Full characterization of an IncR plasmid harboring qnrS1 recovered from a VIM-11-producing Pseudomonas aeruginosa.

Metallo-ß-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaVIM-11 in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening for MBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrS1; however, both markers were located in different plasmids. The qnrS1-harboring plasmid (pP6qnrS1) was 117945bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrS1, it harbored several aminoglycoside resistance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrS1, plus the first detection of an IncR plasmid in Argentina.

First detection of CMY-2 plasmid mediated ß-lactamase in Salmonella Heidelberg in South America.

Salmonella enterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries. Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the ß-lactam resistance profile. S. Heidelberg contained a 97kb plasmid belonging to the Inc N group harboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization. The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identified the first CMY-2 producing isolate of S. Heidelberg in Argentina and even in South America.

Plasmid-Encoded AmpC (pAmpC) in Enterobacteriaceae: epidemiology of microorganisms and resistance markers.

CMY-2 Β-lactamase is an important cause of Β-lactam resistance in Enterobacteriaceae and constitutes the most widespread pAmpC. Although CMY-2 has been previously recognized in our region, the real prevalence and epidemiology of this resistance marker was uncertain. During August-October 2009, we conducted a multicenter, prospective study to determine pAmpC prevalence and to characterize CMY-2 producing Escherichia coli associated plasmids. Plasmid-encoded AmpC prevalence was 0.9 % in enterobacteria in this period, being CMY-2 prevalent and to a lesser extent DHA. Molecular typing of CMY-2- producing Escherichia coli isolates showed several lineages. Moreover, replicon typing of cmy-2- containing plasmids displayed a broad diversity in Inc/cmy-2 links. Therefore, association of cmy-2 with specific transposon elements may be responsible for the spread of this resistance marker in Enterobacteriaceae.

[Antimicrobial susceptibility testing in clinically relevant non-fermenting gram-negative bacilli: recommendations from the Antimicrobial Agents Subcommittee of the Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas, Asociación Argentina de Microbiología]. / Criterios de ensayo, interpretación e informe de las pruebas de sensibilidad a los antibióticos en los bacilos gram negativos no fermentadores de importancia clínica: recomendaciones de la Subcomisión de Antimicrobianos de la Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas, Asociación Argentina de Microbiología.

This document contains the recommendations for antimicrobial susceptibility testing of the clinically relevant non-fermenting gram-negative bacilli (NFGNB), adopted after conforming those from international committees to the experience of the Antimicrobial Agents Subcommittee members and invited experts. This document includes an update on NFGNB classification and description, as well as some specific descriptions regarding natural or frequent antimicrobial resistance and a brief account of associated resistance mechanisms. These recommendations not only suggest the antimicrobial drugs to be evaluated in each case, but also provide an optimization of the disk diffusion layout and a selection of results to be reported. Finally, this document also includes a summary of the different methodological approaches that may be used for detection and confirmation of emerging b-lactamases, such as class A and B carbapenemases.

A 7-year national survey on bacterial resistance in bronchoalveolar lavage from patients hospitalized in Argentina.

The purpose of this study is to undertake a nationwide survey on bacterial resistance in bronchoalveolar lavage (BAL) from patients hospitalized in Argentina. A 2-month point prevalence study was conducted twice yearly (April-May and October-November) from 1997 to 2003 by 36 Argentinean centers. Antimicrobial susceptibility data of the potential pathogens recovered from the BAL (samples containing <1% of squamous epithelial cells and bacterial counts >or=10(4) CFU/mL) of inpatients (i.e., >or=48-h hospital length of stay) with suspected hospital-acquired pneumonia (HAP) were collected on a computerized system (SIR) described previously. The survey was split into 2 periods for comparison purposes, 1997 to 2000 and 2001 to 2003. A total of 752 organisms were included. Staphylococcus aureus was the most frequent species, followed by Acinetobacter spp. and Pseudomonas aeruginosa. In both periods, more than a half of the Klebsiella pneumoniae strains displayed a phenotype of extended-spectrum beta-lactamase producer. A doubling of imipenem-resistant Acinetobacter frequency was shown from the 1st period to the 2nd one (25-48%). More than two-thirds of the S. aureus strains proved to be methicillin resistant in both periods, and a pronounced decrease of resistance rates to trimethoprim/sulfamethoxazole and rifampin was shown in the 2nd period. The present study shows the worrisome increasing bacterial resistance in BAL samples to most available antimicrobial options for treating patients with suspected HAP. Variations over time support the need for systematic tailored surveillance and compel us to establish a rational usage of antimicrobial agents in our country.

[Community-acquired methicillin-resistant Staphylococcus aureus disseminated disease]. / Enfermedad invasora por Staphylococcus aureus meticilino resistente adquirida en la comunidad.

A 21 year old man, previously healthy, presented with subcutaneous nodes consistent with gummas. Ultrasonography disclosed multiple subcutaneous abscesses and images suitable with piomiositis, pleural and pericardium effusion. A puncture-aspirate with fine-needle was performed and produced purulent material, with isolate of Staphylococcus aureus. Antimicrobial susceptibility testing by disk diffusion showed resistant to cefalotin, erythromycin and clindamycin, and susceptibility to trimethoprim-sulfamethoxazole, ciprofloxacin and rifampicin. Methicilin-resistance was confirmed by Staphyslide agglutination testing (Biomérieux). The patient was treated with ciprofloxacin and rifampicin during four weeks, with a good clinical response. The frequency of CA-MRSA infections is increasing, and these are reported in patients without identified predisposing risks leading to failure on empiric therapy for community infections presumed to be due to staphylococcal agents.

Full characterization of an IncR plasmid harboring qnrSI recovered from a VIM-11-producing Pseudomonas aeruginosa

Abstract Metallo-p-lactamases (MBL) producing Pseudomonas aeruginosa isolates have been well characterized. Quinolones are commonly used in the treatment of carbapenem-resistant P. aeruginosa infections; however, data about PMQR in this species are scarce. The objective of this study was to report the simultaneous presence of qnrS and blaV-M-n in P. aeruginosa, and to characterize the qnrS-harboring plasmid. Thirty-eight carbapenem-resistant P. aeruginosa isolates were recovered from a hospital in Buenos Aires during 2012. Screening forMBL was assessed by the double disk synergy test using EDTA and carbapenem discs. Plasmid DNA extraction was performed by a method using phenol-chloroform. PCR followed by sequencing was carried out to determine each MBL and PMQR allele. PCR-BseGI-RFLP was performed to detect aac-(6')-Ib-cr. The gyrA-QRDR was sequenced in those PMQR-harboring isolates. Plasmid incompatibility groups and addiction systems were characterized by PCR. The PMQR-carrying plasmid was sequenced using Illumina technology, annotated using RAST and manually curated. Eleven/38 isolates were VIM producers (blaVIM-2 and blaVIM-11) while 1/38 harbored blaIMP-13. One isolate harbored blaVIM-11 and the PMQR qnrSI; however, both markers were located in different plasmids. The qnrSí-harboring plasmid (pP6qnrS1) was 117 945 bp in size, presented 154 CDS and corresponded to the IncR group. In addition to qnrSI, it harbored several aminoglycoside resis-tance markers. Although pP6qnrS1 was non-conjugative, it presented an oriT which made it possible for this plasmid to be transferable. This is the first report on P. aeruginosa carrying both blaVIM-11 and qnrSI, plus the first detection of an IncR plasmid in Argentina.

Resumen Las quinolonas son comúnmente utilizadas en el tratamiento de las infecciones producidas por Pseudomonas aeruginosa resistentes a carbapenems (PARC); aun así, la información sobre la resistencia a quinolonas mediada por plásmidos (PMQR) en esta especie es escasa. El objetivo de este trabajo fue reportar la presencia simultánea de los genes qnrS y blaVIM-11 en PARC y caracterizar el plásmido portador de qnrS. Durante 2012 se recuperaron 38 PARC en un hospital de Buenos Aires. El tamizaje para detectar producción de metalo-beta-lactamasas (MBL) se llevó a cabo mediante sinergia de doble disco utilizando EDTA y carbapenems. El ADN plasmídico fue extraído utilizando fenolcloroformo. Para determinar los alelos de los genes implicados en la síntesis de MBL y de PMQR, se llevó a cabo PCR-secuenciación. Para la detección de aac-(6')-Ib-cr se realizó PCR-BseGI-RFLP. En aquellos aislamientos portadores de PMQR se secuenció el gen gyrA. Los grupos de incompatibilidad y sistemas de adicción fueron caracterizados por PCR. El plásmido portador de PMQR fue secuenciado completamente y curado manualmente. De 38 aislamientos, 11 fueron productores de VIM (blaVIM-2 y blaVIM-11), mientras que uno contenía blaIMP-13. Si bien un aislamiento fue portador de blaVIM-11 y de qnrSI, dichos marcadores se encontraban en distintos plásmidos. El plásmido portador de qnrSI (pP6qnrS1) presentó un tamaño de 117.945 pby 154 secuencias codificantes (CDS); este correspondió al grupo de incompatibilidad IncR. Además de qnrSI, el plásmido portaba diversos marcadores de resistencia a aminoglucósidos. Aun cuando pP6qnrS1 no resultó conjugativo, presentó un oriT, de modo que posiblemente sea transferible. Este es el primer informe acerca de PARC portadora de blaVIM-11 y de qnrSI en simultáneo, además, es la primera descripción de un plásmido IncR en Argentina.

Argentinean collaborative multicenter study on the in vitro comparative activity of piperacillin-tazobactam against selected bacterial isolates recovered from hospitalized patients.

The in vitro activity of piperacillin-tazobactam and several antibacterial drugs commonly used in Argentinean hospitals for the treatment of severe infections was determined against selected but consecutively isolated strains from clinical specimens recovered from hospitalized patients at 17 different hospitals from 9 Argentinean cities from different geographic areas during the period November 2001-March 2002. Out of 418 Enterobacteriaceae included in the Study 84% were susceptible to piperacillin-tazobactam. ESBLs putative producers were isolated at an extremely high rate since among those isolates obtained from patients with hospital acquired infections 56% of Klebsiella pneumoniae, 32% of Proteus mirabilis and 25% Escherichia coli were phenotypically considered as ESBLs producers Notably P.mirabilis is not considered by for screening for ESBL producers. ESBLs producers were 100% susceptible to imipenem and 70% were susceptible to piperacillin-tazobactam whereas more than 50% were resistant to levofloxacin. The isolates considered as amp C beta lactamase putative producers showed 99% susceptibility to carbapenems while 26.7% were resistant to piperacillin-tazobactam and 38.4% to levofloxacin. Noteworthy only 4% of the Enterobacteriaceae isolates were resistant to amikacin. Piperacillin-tazobactam was the most active agent against Pseudomonas aeruginosa isolates (MIC(90): 128 microg/ml; 78% susceptibility) but showed poor activity against Acinetobacter spp (MIC(90):>256 microg/ml; 21.7% susceptibility). Only 41.7% Acinetobacter spp isolates were susceptible to ampicillin-sulbactam. Piperacillin-tazobactam inhibited 100% of Haemophilus influenzae isolates (MIC(90) < 0.25 microg/ml) but only 16.6% of them were ampicillin resistant. The activity of piperacillin-tazobactam against oxacillin susceptible Staphylococcus aureus or coagulase negative staphylococci was excellent (MIC(90) 2 microg/ml; 100% susceptibility). Out of 150 enterococci 12 isolates (8%) were identified as E.faecium and only three isolates (2%), 2 E.faecium and 1 E.faecalis were vancomycin resistant. All the enterococci isolates were susceptible to linezolid. Piperacillin-tazobactam showed excellent activity (MIC(90) 2 microg/ml; 92% susceptibility). Regarding pneumococci all the isolates showed MICs of 16 microg/ml for piperacillin-tazobactam. Among 34 viridans group streptococci only 67% were penicillin susceptible and 85.2% ceftriaxone susceptible whereas piperacillin-tazobactam was very active (MIC(90) 4 microg/ml).Piperacillin-tazobactam is therefore a very interesting antibacterial drug to be used, preferably in combination (IE: amikacin-vancomycin) for the empiric treatment of severe infections occurring in hospitalized patients in Argentina. Caution must be taken for infections due to ESBL producers considering that the inoculum effect MICs can affect MIC values.

Primera detección de CMY-2 en Salmonella Heidelberg en Sudamérica / First detection of CMY-2 plasmid mediated ß-lactamase in Salmonella Heidelberg in South America

Salmonellaenterica serovar Heidelberg es uno de los principales agentes causantes de salmonelosis en humanos en Estados Unidos y Canadá, sin embargo, resulta infrecuente en los países de Sudamérica y Europa. En este trabajo se caracterizó un aislamiento de S. Heidelberg resistente a oximino-cefalosporinas recuperado de un paciente internaen un hospital de la Ciudad de Buenos Aires. Se evidenció la presencia de un plásmido de 97 kbperteneciente al grupo de incompatibilidad IncN, portador del gen blaCMY-2. ISEcp1 fue localizado corriente arriba de blaCMY-2, promoviendo su expresión y movilización.El aislamiento de S. Heidelberg correspondió al secuenciotipo 15 y en la virotipifi cación se detectó el gen sopE. En este trabajo describimos por primera vez la producción de CMY-2 en una cepa de S. Heidelberg en nuestro país y América Latina

Salmonellaenterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries.Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the ß-lactam resistance profi le. S. Heidelberg contained a 97 kb plasmid belonging to the Inc N groupharboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization.The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identifi ed the fi rst CMY-2 producing isolate of S. Heidelberg in Argentina and even in South Americ

Criterios de ensayo, interpretación e informe de las pruebas de sensibilidad a los antibióticos en los bacilos gram negativos no fermentadores de importancia clínica: recomendaciones de la Subcomisión de Antimicrobianos de la Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas, Asociación Argentina de Microbiología / Antimicrobial susceptibility testing in clinically relevant non-fermenting gram-negative bacilli: recommendations from the Antimicrobial Agents Subcommittee of the Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas, Asociación Argentina de Microbiología

En este documento se dan a conocer una serie de recomendaciones para el ensayo, la lectura, la interpretación y el informe de las pruebas de sensibilidad a los antimicrobianos para los bacilos gram negativos no fermentadores (BGNNF) que se aíslan en humanos. Se adoptaron como base las recomendaciones internacionales, las de la Subcomisión de Antimicrobianos de la Sociedad Argentina de Bacteriología, Micología y Parasitología Clínicas y las de un grupo de expertos invitados. Se incluye, además, la nomenclatura actualizada de los BGNNF y la descripción de algunas de sus características individuales, de sus resistencias naturales o habituales a los antimicrobianos de uso clínico y de los mecanismos responsables de tales resistencias. También se indican los agentes antimicrobianos que se deberían ensayar frente a las distintas especies, con la especificación de cuáles deberían ser informados, y su ubicación estratégica en las placas de cultivo para poder detectar los mecanismos de resistencia más frecuentes y relevantes. Por último, se detallan los métodos de detección y de confirmación fenotípica de la presencia de b-lactamasas emergentes en Argentina, como las carbapenemasas clases A y B.

This document contains the recommendations for antimicrobial susceptibility testing of the clinically relevant non-fermenting gram-negative bacilli (NFGNB), adopted after conforming those from international committees to the experience of the Antimicrobial Agents Subcommittee members and invited experts. This document includes an update on NFGNB classification and description, as well as some specific descriptions regarding natural or frequent antimicrobial resistance and a brief account of associated resistance mechanisms. These recommendations not only suggest the antimicrobial drugs to be evaluated in each case, but also provide an optimization of the disk diffusion layout and a selection of results to be reported. Finally, this document also includes a summary of the different methodological approaches that may be used for detection and confirmation of emerging b-lactamases, such as class A and B carbapenemases.

Enfermedad invasora por Staphylococcus aureus meticilino: resistente adquirida en la comunidad / Community-acquired methicillin-resistant Staphylococcus aureus disseminated disease

Se presenta un caso de enfermedad invasora por Staphylococcus aureus meticilino resistente (SAMR)adquirido en la comunidad (SAMR-c). Paciente varón de 21 años, previamente sano, que consultó por nódulos subcutáneos compatibles con gomas. La ultrasonografía reveló múltiples abscesos subcutáneos e imágenes consistentes con piomiositis, además de derrame pleural y pericárdico. En el cultivo del material purulentoobtenido por punción-aspiración con aguja fina de las lesiones se aisló S. aureus. El antibiograma por difusiónmostró resistencia a cefalotina, eritromicina y clindamicina, y sensibilidad a trimetroprima-sulfametoxazol, ciprofloxacina y rifampicina. La meticilino resistencia se confirmó por aglutinación con partículas de látex sensibilizadas con anticuerpos monoclonales dirigidos contra la proteína ligadora de penicilina 2A. El paciente fue tratado con ciprofloxacina y rifampicina durante cuatro semanas, con evolución favorable. La frecuencia de infecciones por SAMR-c está en aumento, observándose en personas sin factores de riesgo aparentes. Esto llevaría a fracaso en el tratamiento empírico para infecciones de la comunidad en los que se presume etiología estafilocócica

A 21 year old man, previously healthy, presented with subcutaneous nodes consistent with gummas. Ultrasonography disclosed multiple subcutaneous abscesses and images suitable with piomiositis, pleural and pericardium effusion. A puncture-aspirate with fine-needle was performed and produced purulent material, with isolate of Staphylococcus aureus. Antimicrobial susceptibility testing by disk diffusion showed resistant to cefalotin, erythromycin and clindamycin, and susceptibility to trimethoprim-sulfamethoxazole, ciprofloxacin and rifampicin. Methicilin-resistance was confirmed by Staphyslide agglutination testing (Biomérieux). The patient was treatedwith ciprofloxacin and rifampicin during four weeks, with a good clinical response. The frequency of CA-MRSAinfections is increasing, and these are reported in patients without identified predisposing risks leading to failureon empiric therapy for community infections presumed to be due to staphylococcal agent