Correspondence: C. Gagliotti, Agenzia Sanitaria e Sociale Regionale Emilia-Romagna, Viale Aldo Moro 21, 40127 Bologna, Italy. Tel: � 39 051 5277042. Fax: � 39 051 5277053. E-mail: cgagliotti@regione.emilia-romagna.it

(Received 10 May 2011 ; accepted 30 June 2011 )

Introduction

Staphylococcus aureus is a leading cause of commu-nity-acquired infections (CAI) and healthcare-associ-ated infections (HAI), such as bacteraemia and

pneumonia. Methicillin-resistant S. aureus (MRSA), a multidrug-resistant microorganism causing severe infections, is of particular concern due to its capacity for transmission in hospital settings. The proportion

ORIGINAL ARTICLE

Staphylococcus aureus in a northern Italian region: Phenotypic and molecular characterization

CARLO GAGLIOTTI 1 , MONICA MONACO 2 , CARLA SABIA 3 , RAFFAELE GARGIULO 4 , MARIO SARTI 4 , ANDREA SANCHINI 2 , MASSIMILIANO MARCHI 1 , SIMONE AMBRETTI 5 , FABIO CALANCA 6 , CARMELINA CARILLO 7 , ANTONIO PAOLO CIPOLLONI 8 , MASSIMO CONFALONIERI 9 , CLAUDIA DI CARLO 10 , MARIA FEDERICA PEDNA 11 , LIDIA RICCI 12 , MARIA RITA ROSSI 13 , SILVIA STORCHI INCERTI 14 , GIOVANNA TESTA 15 , CLAUDIA VENTURELLI 16 , ANNALISA PANTOSTI 2 & MARIA LUISA MORO 1

From the 1 Agenzia Sanitaria e Sociale Regionale Emilia-Romagna, Bologna, 2 Istituto Superiore di Sanit à , Rome, 3 Universit à degli Studi di Modena e Reggio-Emilia, Dipartimento di Scienze Biomediche, Modena, 4 AUSL Modena, Nuovo Ospedale di Modena S. Agostino-Estense, Baggiovara (MO), 5 Azienda Ospedaliero-Universitaria di Bologna, Policlinico S. Orsola-Malpighi, Bologna, 6 AUSL Bologna, Ospedale Maggiore, Bologna, 7 AUSL Ferrara, Ospedale Mazzolani Vandini, Argenta (FE), 8 AUSL Cesena, Ospedale Bufalini, Cesena, 9 AUSL Piacenza, Ospedale Guglielmo da Saliceto, Piacenza, 10 AUSL Imola, Ospedale S. Maria della Scaletta, Imola (BO), 11 AUSL Ravenna, Ospedale S. Maria Delle Croci, Ravenna, 12 Azienda Ospedaliera di Reggio Emilia, Arcispedale S. Maria Nuova, Reggio Emilia, 13 Azienda Ospedaliero-Universitaria di Ferrara, Arcispedale S. Anna, Ferrara, 14 AUSL Reggio-Emilia, Ospedale Civile di Guastalla, Guastalla (RE), 15 AUSL Rimini, Ospedale Infermi, Rimini, and 16 Microbiology and Virology Laboratory, Policlinico Hospital of Modena, Modena, Italy

Abstract Background: Staphylococcus aureus is a leading cause of community-acquired infections and healthcare-associated infec-tions. Epidemiological data are useful for understanding the dynamics of the diffusion of this pathogen, and to plan control activities and monitor their effi cacy. Methods: S. aureus isolates were collected in 13 public hospital laboratories of Emilia-Romagna (northern Italy region) during February – March 2009; phenotypic and molecular characterizations of these iso-lates were performed. Results: The study sample included 267 isolates, 57 from blood, 81 from respiratory tract, and 129 from wounds; 106 (40%) were methicillin-resistant S. aureus (MRSA). MRSA showed a limited number of circulating clones with 2 predominant spa types – t008 and t041 – accounting for 36% and 27% of MRSA isolates, respectively. The t041 type had a higher prevalence of antimicrobial resistance compared to other spa types and accounted for most of the retrieved hetero-vancomycin-intermediate S. aureus (h-VISA), while t008 was more frequently detected in non-hospital isolates. A higher degree of genetic diversity was observed in methicillin-susceptible S. aureus (MSSA), with no predominant clones and low prevalence of antimicrobial resistance. The occurrence of community-acquired MRSA infection appears to be rare in Emilia-Romagna. Conclusions: In contrast to previous studies reporting Italian data, t008 was the most frequent spa type among MRSA isolates in Emilia-Romagna. The prevalence of antimicrobial resistance of different MRSA spa types could infl uence their ability to cause infections with hospital onset. The presence of only 2 major MRSA clones circulating in Emilia-Romagna increases the chances that a regional strategy aimed at MRSA prevention will be effective.

Keywords: Methicillin resistance , molecular biology , Staphylococcus aureus , typing

Scandinavian Journal of Infectious Diseases, 2012; 44: 24–28

ISSN 0036-5548 print/ISSN 1651-1980 online © 2012 Informa HealthcareDOI: 10.3109/00365548.2011.603744

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Staphylococcus aureus phenotyping and genotyping 25

of MRSA among all S. aureus isolates varies widely in Europe from 1% to 50% [1]. According to data from the annual report of the European Antibiotic Resistance Surveillance Network (EARS-Net), MRSA strains accounted for 37% of the invasive S. aureus isolates in Italian hospitals in 2009, one of the highest rates in Europe [1]. Most MRSA strains caus-ing infections belong to a limited number of circulat-ing clones and are often multidrug-resistant, being commonly resistant to macrolides, aminoglycosides, and fl uoroquinolones, in addition to beta-lactam antibiotics. Furthermore, the recent emergence of reduced susceptibility to glycopeptides, in particular vancomycin, has led to the worldwide appearance of vancomycin-resistant S. aureus (VRSA), vancomy-cin-intermediate S. aureus (VISA), and hetero-vancomycin-intermediate S. aureus (h-VISA) [2,3].

Efforts to reduce the occurrence of MRSA infec-tions in hospitals have been implemented in many countries in Europe and other parts of the world [4 – 11]. An important prerequisite to planning con-trol activities and assessing their effi cacy in reducing the spread of S. aureus, is the availability of local epidemiological data, including the rate of infection and prevalence of MRSA. Additional information on the genetic structure of S. aureus populations is regarded as useful in understanding the dynamics of the diffusion of this pathogen [12 – 15].

Emilia-Romagna is a northern Italian region of 4.3 million inhabitants with well-established activi-ties for the surveillance and control of HAI. The regional system for surveillance of antimicrobial resistance provided epidemiological data showing a stable trend of S. aureus infections with a high prev-alence of MRSA (40.5% in isolates from blood cul-tures in 2008) for the period 2005 – 2008 [16]. The objective of this study was to provide phenotypic and genotypic characterizations of the S. aureus isolates in the Emilia-Romagna region.

Materials and methods

All consecutive S. aureus isolates from blood, respi-ratory tract (bronchoalveolar lavage, tracheoaspi-rate, and sputum), and wounds were collected in 13 public hospital laboratories in Emilia-Romagna during February – March 2009. Duplicates by patient were excluded. Data on patient characteristics (age and gender) and patient location at the time of sample collection (hospital, outpatient clinic, nurs-ing home, other site) were recorded on a study-specifi c form.

The isolates collected by the participating labo-ratories were sent to the regional study coordination laboratory (Nuovo Ospedale S. Agostino-Estense,

Modena) where they were tested for antimicrobial susceptibility using an automated system (Vitek2, card AST-GP 580, bioM é rieux) and for the presence of the mec A gene by multiplex polymerase chain reac-tion (PCR), as previously described [17]. The Clinical and Laboratory Standards Institute (CLSI) [18] breakpoints were applied. Susceptibility testing for vancomycin, teicoplanin, tigecycline, linezolid, and daptomycin was performed by Etest. Vancomycin minimum inhibitory concentrations (MICs) were confi rmed by broth microdilution method. The D-test was used to identify inducible resistance to clindamy-cin in all isolates showing resistance to erythromycin and susceptibility to clindamycin.

Methicillin-susceptible S. aureus (MSSA) samples from blood and all MRSA isolates were sent to the national reference laboratory (Istituto Superiore di Sanit à , Rome) for further phenotypic and molecular analysis, including the detection of Panton – Valentine leukocidin (PVL) toxin genes and the amplifi cation of the polymorphic region of the protein A gene ( spa typ-ing). The Macro Etest (MET) for detection of h-VISA and staphylococcal cassette chromosome mec (SCC -mec ) typing were performed on MRSA only. Multilo-cus sequence typing (MLST) was assayed on selected strains.

STATA 10.0 (Stata Corp., College Station, TX, USA) was used for the statistical analysis. Univariate and bivariate analyses were performed using logistic regression.

Results

The study sample collected by 13 laboratories included 267 isolates: 57 from blood, 81 from respiratory tract, and 129 from wounds. The number of isolates per submitting laboratory ranged from 1 to 34 (median 18). The proportion of MRSA, defi ned by the pres-ence of the mec A gene, was 40% (106 of 267 isolates), with differences according to source, patient location at the time of microbiological sample collection, patient age, and gender (Table I).

By spa typing, MRSA isolates included 2 predom-inant types: t008 and t041. The t008 type accounted for 36%, 29%, and 44% of all MRSA isolates from blood, respiratory tract, and wounds, while t041 accounted for 28%, 29%, and 25% of these isolates, respectively. In contrast, no spa types occurred in more than 10% of MSSA isolated from blood culture. The total number of different spa types in MRSA isolates from blood was 11 and in MSSA isolates from blood was 25 (Figure 1). Two MRSA strains of the most frequent spa types in the study sample (t008 and t041) were sequenced by MLST: the t008 strain was ST8, while the t041 strain was ST228, belonging to clonal

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26 C. Gagliotti et al.

complexes (CC) 8 and 5, respectively. MRSA isolates spa -typed as t008 were signifi cantly less frequent in hospital (27.1%) compared to other patient locations (46.8%), while isolates belonging to t041 were simi-larly distributed in hospital and other patient locations (25.4% and 29.8%, respectively). Thirteen of 22 non-hospital t008 MRSA isolates were from wounds, while

2 and 7 isolates were from blood and respiratory tract, respectively. Even taking into account the isolate source (blood, wound, tracheoaspirate, bronchoalveo-lar lavage, or sputum) by bivariate model, t008 was still signifi cantly less frequent in hospital compared to other patient locations.

The t041 strain showed higher prevalence of anti-microbial resistance for single antibiotics when com-pared to other spa types. These differences were more evident for gentamicin, rifampicin, erythromycin, and clindamycin. Resistance to clindamycin in t041 iso-lates (89.7%) was always constitutive, while it was mainly inducible in MRSA isolates belonging to t008 (50% inducible versus 5.3% constitutive resis-tance) and in MSSA isolates (7.5% inducible ver-sus 0.6% constitutive resistance). Considering 7 antibiotics – gentamicin, levofl oxacin, rifampicin, tetracycline, erythromycin, clindamycin, and sul-famethoxazole/trimethoprim – that represent 7 dif-ferent antibiotic classes, the average number of antimicrobials to which the isolates were resistant was 0.4 for MSSA, while among MRSA it was 2.6 and 3.9 for t008 and t041, respectively. In MRSA isolates belonging to spa types other than t008 and t041, the average number of resistances was 3.1 (Table II). Twenty-seven h-VISA of 106 MRSA (25%) were detected by MET: 20 belonged to t041, 0 to t008, and 7 to other spa types.

All isolates included in the study were suscepti-ble to vancomycin, daptomycin, tigecycline, and linezolid. SCC mec types IV and I were the most fre-quent among the 106 MRSA isolates: 52% and 42%, respectively. The vast majority of isolates (37 of 38) belonging to t008 contained SCC mec type IV, while all 29 t041 isolates contained SCC mec type I.

Table I. Proportion of methicillin-resistant Staphylococcus aureus by isolate and patient characteristics.

MRSAMSSA

n Total

n n %

All isolates 106 39.7 161 267Patient location at time of sample collection

Hospital 59 45.0 72 131Outpatient clinic 31 29.0 76 107Nursing home 11 57.9 8 19Other/unknown 5 50.0 5 10

Isolate sourceBlood 25 43.9 32 57Wound 36 27.9 93 129Tracheoaspirate 23 52.3 21 44Bronchoalveolar

lavage15 68.2 7 22

Sputum 7 46.7 8 15Age category (y)

0 – 19 4 12.5 28 3220 – 39 13 31.7 28 4140 – 59 12 30.8 27 3960 – 79 45 45.5 54 99 � 80 30 58.8 21 51Unknown 2 40.0 3 5

GenderMale 76 43.4 99 175

MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive Staphylococcus aureus.

t041t008

t008 t041

t008

t008 t041

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Wound: MRSA(isolates=36;

spa types=10)

*Respiratory tract:MRSA (isolates=45;

spa types=19)

Blood: MSSA(isolates=32;

spa types=25)

Blood: MRSA(isolates=25;

spa types=11)

Figure 1. spa type distribution by methicillin susceptibility and isolate source. ∗ Tracheoaspirate, bronchoalveolar lavage, and sputum.

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Staphylococcus aureus phenotyping and genotyping 27

Of 138 isolates tested (including all MRSA and MSSA from blood), only 2 isolates harboured PVL toxin genes: 1 community-acquired MRSA (CA-MRSA) strain from the wound sample of a 64-y-old outpatient ( spa type t044, SCC mec IV, MLST ST80), susceptible to all antibiotics listed in Table II except for tetracycline; 1 MSSA strain from the blood culture of a 54-y-old hospital inpa-tient ( spa type t127), susceptible to all antibiotics considered.

Discussion

A limited number of spa types were identifi ed among the MRSA included in this study, with 2 predominant types (t008 and t041) accounting for almost two-thirds of all MRSA isolates (Figure 1). These fi ndings indicate that few MRSA clones circulate in Emilia-Romagna, and that they mainly harbour SCC mec types IV and I and belong to CCs 8 and 5. The occur-rence of CA-MRSA infection appears to be rare in Emilia-Romagna, as previously observed in other Ital-ian and European studies [13,14], with only 1 strain of 106 MRSA tested; this strain ( spa type t044, SCC -mec IV, MLST ST80), was PVL-positive and belonged to the European clone CC80. The prevalence of anti-microbial resistance in MRSA is associated with spa types, being higher in t041 compared to other spa types; t041 also included most h-VISA isolates (20 of 27; 74%). This result confi rms that t041 strains show h-VISA phenotype in a high percentage compared to other spa types, as previously reported [14]. More-over, among t041 the rate of h-VISA increased from 50% in the period 2006 – 2007 to 74% in this study. Though limited to the t041 cluster, the increase in h-VISA is worrisome, because it represents a reduction in therapeutic options for clinicians. Compared to MRSA, MSSA showed a higher degree of genetic diversity and low prevalence of antimicrobial resistance

(Table II). One MSSA isolate of 32 isolates from blood culture was PVL-positive.

Previous studies reporting Italian data have indi-cated t041 as the most common MRSA spa type [13,14], while in this study t008 was the most frequent spa type among MRSA isolates in Emilia-Romagna, followed by t041. One possible explana-tion for the difference observed in spa type distribution is the sampling method used in this study, where MRSA isolates from outside of hospital (e.g. outpatient clinic or nursing home) had higher weight compared to previous reports and accounted for about half of the study sample. Also, taking into account the source (blood, wound, tracheoaspirate, bronchoalveolar lavage, or sputum), the non-hospital isolates belonged to t008 signifi cantly more often than those collected in hospital. According to these fi ndings, it could be hypothesized that t008 isolates, compared to other MRSA isolates with higher prev-alence of antimicrobial resistance, have a less pro-nounced selective advantage in hospital where patients are commonly exposed to antibiotics for therapy or prophylaxis.

The main limitation of this study is the lack of data on previous in-hospital stays of patients and on the length of hospitalization before S. aureus detec-tion. Therefore, it was not possible to identify samples collected outside hospital from recently hospitalized patients and differentiate between hospitalized patients with early and late S. aureus detection. The strength of the study is the method of isolate collec-tion, which did not focus only on invasive infection and samples collected in hospital. This made it pos-sible to describe the distribution of spa types in MRSA isolates by source and by patient location at the time of microbiological sample collection (hospital, outpa-tient clinic, nursing home, other site).

In conclusion, the fi ndings of this study confi rm that a limited number of genotypes compose the

Table II. Antimicrobial resistance by methicillin susceptibility and spa type.

% Resistant

spa typeNo. of isolates GEN LEV RIF TET ERY

CLI

SXTNo. of

resistances c Const a Ind b

MSSA All types 161 8.7 5.0 0.6 5.0 8.1 0.6 7.5 2.5 0.4MRSA t008 38 42.1 100 2.6 0 55.3 5.3 50.0 2.6 2.6

t041 29 89.7 96.6 20.7 0 89.7 89.7 0 0 3.9Other types 39 46.2 89.7 10.3 15.4 74.4 46.2 28.2 2.6 3.1

GEN, gentamicin; LEV, levofl oxacin; RIF, rifampicin; TET, tetracycline; ERY, erythromycin; CLI, clindamycin; SXT, sulfamethoxazole/trimethoprim. a Constitutive resistance. b Inducible resistance. c Average number of antimicrobials (among the 7 included) to which the isolates are resistant.

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28 C. Gagliotti et al.

MRSA population, with the predominance of 2 spa types – t008 and t041 – as already observed in other surveys reporting Italian data [13,14]. In contrast, the MSSA population shows greater genetic diversity. The occurrence of t008 MRSA appears to be less frequent in hospital, possibly because of the lower prevalence of antimicrobial resistance in this spa type compared to other MRSA isolates. Infections caused by CA-MRSA are rare in Emilia-Romagna.

Further studies are needed to describe in detail the genetic structure of MRSA in non-hospital patients, such as residents of nursing homes, in Emilia-Romagna. The limited number of MRSA clones cir-culating in Emilia-Romagna is an advantage for controlling the transmission of this microorganism and increases the chances that a regional strategy aimed at MRSA prevention will be effective, as already observed in other settings [4 – 11].

Acknowledgements

This study was supported in part by a grant from the Italian Ministry of Health (CCM) “ Valutazi-one della diffusione nosocomiale dello Staphylo-coccus aureus resistente alla meticillina; stesura di raccomandazioni ” .

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

References

European Centre for Disease Prevention and Control [1] (ECDC). Antimicrobial resistance surveillance in Europe 2009. Annual Report of the European Antimicrobial Resist-ance Surveillance Network (EARS-Net). Stockholm: ECDC; 2010. Jones RN. Key considerations in the treatment of compli-[2] cated staphylococcal infections. Clin Microbiol Infect 2008;14(Suppl 2):3 – 9. Perichon B, Courvalin P. VanA-type vancomycin-resistant [3] Staphylococcus aureus. Antimicrob Agents Chemother 2009;53:4580 – 7. Recent trends in antimicrobial resistance among Strepto-[4] coccus pneumoniae and Staphylococcus aureus isolates: the French experience. Euro Surveill 2008;13(46). pii: 19035.

Ellingson K, Muder RR, Jain R, Kleinbaum D, Feng PJ, Cun-[5] ningham C, et al. Sustained reduction in the clinical incidence of methicillin-resistant Staphylococcus aureus colonization or infection associated with a multifaceted infection control intervention. Infect Control Hosp Epidemiol 2011;32:1 – 8. Gagliotti C, Balode A, Baquero F, Degener J, Grundmann H, [6] Gur D, et al. Escherichia coli and Staphylococcus aureus: bad news and good news from the European Antimicrobial Resist-ance Surveillance Network (EARS-Net, formerly EARSS), 2002 to 2009. Euro Surveill 2011;16(11). pii: 19819. Jain R, Kralovic SM, Evans ME, Ambrose M, Simbartl LA, [7] Obrosky DS, et al. Veterans Affairs initiative to prevent methicillin-resistant Staphylococcus aureus infections. N Engl J Med 2011;364:1419 – 30. Kallen AJ, Mu Y, Bulens S, Reingold A, Petit S, Gershman K, [8] et al. Health care-associated invasive MRSA infections, 2005 – 2008. JAMA 2010;304:641 – 8. Lee AS, Huttner B, Harbarth S. Control of methicillin-[9] resistant Staphylococcus aureus. Infect Dis Clin North Am 2011;25:155 – 79. Pearson A, Chronias A, Murray M. Voluntary and mandatory [10] surveillance for methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) bacteraemia in England. J Antimicrob Chemother 2009;64(Suppl 1):i11 – 7. Struelens MJ, Monnet DL. Prevention of methicillin-resistant [11] Staphylococcus aureus infection: is Europe winning the fi ght? Infect Control Hosp Epidemiol 2010;31(Suppl 1):S42 – 4. Budimir A, Deurenberg RH, Bosnjak Z, Stobberingh EE, Cetk-[12] ovic H, Kalenic S. A variant of the Southern German clone of methicillin-resistant Staphylococcus aureus is predominant in Croatia. Clin Microbiol Infect 2010;16:1077 – 83. Grundmann H, Aanensen DM, van den Wijngaard CC, [13] Spratt BG, Harmsen D, Friedrich AW. Geographic distribu-tion of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis. PLoS Med 2010;7:e1000215. Monaco M, Sanchini A, Grundmann H, Pantosti A. Vanco-[14] mycin-heteroresistant phenotype in invasive methicillin-re-sistant Staphylococcus aureus isolates belonging to spa type 041. Eur J Clin Microbiol Infect Dis 2010;29:771 – 7. Johnson AP. Methicillin-resistant Staphylococcus aureus: the [15] European landscape. J Antimicrob Chemother 2011;66(Suppl 4):iv43 – 8. Pan A, Buttazzi R, Capatti C, Pedna F, Resi D, Sarti M, et al. [16] Sorveglianza dell ’ Antibioticoresistenza e Uso di Antibiotici Sistemici in Emilia-Romagna. Rapporto 2008. (Collana Dossier, No. 201). Bologna, Italy: Agenzia Sanitaria e Sociale Regionale Emilia-Romagna; 2010. Salisbury SM, Sabatini LM, Spiegel CA. Identifi cation of [17] methicillin-resistant staphylococci by multiplex polymerase chain reaction assay. Am J Clin Pathol 1997;107:368 – 73. Clinical and Laboratory Standards Institute (CLSI). Per-[18] formance standards for antimicrobial susceptibility testing. Supplement M100-S19. Wayne, PA: CLSI; 2009.

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