Changing Italian nosocomial-community trends and heteroresistance in Staphylococcus aureus from bacteremia and endocarditis

F. Campanile 1 2 D. Bongiorno 1 2 M. Falcone 1 2 F. Vailati 1 2 M. B. Pasticci 1 2 M. Perez 1 2 A. Raglio 1 2 F. Rumpianesi 1 2 C. Scuderi 1 2 F. Suter 1 2 M. Venditti 1 2 C. Venturelli 1 2 V. Ravasio 1 2 M. Codeluppi 1 2 S. Stefani 1 2 0 ) Department of Bio-Medical Sciences, Section of Microbiology, University of Catania , Via Androne 81, 95124 Catania (I), Italy 1 F. Suter USC Malattie infettive, AO Ospedali Riuniti , Bergamo (I), Italy 2 M. B. Pasticci Infectious Disease Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia , Perugia, Italy Bloodstream infections due to Staphylococcus aureus (BSI) are serious infections both in hospitals and in the community, possibly leading to infective endocarditis (IE). The use of glycopeptides has been recently challenged by various forms of low-level resistance. This study evaluated the distribution of MSSA and MRSA isolates from BSI and IE in 4 Italian hospitals, their antibiotic susceptibilityfocusing on the emergence of hVISAand genotypic relationships. Our results demonstrate that the epidemiology of MRSA is changing versus different STs possessing features between community-acquired (CA)and hospital-acquired (HA)-MRSA groups; furthermore, different MSSA isolated from BSI and IE were found, with the same backgrounds of the Italian CA-MRSA. The hVISA phenotype was very frequent (19.5%) and occurred more frequently in isolates from IE and in both the MSSA and MRSA strains. As expected, hVISA were detected in MRSA with vancomycin minimum inhibitory concentrations (MICs) of 1-2 mg/l, frequently associated with the major SCCmec I and II nosocomial clones; this phenotype was also detected in some MSSA strains. The few cases of MR-hVISA infections evaluated in our study demonstrated that 5 out of 9 patients (55%) receiving a glycopeptide, died. Future studies are required to validate these findings in terms of clinical impact. - Introduction Staphylococcus aureus has become an increasing cause of nosocomial and community acquired bloodstream infections (BSI), possibly leading to infective endocarditis (IE), with a high risk of mortality despite aggressive therapy [1, 2]. Since 1990, the incidence of S.aureus bacteremia has increased because of the extensive use of indwelling intravenous catheters. Predisposing factors for S. aureus infections include severe underlying conditions, prolonged hospital stay, previous antibiotic treatment and nasal carriage. In this context, the emergence of methicillinresistant S.aureus (MRSA) is a major clinical challenge, particularly for the poor outcome related to such serious infections, and for the increasing therapeutic failures. In fact, the use of glycopeptides has been recently challenged by various forms of reduced-susceptibility (VISA and hVISA phenotypes), with consequential effects on vancomycin efficacy in MRSA bacteremia and endocarditis [35]. Until now, only a few studies have compared the clinical and molecular features of MSSA versus MRSA in patients with infective endocarditis or bacteremia [6 12]. Additional knowledge could be useful to understand and correlate the impact of specific genotypic markers with clinical outcomes. This study was undertaken to evaluate MSSA and MRSA distribution in strains from BSI and IE isolated in four Italian hospitals, in order to evaluate their genotypic relationship, pvl gene distribution, antibiotic susceptibility patterns and presence of hVISA strains. Materials and methods Microbial population and epidemiological correlations The microbial population consisted of 128 S. aureus clinical isolates, belonging to 76 patients with definite S. aureus IE, according to the modified Duke criteria [13], and 52 patients with definite BSI. Patients were admitted to four Italian hospitals (Modena, Bergamo, Perugia, and Rome) between 2007 and 2009. The S. aureus isolates, randomly selected (multiple isolates from the same patient and from other patients at the same time in the same ward were excluded) among all S. aureus isolates, were sent to our laboratory for further characterizations. Infection classification was performed as follows: nosocomial infection was defined as an IE developing in a patient hospitalized for >48 h before the onset of signs and symptoms consistent with IE, and non-nosocomial health-care-associated infection was defined as an IE diagnosed within 48 h of admission in an outpatient with extended health-care contact. Persistent bacteremia was defined as >3 days of bacteremia despite receipt of an antibiotic to which the isolate was susceptible in vitro [14]. Microbiological characterization Both groups of strains (BSI and IE) were all isolated from blood cultures. All staphylococci were re-identified at the species level by the catalase test, the S. aureus agglutination test (Staphylase Test; Oxoid, Basingstoke, Hampshire, UK) and biochemical tests (API-Staph system; bioMrieux, Bagno a Ripoli, FI, Italy). Methicillin resistance was evaluated by the cefoxitin disk diffusion method and correlated with the presence of the mecA gene [1517]. Antimicrobial susceptibility was determined by the disk diffusion method, according to CLSI guidelines [15]. All isolates were tested against a panel of nine antimicrobial agents as follows: ampicillin1 g, ciprofloxacin5 g, chloramphenicol30 g, gentamicin10 g, erythromycin 15 g, clindamycin2 g, trimethoprim-sulfamethoxazole 25 g, rifampin5 g, and tetracycline30 g (Oxoid, Milan, Italy). In vitro susceptibility testing for vancomycin (Sigma Chemical, St. Louis, MO, USA), teicoplanin, quinupristin/dalfopristin (Aventis, West Malling, UK), linezolid (Pfizer, Groton, CT, USA), tigecycline (Wyeth Pharmaceuticals, Collegeville, PA, UK) and daptomycin (Novartis, Basel, Switzerland) was further performed by the broth microdilution method to determine the minimum inhibitory concentrations (MICs), following the CLSI guidelines. The EUCAST guidelines were also used for comparison [15, 16]. Heteroresistance to glycopeptides was screened using the macro Etest (bioMrieux), and confirmed by the reference PAP/AUC method. S. aureus Mu3 (hVISA), Mu50 (VISA), and ATCC 29213 were used as control strains, as previously described [18]. Molecular characterization of all strains was conducted by PCR of mecA and pvl genes, SCCmec-typing, and MLST; PFGE was also used only to define possible relationships among the isolates. All techniques were performed as previously described [17]. MLST was performed on all MRSA strains and on a selection of MSSA isolates (n = 50), based on phenotypic, genotypic, and susceptibility testing differences (http://saureus.mlst.net/). Clinical data on therapies for hVISA infections All centers were asked to provide clinical data on the treatment and outcome of patients with hVISA infections. Overall, complete data were available for 20 out of 25 patients. Strain characteristics and infections Overall, MSSA isolates were predominant, both in BSI and (...truncated)