Significant increase of CTX-M-15-ST131 and emergence of CTX-M-27-ST131 Escherichia coli high-risk clones causing healthcare-associated bacteraemia of urinary origin in Spain (ITUBRAS-2 project).

OBJECTIVES: To assess the microbiological characteristics of Escherichia coli causing healthcare-associated bacteraemia of urinary origin (HCA-BUO) in Spain (ITUBRAS-2 project), with particular focus on ESBL producers and isolates belonging to ST131 high-risk clone (HiRC). Clinical characteristics and outcomes associated with ST131 infection were investigated. METHODS: A total of 222 E. coli blood isolates were prospectively collected from patients with HCA-BUO from 12 tertiary-care hospitals in Spain (2017-19). Antimicrobial susceptibility and ESBL/carbapenemase production were determined. ST131 subtyping was performed. A subset of 115 isolates were selected for WGS to determine population structure, resistome and virulome. Clinical charts were reviewed. RESULTS: ESBL-producing E. coli prevalence was 30.6% (68/222). ST131 represented 29.7% (66/222) of E. coli isolates and accounted for the majority of ESBL producers (46/68, 67.6%). The C2/H30-Rx subclone accounted for most ST131 isolates (44/66) and was associated with CTX-M-15 (37/44) and OXA-1 enzymes (27/44). Cluster C1-M27 was identified in 4/10 isolates belonging to subclade C1/H30-R1 and associated with CTX-M-27. Additionally, ST131 isolates showed a high content of other acquired resistance genes, and clade C/ST131 isolates carried characteristic QRDR mutations. They were categorized as uropathogenic E. coli and had higher aggregate virulence scores. ST131 infection was associated with more complex patients, prior use of cephalosporins and inadequate empirical treatment but was not associated with worse clinical outcomes. CONCLUSIONS: ST131 HiRC is the main driver of ESBL-producing E. coli causing HCA-BUO in Spain, mainly associated with the expansion of subclade CTX-M-15-C2/H30-Rx and the emergence of CTX-M-27-C1/H30-R1 (Cluster C1-M27).

In Vitro Models for the Study of the Intracellular Activity of Antibiotics.

Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine the antibiotics which are the most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of preopsonized bacteria by eukaryotic cells, (2) elimination of noninternalized bacteria with gentamicin, (3) incubation of infected cells with antibiotics, and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample protein content. The use of strains expressing fluorescent proteins under the control of an inducible promoter allows to follow intracellular bacterial division at the individual level and therefore to monitor bacterial persisters that do not multiply anymore.

Penicillin susceptibility among invasive MSSA infections: a multicentre study in 16 Spanish hospitals.

OBJECTIVES: To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates. METHODS: A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018-19 (6-12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type. RESULTS: The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton-Valentine leucocidin ('PVL') positive. CONCLUSIONS: A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis.

Prevalence and Genetic Characteristics of Staphylococcus aureus CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade.

BACKGROUND: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. OBJECTIVES: To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. METHODS: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6-12 months in 17 Spanish hospitals (2018-2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. RESULTS: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin-inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005). CONCLUSION: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis.

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

In Vitro and In Vivo Antibacterial Activity of Gliotoxin Alone and in Combination with Antibiotics against Staphylococcus aureus.

Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired and community infections and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin (GT) is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that GT shows antimicrobial activity against S. aureus in vitro, albeit its efficacy against multidrug-resistant strains such asMRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimedto evaluate the antibiotic efficacy of GT as monotherapy or in combination with other therapeutics against MRSA in vitro and in vivo using a Caenorhabditis elegans infection model.

Innominate Artery Dissection Caused by Video-Mediastinoscopy.

Mediastinoscopy is considered a safe technique to biopsy mediastinal lesions. Among its complications, vascular ones are the most common. We present a rare case of intimal dissection of the innominate artery during the performance of a mediastinoscopy that caused an ischemic attack from which the patient recovered completely without long-term sequelae. We analyze the possible causes and risk factors of this complication.

Mechanisms of Linezolid Resistance Among Enterococci of Clinical Origin in Spain-Detection of optrA- and cfr(D)-Carrying E. faecalis.

The mechanisms of linezolid resistance among 13 E. faecalis and 6 E. faecium isolates, recovered from six Spanish hospitals during 2017-2018, were investigated. The presence of acquired linezolid resistance genes and mutations in 23S rDNA and in genes encoding for ribosomal proteins was analyzed by PCR and amplicon sequencing. Moreover, the susceptibility to 18 antimicrobial agents was investigated, and the respective molecular background was elucidated by PCR-amplicon sequencing and whole genome sequencing. The transferability of the linezolid resistance genes was evaluated by filter-mating experiments. The optrA gene was detected in all 13 E. faecalis isolates; and one optrA-positive isolate also carried the recently described cfr(D) gene. Moreover, one E. faecalis isolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all six E. faecium isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. The fexA gene was located upstream of the optrA gene in 12 of the E. faecalis isolates. Moreover, an erm(A)-like gene was located downstream of optrA in two isolates recovered from the same hospital. The optrA gene was transferable in all but one E. faecalis isolates, in all cases along with the fexA gene. The cfr(D) gene was not transferable. The presence of optrA and mutations in the 23S rDNA are the main mechanisms of linezolid resistance among E. faecalis and E. faecium, respectively. We report the first description of the cfr(D) gene in E. faecalis. The presence of the optrA and cfr(D) genes in Spanish hospitals is a public health concern.

Comparison of several Real-Time PCR Kits versus a Culture-dependent Algorithm to Identify Enteropathogens in Stool Samples.

This study aims to validate the current diagnostic method for the clinical detection of gastroenteritis. We analyzed 400 stool samples to detect three of the most common enteropathogens: Salmonella spp., Campylobacter spp., and Yersinia enterocolitica. All specimens were tested with a routine clinical diagnosis algorithm and with five real-time PCR assays. A total of 98 specimens (24.5%) were positive for enteropathogens. We found 24 samples positive for Salmonella enterica, 71 positive for Campylobacter spp., and 4 positive for Yersinia enterocolitica. All evaluated methods exhibited a good performance in identifying Salmonella and Yersinia enterocolitica, being the highest positive percent agreement (PPA) value of 95.8% and 100%, respectively. The clinical algorithm showed the highest PPA value identifying Salmonella, due to the enrichment in selenite broth. However, the evaluated methods showed notable differences in the identification of Campylobacter species, obtaining a wide range of PPA values: 59.2%-100%. The clinical algorithm showed the lowest PPA value since it was only able to detect Campylobacter jejuni and Campylobacter coli species. This study revealed the importance of implementing the real-time PCR technique in a clinical algorithm: it improved the accuracy of the diagnosis and provided results in a shorter time compared to routine clinical methods.

Epidemiology of MRSA CC398 in hospitals located in Spanish regions with different pig-farming densities: a multicentre study.

BACKGROUND: Tetracycline resistance (TetR) is a marker of livestock-associated MRSA of lineage CC398. OBJECTIVES: To determine the MRSA CC398 prevalence among TetR-MRSA recovered in Spanish hospitals located in regions with different pig-farming densities, and the influence of pig density as a key risk factor for its acquisition. METHODS: TetR-MRSA isolates (n = 232) recovered from clinical and epidemiological samples during January-June 2016 in 20 hospitals in 13 regions with different pig-farming densities were analysed. MRSA CC398 identification, detection of spa types, methicillin resistance genes and immune evasion cluster (IEC) genes were performed by PCR/sequencing. Statistical analyses were performed to establish the relationships between MRSA CC398 prevalence and pig density. RESULTS: The global MRSA prevalence was 29.7% (6.9% TetR-MRSA/MRSA), with 137 CC398 isolates recovered, representing 4.1% of total MRSA and 59.1% of TetR-MRSA. Among MRSA CC398, 16 different spa types were recorded (t011: 72.3%), and all but two strains were IEC negative. Higher pig-density regions were associated with significant MRSA CC398 increases in hospitals located in adjacent regions (P < 0.001). Linear regression models explained the relationships between MRSA CC398 and pig density (P < 0.001), with an increase of 6.6 MRSA CC398 cases per 100 MRSA per increase of 100 pigs/km2 in a region. CONCLUSIONS: High pig density leads to a significant increase in MRSA CC398 in hospitals in Spain, and its combination with a high human population could help its dissemination. In Spain, the prevalence of the zoonotic CC398 lineage is closely related to pig-farming density; therefore, specific tools could be implemented in order to detect its dissemination.

Characterisation of VIM-2-producing Pseudomonas aeruginosa isolates from lower tract respiratory infections in a Spanish hospital.

To analyse the antimicrobial phenotype, carbapenem mechanisms, integrons, virulence factors and molecular typing of 164 Pseudomonas aeruginosa isolates recovered from lower tract respiratory samples in a Spanish hospital (1 year) as well as the patients' clinical data. Susceptibility testing to 12 antipseudomonal agents was determined by microdilution and metallo-beta-lactamase (MBL) phenotype by double disc method. The oprD gene was studied by PCR, sequencing and comparison with P. aeruginosa PAO1 sequence. Detection and characterisation of MBLs, class 1, 2 and 3 integrons, and virulence genes were studied by PCR and sequencing. The prevalence of carbapenem-resistant P. aeruginosa (CRPA) was 26.8%. MBL phenotype was detected in 52.3% CRPA, and all of them were disseminated throughout the intensive care unit. Most of the MBL-carrying patients presented respiratory disease, mechanical ventilation, tracheostomy, bacteraemia, ≥ 30 hospitalisation days and previous treatment with carbapenems and/or ≥ 3 different antimicrobial families. The blaVIM-2 gene was the unique MBL encoding gene and was detected inside class 1 integrons. The class 1 integrons detected in 39 strains (23.8%) were associated with aminoglycosides (aadB, aadA1, aadA6, aacA4, aac(3)-I) and carbapenems resistance genes (blaVIM-2). The aac(3)-I + aadA1 and blaVIM-2 arrangements were the most prevalent ones. Thirty-one different PFGE patterns and 4 STs (ST175, ST235, ST253, ST973) were detected among the 39 intI1-positive isolates, being ST235 the most frequent. CRPA showed a great variety of alterations in oprD gene. The exoU+/exoS- genotype was detected in 82.6% of blaVIM-2-producing strains (ST235) and the exoU-/exoS+ in the remaining 17.4% (ST973).

Pseudomonas aeruginosa Isolates from Spanish Children: Occurrence in Faecal Samples, Antimicrobial Resistance, Virulence, and Molecular Typing.

Pseudomonas aeruginosa is a major opportunistic human pathogen, responsible for nosocomial infections and infections in patients with impaired immune systems. Little data exist about the faecal colonisation by P. aeruginosa isolates in healthy humans. The occurrence, antimicrobial resistance phenotype, virulence genotype, and genetic lineages of P. aeruginosa from faecal samples of children from two different Spanish regions were characterised. Seventy-two P. aeruginosa were isolated from 1,443 faecal samples. Low antimicrobial resistance levels were detected: ceftazidime (8%), cefepime (7%), aztreonam (7%), gentamicin (3%), ciprofloxacin (1%), and imipenem (1%); susceptibility to meropenem, amikacin, tobramycin, levofloxacin, and colistin. Four multidrug-resistant strains were found. Important differences were detected between both geographical regions. Forty-one sequence types were detected among the 48 tested strains. Virulence and quorum sensing genes were analysed and 13 virulotypes were detected, being 26 exoU-positive strains. Alteration in protein OprD showed eight different patterns. The unique imipenem-resistant strain showed a premature stop codon in OprD. Intestinal colonisation by P. aeruginosa, mainly by international clones (as ST244, ST253, and ST274), is an important factor for the systemic infections development and the environmental dissemination. Periodic active surveillance is useful to identify these community human reservoirs and to control the evolution of antibiotic resistance and virulence activity.

Persistencia de un clon ST6 de Enterococcus faecalis con genotipo vanB2 en dos hospitales de Aragón / Persistence of a ST6 clone of Enterococcus faecalis genotype vanB2 in two Hospitals in Aragon (Spain)

Introducción: Con el objetivo de estudiar la evolución del brote por Enterococcus faecalis ST6 genotipo vanB2 descrito en 2009-2010 en 3 hospitales de Zaragoza, se caracterizaron todos los aislados clínicos E.faecalis resistentes a vancomicina obtenidos entre 2011 y 2013 en dichos hospitales. Métodos: Caracterización molecular de los aislados y estudio de su relación clonal por electroforesis en campos pulsados. Revisión de las historias clínicas de los pacientes. Resultados: Se detectaron 79 aislados E.faecalis genotipo vanB2 de 73 pacientes de 2 de los 3 hospitales analizados, la mayoría de origen urinario. El 46,5% de los casos fueron nosocomiales. La distribución según servicios hospitalarios mostró gran variabilidad, no pudiéndose identificar una fuente de infección común. Todas las cepas fueron multirresistentes (vancomicina, eritromicina, tetraciclina, ciprofloxacino, estreptomicina, gentamicina, kanamicina) y pertenecieron al clon ST6. El 93,7% eran indistinguibles al clon del inicio del brote o subtipos estrechamente relacionados. Conclusión: El brote se mantiene constante en los 3 años posteriores a su descripción, lo que señala la necesidad de mantener un control activo que limite la emergencia y diseminación de clones resistentes a vancomicina (AU)

Introduction: In order to study the evolution of the outbreak that occurred between 2009 and 2010 in 3 hospitals in Zaragoza, all vancomycin-resistant clinical Enterococcus faecalis isolates identified between 2011 and 2013 at these hospitals were characterised. Methods: Molecular characterisation of the isolates and analysis of their clonal relationships was performed using pulsed field electrophoresis, along with a retrospective review of the patient records. Results: A total of 79 vancomycin-resistant E.faecalis isolates with genotype vanB2 of 73 patients were recovered in 2 of the 3 hospitals, most of them from urine specimens. About 46% of the cases were nosocomial. Distribution of the isolates among hospital services demonstrated high variability, making it difficult to predict a common source of infection. All the strains were multiresistant (vancomycin, erythromycin, tetracycline, ciprofloxacin, streptomycin, gentamicin, kanamycin) and belonged to lineage ST6. Seventy-four isolates (93.7%) were identical or closely related to the dominant one in the origin of the outbreak. Conclusion: The outbreak remains constant over three years after being initially described, indicating the need to implement an active control in order to limit the emergence and spread of vancomycin-resistant clones (AU)

Persistence of a ST6 clone of Enterococcus faecalis genotype vanB2 in two Hospitals in Aragon (Spain). / Persistencia de un clon ST6 de Enterococcus faecalis con genotipo vanB2 en dos hospitales de Aragón.

INTRODUCTION: In order to study the evolution of the outbreak that occurred between 2009 and 2010 in 3 hospitals in Zaragoza, all vancomycin-resistant clinical Enterococcus faecalis isolates identified between 2011 and 2013 at these hospitals were characterised. METHODS: Molecular characterisation of the isolates and analysis of their clonal relationships was performed using pulsed field electrophoresis, along with a retrospective review of the patient records. RESULTS: A total of 79 vancomycin-resistant E.faecalis isolates with genotype vanB2 of 73 patients were recovered in 2 of the 3 hospitals, most of them from urine specimens. About 46% of the cases were nosocomial. Distribution of the isolates among hospital services demonstrated high variability, making it difficult to predict a common source of infection. All the strains were multiresistant (vancomycin, erythromycin, tetracycline, ciprofloxacin, streptomycin, gentamicin, kanamycin) and belonged to lineage ST6. Seventy-four isolates (93.7%) were identical or closely related to the dominant one in the origin of the outbreak. CONCLUSION: The outbreak remains constant over three years after being initially described, indicating the need to implement an active control in order to limit the emergence and spread of vancomycin-resistant clones.

Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clones with high-level mupirocin resistance.

A high proportion of methicillin-resistant Staphylococcus aureus isolates recovered in one year period showed high-level mupirocin-resistance (HLMUPR-MRSA) in our environment (27.2%). HLMUPR-MRSA isolates were mainly collected from skin and soft tissue samples, and diabetes was the main related comorbidity condition. These isolates were more frequently found in vascular surgery. HLMUPR-MRSA was more resistant to aminoglycosides than mupirocin-susceptible MRSA, linked to the presence of bifunctional and/or nucleotidyltransferase enzymes with/without macrolide resistance associated with the msr(A) gene. Most of HLMUPR-MRSA isolates belonged to ST125/t067. Nine IS257-ileS2 amplification patterns (p3 was the most frequent) were observed in HLMUPR-MRSA isolates, suggesting the presence of several mupirocin-resistance-carrying plasmids in our environment and promoting the emergence of mupirocin resistance. The presence of the same IS257-ileS2 amplification pattern p3 in 65% of HLMUPR-MRSA, all of them ST125/t067, suggests a clonal spread in our hospital and community environment which could explain the high prevalence of HLMUPR-MRSA during the study period. An outbreak situation or an increase in mupirocin consumption was not observed.

In Vitro Models for the Study of the Intracellular Activity of Antibiotics.

Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine which antibiotics are most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of pre-opsonized bacteria by eukaryotic cells; (2) elimination of non-internalized bacteria with gentamicin; (3) incubation of infected cells with antibiotics; and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample protein content.

Antimicrobial resistance, virulence factors and genetic lineages of hospital-onset methicillin-resistant Staphylococcus aureus isolates detected in a hospital in Zaragoza / Resistencia a antibióticos, factores de virulencia y líneas clonales de Staphylococcus aureus resistente a la meticilina de origen hospitalario en un hospital de Zaragoza

INTRODUCTION: MRSA population dynamics is undergoing significant changes, and for this reason it is important to know which clones are circulating in our nosocomial environment. MATERIALS AND METHODS: A total of 118 MRSA isolates were collected from clinical samples from patients with previous hospital or healthcare contact (named as hospital-onset MRSA (HO-MRSA)) during a one year period. Susceptibility testing was performed by disk diffusion and microdilution. The presence of resistance genes and virulence factors were tested by PCR. All isolates were typed by SCCmec, spa and agr typing. PFGE and MLST were applied to a selection of them. RESULTS: Eighty-three HO-MRSA isolates (70.3%) were resistant to any antibiotic included in the macrolide-lincosamide-streptogramin B group. Among these isolates, the M phenotype was the most frequent (73.5%). One hundred and seven of HO-MRSA isolates (90.7%) showed aminoglycoside resistance. The combination aac(6')-Ie-aph(2'')-Ia + ant(4')-Ia genes was the most frequent (22.4%). Tetracycline resistance rates in HO-MRSA isolates were low (3.4%), although a high level of mupirocin resistance was observed (25.4%). Most of the HO-MRSA isolates (approximately 90%) showed SCCmec type IVc and agr type II. Fifteen unrelated pulsotypes were identified. CC5 was the most prevalent (88.1%), followed by CC8 (5.9%), CC22 (2.5%), CC398 (2.5%) and CC1 (0.8%). CONCLUSION: CC5/ST125/t067 lineage was the most frequent. This lineage was related to aminoglycoside resistance, and to a lesser extent, with macrolide resistance. The presence of international clones as EMRSA-15 (CC22/ST22), European clones as CC5/ST228, community clones related to CC1 or CC8 and livestock associated clones, as CC398, were observed in a low percentage

INTRODUCCIÓN: Las dinámicas poblacionales de SARM están experimentando cambios significativos en los últimos tiempos. Por ello es importante conocer qué líneas clonales circulan en nuestro ambiente hospitalario. MATERIALES Y MÉTODOS: Durante un año, se seleccionaron 118 SARM de muestras clínicas de pacientes con contacto previo con el ambiente hospitalario (SARM de origen hospitalario [SARM-OH]). Las pruebas de sensibilidad se realizaron mediante difusión con discos y microdilución. La presencia de genes de resistencia y factores de virulencia fueron estudiados mediante PCR. Se estableció el tipo de SCCmec, spa y agr en todos los aislados, y en una selección se estudió su relación genética por PFGE y MLST. RESULTADOS: Ochenta y tres SARM-OH (70,3%) fueron resistentes a al menos un antibiótico del grupo de los macrólidos-lincosamidas-estreptograminas B. Entre estos, el fenotipo M fue el más frecuente (73,5%). Ciento siete aislamientos (90,7%) mostraron resistencia a aminoglucósidos. La combinación aac(6')-Ieaph( 2'')-Ia + ant(4')-Ia fue la más frecuente (22,4%). Las tasas de resistencia a tetraciclinas detectadas fueron bajas (3,4%). Se observó un 25,4% de resistencia de alto nivel a mupirocina. Aproximadamente un 90% de SARM-OH mostraron SCCmec tipo IVc y agr tipo II. Se identificaron 15 pulsotipos no relacionados. El CC5 fue el más prevalente (88,1%) seguido de CC8 (5,9%), CC22 (2,5%), CC398 (2,5%) y CC1 (0,8%). CONCLUSIÓN: La línea clonal CC5/ST125/t067 fue la más habitual. Esta línea se relacionó con resistencia a aminoglucósidos, y, en menor medida, con macrólidos. La presencia de clones internacionales como EMRSA-15 (CC22/ST22), clones europeos como CC5/ST228, clones comunitarios relacionados con CC1 o CC8 y clones asociados al ganado, como el CC398, se observaron en un bajo porcentaje

Antimicrobial resistance, virulence factors and genetic lineages of hospital-onset methicillin-resistant Staphylococcus aureus isolates detected in a hospital in Zaragoza.

INTRODUCTION: MRSA population dynamics is undergoing significant changes, and for this reason it is important to know which clones are circulating in our nosocomial environment. MATERIALS AND METHODS: A total of 118 MRSA isolates were collected from clinical samples from patients with previous hospital or healthcare contact (named as hospital-onset MRSA (HO-MRSA)) during a one year period. Susceptibility testing was performed by disk diffusion and microdilution. The presence of resistance genes and virulence factors were tested by PCR. All isolates were typed by SCCmec, spa and agr typing. PFGE and MLST were applied to a selection of them. RESULTS: Eighty-three HO-MRSA isolates (70.3%) were resistant to any antibiotic included in the macrolide-lincosamide-streptogramin B group. Among these isolates, the M phenotype was the most frequent (73.5%). One hundred and seven of HO-MRSA isolates (90.7%) showed aminoglycoside resistance. The combination aac(6')-Ie-aph(2″)-Ia+ant(4')-Ia genes was the most frequent (22.4%). Tetracycline resistance rates in HO-MRSA isolates were low (3.4%), although a high level of mupirocin resistance was observed (25.4%). Most of the HO-MRSA isolates (approximately 90%) showed SCCmec type IVc and agr type II. Fifteen unrelated pulsotypes were identified. CC5 was the most prevalent (88.1%), followed by CC8 (5.9%), CC22 (2.5%), CC398 (2.5%) and CC1 (0.8%). CONCLUSION: CC5/ST125/t067 lineage was the most frequent. This lineage was related to aminoglycoside resistance, and to a lesser extent, with macrolide resistance. The presence of international clones as EMRSA-15 (CC22/ST22), European clones as CC5/ST228, community clones related to CC1 or CC8 and livestock associated clones, as CC398, were observed in a low percentage.

Detection of carbapenemase-producing Enterobacteriaceae in various scenarios and health settings.

Detection of carbapenemase-producing Enterobacteriaceae (CPE) is an important task at microbiology laboratories in hospitals. As the prevalence of CPE is increasing worldwide, the implementation of phenotypically based screening as well as confirmatory procedures to detect CPE are important for microbiologists. In addition to detection of carbapenem hydrolysis, the inhibition of activity against a carbapenem in the presence of several inhibitor compounds specific to class A, B, or class C beta-lactamases is a useful method to confirm the presence of carbapenemases in bacterial isolates. There is also a proteomic approach that compares the MALDI-TOF spectrum generated by the intact carbapenem (non-hydrolyzed) with that obtained after hydrolysis of the beta-lactam ring by beta-lactamase to reveal the presence of carbapenemases in bacterial isolates. Proteomic methods will probably be more frequently implemented in laboratories in the near future. Finally, molecular methods to directly or indirectly detect the presence of a carbapenemase genes are increasingly being used in microbiology laboratories. One of the main advantages of these methods is their speed, and also that they can be used directly with the clinical sample without the need for an isolated bacterial colony. Multiplex PCR, real-time PCR, DNA microarrays and pyrosequencing are some examples of molecular-based tests. Their main disadvantage is their cost, although prices are going down as the range of services increases. Surveillance of carriers is also an important task for infection control purposes. In this case, commercially available chromogenic medium supplemented with low carbapenem concentrations has shown an excellent ability to detect CPE. Moreover, molecular methods to detect specific carbapenemase genes directly from rectal swabs, stools, or other colonization sources have had excellent results.