This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% ( = 101) of the XDR isolates, distantly followed by ST244 ( = 16), ST253 ( = 12), ST235 ( = 8), and ST111 ( = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital ( = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

Group A streptococcus (GAS) has been described as an emerging cause of severe invasive infections. A retrospective hospital-based study was conducted, including GAS isolates causing invasive or non-invasive infections from January 1999 to June 2003 in Barcelona. Demographic and clinical information on the invasive cases was obtained from medical files. GAS isolates collected from 27 patients with invasive infections and 99 patients with non-invasive infections were characterized by emm type and subtype, superantigen (SAg) gene profile (speA-C, speF-J, speL, speM, ssa and smeZ), allelic variants of speA and smeZ genes, antibiotic susceptibility and genetic resistance determinants. The most prevalent emm type was emm1 (17?5 %), followed by emm3 (8?7 %), emm4 (8?7 %), emm12 (7?1 %) and emm28 (7?1 %). The smeZ allele and SAg gene profiles were closely associated with the emm type. The speA2, speA3 and speA4 alleles were found in emm1, emm3 and emm6 isolates, respectively. Overall, 27?8, 25?4 and 11?9 % of isolates were resistant to erythromycin, tetracycline or both agents, respectively. Reduced susceptibility to ciprofloxacin and levofloxacin (MIC 2-4 mg ml "1 ) was found in 3?2 % of isolates. mef(A)-positive emm types 4, 12 and 75, and erm(B)-positive emm types 11 and 25 were responsible for up to 80 % of the erythromycin-resistant isolates. No significant differences in emm-type distribution, SAg gene profile or resistance rates were found between invasive and non-invasive isolates. The SAg and antibiotic resistance genes appeared to be associated with the emm type and were independent of the disease type. INTRODUCTIONGroup A streptococcus (GAS) is a human pathogen that is responsible for a wide array of infections, varying in severity from acute pharyngitis and impetigo to severe invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS) (Cunningham, 2000).The resurgence and persistence of severe forms of GAS diseases reported since the mid 1980s (Hoge et al., 1993;Stevens, 2002) has motivated intensive research on epidemiological, microbiological and clinical aspects of invasive GAS disease. Several factors have been considered to explain differences in disease frequency and severity, including changes in the virulence of the bacterium (Banks et al., 2002;Musser et al., 1993) and the role of host immunity (Å kesson et al., 2004;Basma et al., 1999;Kotb et al., 2002; NorrbyTeglund et al., 2000).Among the many factors involved in the virulence of the pathogen, the M protein and a group of exotoxins known as streptococcal superantigens (SAgs) have received considerable attention. Sequence analysis of the emm gene (encoding the M protein) has become an important surveillance tool for investigating the dynamics of GAS infection and more than 150 emm gene sequence types and several emm subtypes have been documented (Facklam et al., 2002;Li et al., 2003).SAgs are thought to contribute to the pathogenesis of severe GAS infections by virtue of their potent immunostimulatory activ...

A total of 788 clinical Enterobacteriaceae were collected to describe the aminoglycoside-modifying genes (AME genes) and to characterize the plasmids that carry these genes. Among the 788 strains collected, 330 (41.8%) were aminoglycoside-resistant: 264 Escherichia coli (80%), 33 Proteus mirabilis (10%), 10 Klebsiella pneumoniae (3%), six K. oxytoca (1.8%), five Enterobacter cloacae (1.5%), three Morganella morganii (0.9%), three Providencia stuartii (0.9%), two Salmonella enterica (0.6%), and one each Citrobacter freundii, C. koseri, Proteus vulgaris, and Shigella sonnei. The most affected aminoglycoside was streptomycin (92.7%), followed by kanamycin (26.3%), gentamicin (18%), tobramycin (16.9%), netilmicin (3.6%), and amikacin (1.5%). The AME genes found were aph(3″)-Ib (65.4%), ant(3″)-Ia (37.5%), aph(3')-Ia (13.9%), aac(3)-IIa (12.4%), aac(6')-Ib (4.2%), ant(2″)-Ia (3.6%), and aph(3')-IIa (1.2%). Thirty-four percent of the strains showed more than one enzyme. The most frequent association was ant(3″)-Ia plus aph(3″)-Ib (35 strains). From 66 selected AME genes, 24 were plasmid located: 12 aac(3)-IIa, six aph(3')-Ia, three ant(3″)-Ia, two ant(2″)-Ia, and one aac(6')-Ib. These genes were located in plasmids belonging to incompatibility groups F, FIA, FIB, or HI2. In conclusion, the AME genes involved in aminoglycoside-clinical resistance were aac(3)-IIa, aac(6')-Ib, and ant(2″)-Ia, genes that confer resistance to tobramycin, gentamicin, and amikacin.

mWe conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC > 32/16 g/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC Escherichia coli is an important etiologic agent for both nosocomial-and community-acquired infections in humans (9, 13, 22). Amoxicillin-clavulanate (AMC) is one of the most widely used antibiotics in many countries (3,11,15). In Spain, a 34.7% increase in community use of AMC was recorded from 2000 to 2006 (20). Recently, blood isolates of E. coli nonsusceptible to AMC increased from 9.3% (2003) Enzymatic mechanisms of E. coli resistance to AMC include hyperproduction of plasmid-mediated class A ␤-lactamases such as TEM-1 and SHV-1 (18, 31), plasmid-mediated AmpC-type ␤-lactamase (p-AmpC) (21), chromosomal AmpC ␤-lactamase (c-AmpC) (21), production of inhibitor-resistant TEM (IRT) ␤-lactamases (17, 27), plasmid-mediated ␤-lactamase OXA-1 (32), and complex mutant TEM (CMT) enzymes than combine IRT-and extended-spectrum ␤-lactamase (ESBL)-type substitutions (26).In spite of the significant increase in AMC use in the last several years, there is little recent information available about the prevalence of AMC resistance mechanisms in E. coli; most previous studies analyzed strains isolated more than 10 years ago from single hospitals in the United States (12, 28), France (16), and Spain (19,24). Accordingly, the aim of this prospective Spanish national multicenter study was to investigate the epidemiology and mechanisms of AMC resistance in clinical isolates of E. coli causing both community and nosocomial infections. MATERIALS AND METHODS Study design and bacterial isolates.A prospective multicenter study was designed to obtain E. coli isolates resistant to AMC (MIC of Ն 32/16 g/ml and/or disk inhibition zone of Յ13 mm according to the Clinical and Laboratory Standards Institute [CLSI]) (7) from clinical samples col-

The prevalence of ESBL-producing strains in stool samples was higher than that observed in clinical samples from the same period. The different types of ESBLs found were similar in both contexts. The most prevalent ESBLs were the CTX-M-related enzymes, with nine different types, followed by SHV-12.