rapidly. In the early 1940s, before the introduction of penicillin and the treatment of S. aureus infections with this antibiotic, the mortality rate of patients with S. aureus infection was approximately 80% [1]. A few years later, penicillin was introduced for medical purposes, and penicillin-resistant strains emerged, first in clinical settings and then in the community. The progressive introduction of new antimicrobial agents has been a powerful factor triggering MRSA to develop resistance mechanisms. The acquisition of meca generated the first MRSA strain [1]. This gene is located in a genetic determinant named staphylococcal chromosome cassette mec (SCCmec). Several types of SCCmec have been distinguished, from I to XI, differing in size and content [2]. Some cassettes can confer resistance to b-lactam antibiotics only; some others are able to confer multiple resistance determinants, owing to integrated plasmids or transposons [1]. The original criteria for distinguishing between hospital-acquired and community-acquired infections were suggested by the CDC, and were based on a defined group of risk factors, such as hospitalization, invasive clinical practices, and staying in long-term-care facilities [1]. From the late 1990s, many reports appeared concerning MRSA colonization and infection involving patients lacking the previously described risk factors. Community-acquired MRSA Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases

2 CMI Cocchi et al. Transmission of PVL-positive CA-MRSA clone 1159 (CA-MRSA) clones were spreading worldwide. Recently, CA-MRSA has started to replace hospital-acquired MRSA (HA-MRSA) in hospital settings, especially in areas with a high prevalence of CA-MRSA [1]. On the basis of the differences in SCCmec genomic islands, it was possible to define differences between CA-MRSA and HA-MRSA [1], and to obtain a likely picture of MRSA epidemiology. CA-MRSA strains are usually characterized by a smaller SCCmec genomic island than HA-MRSA, and a different genetic background: CA-MRSA strains belong to unrelated clonal types [1]. Different sizes of SCCmec elements between CA-MRSA and HA-MRSA correspond to different antimicrobial resistance patterns; HA-MRSA strains are more often multidrug-resistant than are CA-MRSA strains, which tend to be more virulent [3]. CA-MRSA can cause a wide range of diseases, from soft tissue infections to highly invasive necrotizing pneumonia, severe sepsis, and necrotizing fasciitis [3], as a consequence of its wide repertoire of transferable toxin genes. Among this plethora of genes, those encoding Panton Valentine leukocidin (PVL) constitute the most consistently present transferable toxin locus in CA-MRSA. PVL is a bi-component pore-forming leukotoxin that was associated with skin and soft tissue infections in 1932 by Panton and Valentine [3]. This toxin is encoded by two contiguous and co-transcribed genes, lukf-pv and luks-pv, which reside in the genomes of several different temperate bacteriophages [4]. Infiltration of such bacteriophages in different lineages of MRSA caused the spread of a polyclonal population of CA-MRSA producing PVL [4]. Several sequence types (STs) of PVL-positive MRSA are known, and some of these strains occur and predominate in certain regions [5], such as USA300 in North America, and ST30-MRSA-IV, South West Pacific clone (SWP) in South America. Moreover, different PVL-positive clones were recently described as ST88 and ST1820 in Tanzania [6], and ST152-MRSA-V or Balkan clone [8] in Serbia. SWP was defined as ubiquitous because of its wide distribution all over the world [7,9 12]. This particular clone has been reported to be responsible for mild and severe infections in Brazil [12,13,32], and also in Europe [2,14]. Strains and Methods Bacterial culture and biochemical identification Pleural drainage specimens and parental nasal samples were cultured by inoculating plates of agar selective for S. aureus (Mannitol Salt 2 Agar; biomerieux, Marcy L Etoile, France), and three strains were obtained, one from each sample. To confirm the species-level identification, the Slidex Staph Plus (Bio-Rad, Hercules, CA, USA) test was performed, and strains were identified with Vitek (Vitek 2; biomerieux). Antimicrobial susceptibility testing The three strains obtained by culture were tested with Vitek (Vitek 2; biomerieux), using a P580card. Antimicrobial susceptibility patterns were determined according to the CLSI recommendations [15]. A panel of antimicrobial agents was tested, including: oxacillin, penicillin G, vancomycin, teicoplanin, linezolid, gentamicin, tobramycin, norfloxacin, levofloxacin, moxifloxacin, erythromycin, clindamycin, tetracycline, rifampicin, fosfomycin, mupirocin, fusidic acid, trimethoprim sulphamethoxazole, and nitrofurantoin. Susceptibility to glycopeptides was evaluated with the Etest macromethod. Intraspecies genotyping DNA extraction was performed according to the manufacturer s instructions with a Nucleo Spin Tissue kit (Macherey-Nagel, D uren, Germany), with lysostaphin at 0.5 mg/ml for the lysis step. The first typing method that we used to assess strain sharing was ERIC-PCR, performed according to the method of Struelens et al. [16]. SCCmec typing The structure of the SCCmec element was determined with the multiplex PCR strategy developed by Oliveira and de Lancastre in 2002 [17]. PVL testing The presence of lukf-pv and luks-pv was tested by means of PCR, according to the protocol of Lina et al. [18]. Multilocus sequence typing (MLST) MLST analysis was performed as previously described [19], and STs were attributed by submitting the DNA sequences obtained to the online MLST database ( Bacterial strains A 3-month-old Brazilian child was admitted to hospital because of respiratory distress and poor feeding. He was affected by necrotizing pneumonia with pyopneumothorax, and a pleural drain was positioned. Parental nasal swabs were collected for infection control purposes. Results The first MRSA strain isolated was from a 3-month-old child from Brazil, and living in Italy with his Brazilian family. The infant was previously healthy, and was admitted to hospital because of respiratory distress and poor feeding; he was

1160 Clinical Microbiology and Infection, Volume 19 Number 12, December 2013 CMI started on ampicillin sulbactam and gentamicin after blood test results suggestive of infection had been obtained.

3 1160 Clinical Microbiology and Infection, Volume 19 Number 12, December 2013 CMI started on ampicillin sulbactam and gentamicin after blood test results suggestive of infection had been obtained. A pleural drain was positioned, from which we were able to isolate an MRSA strain. Parental nasal swabs were collected to highlight the presence of MRSA, both giving positive results. All of these isolates the one from the pleural drain and the nasal strains from parental specimens showed the same antimicrobial susceptibility patterns and the same MIC values, each strain being resistant to b-lactams only, as shown in Table 1. The presence of genes encoding for PVL was tested by means of PCR, and lukf-pv and luks-pv were found in all three isolates. A PCR assay to highlight DNA fingerprint similarities was performed as a first step of intraspecies typing, with the random primers ERIC1R and ERIC2 [16]. All isolates matched as a unique genotype, as shown in Fig. 1. Subsequently, we tested these three isolates to determine which SCCmec and ST they belonged to: SCCmec typing and MLST analysis allowed us to ascribe all isolates to SWP, characterized by SCCmec type IV and ST30. Discussion A report by Huijsdens et al. demonstrated that family members can serve as reservoirs of PVL-positive MRSA and that transmission can occur among them [20]. Perez-Roth et al. recently showed that soft tissue infections can be caused by PVL-positive methicillin-sensitive S. aureus belonging to ST152 and shared by different members of the same family [21]. Our recent finding of an entire family sharing the same SWP strain, one of the ancient CA-MRSA lineages, indicates a worrying TABLE 1. Antimicrobial susceptibility profiles of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates Antimicrobial agent MIC (lg/l) son s pleural drainage father s nasal swab mother s nasal swab Clindamycin Erythromycin Fosfomycin Fusidic acid Gentamicin Levofloxacin Linezolid Moxifloxacin Mupirocin Oxacillin Rifampicin Tigecycline Trimethoprim sulphamethoxazole Vancomycin Penicillin G Tobramycin Teicoplanin Tetracycline Nitrofurantoin FIG. 1. Intraspecies genotyping of methicillin-resistant Staphylococcus aureus (MRSA) from three family members. Lane L: DNA ladder, a mixture of DNA ladders XIII and XIV (Roche Diagnostics, Basel, Switzerland). Lanes 1 3: son s pleural drainage, father s nasal swab, and mother s nasal swab, respectively; ERIC1R primer. Lanes 4 6: son s pleural drainage, father s nasal swab, and mother s nasal swab, respectively; ERIC2 primer. possibility of intrafamilial transmission of an already described epidemic clone. In the past few years, isolation of CA-MRSA has been more frequent, especially in geographical areas with a high prevalence, where these strains have also started to replace HA-MRSA in hospital settings [1]. Because of its virulence, CA-MRSA could be a serious public health problem. In particular, the presence of PVL is an important virulence marker. The clinical sequelae of PVL-positive infections are often described as more severe than those of PVL-negative infections [3]. PVL-positive CA-MRSA can be ascribed to six different genetic lineages [10], to which the presence of PVL genes may have conferred better fitness. Moreover, it is known that the genetic backgrounds of CA-MRSA strains are distinct in different geographical locations, and that different genetic backgrounds can exist within a small area [3]. In Europe, the most frequently isolated CA-MRSA clones are USA300-related and European clone-related, and SWP appears to be sporadically present, as is the Queensland clone (ST93-MRSA-IV) [2]. Concerning PVL producers, the most prevalent CA-MRSA clones are ST80 in Europe, USA300 in the USA, and the ubiquitous SWP. The last of these has been recognized as being responsible for severe infections [13,22,23], as well as intrafamilial transmission [10,22,24 27]. Maternal carriage may cause infant colonization, but this hypothesis has been rarely tested so far [24]. A recent study

4 CMI Cocchi et al. Transmission of PVL-positive CA-MRSA clone 1161 pointed out that colonized mothers shared their strains with children during the first months after birth and, interestingly, one of the most frequent clones was SWP [24]. In this particular case, the child s father had a previous history of recurrent furuncles, allowing the hypothesis that he was the first member of the family to be infected. The mother tested positive for nasal colonization, and the strain found was the same as that in her husband and child, as shown by both ERIC-PCR and MLST and SCCmec typing, suggesting that she may have transmitted the infection to her son. The first step used to compare DNA fingerprints was a rapid method that we chose to assess the presence of a shared genotype. Although this method is less discriminative than pulsed-field gel electrophoresis [16], in these particular circumstances it was a reliable and robust first screening tool. The need to test strains that were strongly suspected of belonging to the same clone, as suggested by antimicrobial susceptibility patterns and the presence of PVL, led us to apply this DNA fingerprinting method to confirm this suspicion. At the same time, these three strains were tested by means of SCCmec typing and MLST, to complete the genotyping, and this analysis highlighted the presence of the already described epidemic clone. We found that all MRSA isolates from samples of the members of this family belong to SWP, which is often described as a ubiquitous MRSA PVL-producing strain, with diffusion especially in Asia and South America. Recent reports have described the Italian situation with regard to CA-MRSA diffusion [28 31]. Sanchini et al. collected 18 strains from all over Italy, and showed that the most frequent clone was USA300, but only one of these tested positive for PVL [28]. In the northern part of Italy, SWP was recently described in unrelated infectious episodes [29]. In the southern part of Italy, recent reports indicate that different CA-MRSA lineages are well represented in Palermo [30,31]. To date, the presence of PVL does not appear to be common in Italy, but the distribution of this peculiar virulence factor in the community should be adequately investigated. Our description of the intrafamilial transmission of an epidemic PVL-positive CA-MRSA clone is one of a few cases that have been described [28,29,31]. Interestingly, this family originated from Brazil, an area endemic for SWP, as well as for other clones, such as USA300 and USA400 [32], suggesting direct importation of this infectious agent. This is important for understanding the changing picture of MRSA epidemiology, strain migration, and the replacement of different clones in different areas. Moreover, it is important to note that a recurrent skin infection that is normally not taken into account may be a serious threat when caused by a PVL-producing strain. The importance of correct antimicrobial treatment of a soft tissue recurrent infection is clear, as the first infected patient was able to transmit the same strain to the infant, with a more severe clinical onset. It has been reported that some individuals are more likely to be carriers than others, so understanding the type of strain that they harbour may be important for the development of decontamination strategies [33]. More aggressive decolonization may be suggested for PVL-producing infecting strains. These data may have considerable implications for strategies for infection control and the treatment of MRSA infections. In particular, this intrafamilial transmission suggests the need for more careful molecular surveillance of staphylococcal infections and subsequent antimicrobial treatment. Transparency Declaration The Authors declare no conflicts of interest. References 1. Deurenberg RH, Stobberingh EE. The molecular evolution of hospitaland community-associated methicillin-resistant Staphylococcus aureus. Curr Mol Med 2009; 9: Rolo J, Miragaia M, Turlej-Rogacka A et al. High genetic diversity among community-associated Staphylococcus aureus in Europe: results from a multicenter study. PLoS ONE 2012; 7: e Boyle-Vavra S, Daum RS. Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton Valentine leukocidin. Lab Invest 2007; 87: Boakes E, Kearns AM, Ganner M, Perry C, Hill RL, Ellington MJ. Distinct bacteriophages encoding Panton Valentine leukocidin (PVL) among international methicillin-resistant Staphylococcus aureus clones harboring PVL. J Clin Microbiol 2011; 49: 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: e Moremi N, Mshana SE, Kamugisha E et al. Predominance of methicillin resistant Staphylococcus aureus ST88 and new ST1797 causing wound infection and abscesses. J Infect Dev Ctries 2012; 6: HetemDJ,WesthH,BoyeK,JarløvJO,BontenMJ,BootsmaMC.Nosocomial transmission of community-associated methicillin-resistant Staphylococcus aureus in Danish hospitals. J Antimicrob Chemother 2012; 67: Cirkovic I, Sorum M, Radenkovic D, Svabic VlahovicM, Larsen AR. National surveillance reveals the first findings of Panton Valentine leukocidin positive methicillin-resistant Staphylococcus aureus in Serbia. J Med Microbiol 2012; Orendi JM, Coetzee N, Ellington MJ et al. Community and nosocomial transmission of Panton Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus: implications for healthcare. J Hosp Infect 2010; 75: Robinson DA, Kearns AM, Holmes A et al. 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5 1162 Clinical Microbiology and Infection, Volume 19 Number 12, December 2013 CMI 13. Rozenbaum R, Sampaio MG, Batista GS et al. The first report in Brazil of severe infection caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Braz J Med Biol Res 2009; 8: Ali H, Nash JQ, Kearns AM et al. Outbreak of a South West Pacific clone Panton Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus infection in a UK neonatal intensive care unit. J Hosp Infect 2012; 80: Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: nineteenth Informational Supplement M100-S19. Wayne, PA: CLSI, Struelens MJ, Bax R, Deplano A, Quint WG, Van Belkum A. Concordant clonal delineation of methicillin-resistant Staphylococcus aureus by macrorestriction analysis and polymerase chain reaction genome fingerprinting. J Clin Microbiol 1993; 31: Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2002; 46: Lina G, Piemont Y, Godail-Gamot F et al. Involvement of Panton Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999; 29: Enright MC, Day NP, 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: Huijsdens XW, van Santen-Verheuvel MG, Spalburg E et al. Multiple cases of familial transmission of community-acquired methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2006; 44: Perez-Roth E, Alcoba-Florez J, Lopez-Aguilar C, Gutierrez-Gonzalez I, Rivero-Perez B, Mendez-Alvarez S. Familial furunculosis associated with community-acquired leukocidin-positive methicillin-susceptible Staphylococcus aureus ST152. J Clin Microbiol 2010; 48: Pozzi Langhi SA, Robinson JO, Pearson JC, Christiansen KJ, Coombs GW, Murray RJ. Intrafamilial transmission of methicillin-resistant Staphylococcus aureus. Emerg Infect Dis 2009; 15: Scribel LV, Silva-Carvalho MC, Souza RR et al. Clinical and molecular epidemiology of methicillin-resistant Staphylococcus aureus carrying SCCmecIV in a university hospital in Porto Alegre, Brazil. Diagn Microbiol Infect Dis 2009; 65: Chatzakis E, Scoulica E, Papageorgiou N, Maraki S, Samonis G, Galanakis E. Infant colonization by Staphylococcus aureus: role of maternal carriage. Eur J Clin Microbiol Infect Dis 2011; 30: Catho G, Gillet Y, Dumitrescu O et al. Intrafamilial transmission of Staphylococcus aureus Panton Valentine leukocidin responsible for two cases of neonatal necrotizing pneumonia. Arch Pediatr 2011; 18: Carre N, Herbreteau N, Askeur N et al. Outbreak of skin infections due to Staphylococcus aureus carrying Panton Valentine leukocidin genes in pupils and their relatives. Med Mal Infect 2011; 41: Frick MA, Moraga-Llop FA, Bartolome Ret al. Community-acquired methicillin-resistant Staphylococcus aureus infections in children. Enferm Infecc Microbiol Clin 2010; 28: Sanchini A, Campanile F, Monaco M et al. DNA microarray-based characterisation of Panton Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from Italy. Eur J Clin Microbiol Infect Dis 2011; 30: Aschbacher R, Pichon B, Spoladore G et al. High clonal heterogeneity of Panton Valentine leukocidin-positive meticillin-resistant Staphylococcus aureus strains from skin and soft-tissue infections in the Province of Bolzano, Northern Italy. Int J Antimicrob Agents 2012; 39: Mammina C, Cala C, Bonura C et al. Polyclonal non multiresistant Staphylococcus aureus isolates from clinical cases of infection occurring in Palermo, Italy, during a one-year surveillance period. Ann Clin Microbiol Antimicrob 2012; 11: Giuffre M, Cipolla D, Bonura C et al. Epidemic spread of ST1-MRSA-IVa in a neonatal intensive care unit, Italy. BMC Pediatr 2012; 12: Carvalho KS, Mamizuka EM, Gontijo Filho PP. Methicillin/oxacillin-resistant Staphylococcus aureus as a hospital and public health threat in Brazil. Braz J Infect Dis 2010; 14: Thomsen I, McKenna BD, Saye EJ, Jimenez N, Edwards KM, Creech CB. Molecular distinctions exist between community-associated methicillin-resistant Staphylococcus aureus colonization and disease-associated isolates in children. Pediatr Infect Dis J 2011; 5:

Research Article Genotyping of Methicillin Resistant Staphylococcus aureus Strains Isolated from Hospitalized Children

International Pediatrics, Article ID 314316, 4 pages http://dx.doi.org/10.1155/2014/314316 Research Article Genotyping of Methicillin Resistant Staphylococcus aureus Strains Isolated from Hospitalized