Multiyear, Multinational Survey of the Incidence and Global Distribution of Metallo-ß-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa.

Metallo-ß-lactamases (MBLs) hydrolyze all classes of ß-lactams except monobactams and are not inhibited by classic serine ß-lactamase inhibitors. Gram-negative pathogens isolated from patient infections were collected from 202 medical centers in 40 countries as part of a global surveillance study from 2012 to 2014. Carbapenem-nonsusceptible Enterobacteriaceae and Pseudomonas aeruginosa were characterized for bla genes encoding VIM, IMP, NDM, SPM, and GIM variants using PCR and sequencing. A total of 471 MBL-positive isolates included the following species (numbers of isolates are in parentheses): P. aeruginosa (308), Klebsiella spp. (85), Enterobacter spp. (39), Proteeae (16), Citrobacter freundii (12), Escherichia coli (6), and Serratia marcescens (5) and were submitted by sites from 34 countries. Of these, 69.6% were collected in 9 countries (numbers of isolates are in parentheses): Russia (72), Greece (61), Philippines (54), Venezuela (29), and Kuwait, Nigeria, Romania, South Africa, and Thailand (20 to 25 isolates each). Thirty-two different MBL variants were detected (14 VIM, 14 IMP, and 4 NDM enzymes). Seven novel MBL variants were encountered in the study, each differing from a previously reported variant by one amino acid substitution: VIM-42 (VIM-1 [V223I]), VIM-43 (VIM-4 [A24V]), VIM-44 (VIM-2 [K257N]), VIM-45 (VIM-2 [T35I]), IMP-48 (IMP-14 [I69T]), IMP-49 (IMP-18 [V49F]), and NDM-16 (NDM-1 [R264H]). The in vitro activities of all tested antibiotics against MBL-positive Enterobacteriaceae were significantly reduced with the exception of that of aztreonam-avibactam (MIC90, 0.5 to 1 µg/ml), whereas colistin was the most effective agent against MBL-positive P. aeruginosa isolates (>97% susceptible). Although the global percentage of isolates encoding MBLs remains relatively low, their detection in 12 species, 34 countries, and all regions participating in this surveillance study is concerning.

Host-specific enzyme-substrate interactions in SPM-1 metallo-ß-lactamase are modulated by second sphere residues.

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-ß-lactamases (MßLs), enzymes able to hydrolyze most ß-lactam antibiotics. SPM-1 is an MßL produced only by P. aeruginosa, while other MßLs are found in different bacteria. Despite similar active sites, the resistance profile of MßLs towards ß-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MßLs in that it contains "atypical" second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards ß-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.

Combination therapy for carbapenem-resistant Gram-negative bacteria.

The emergence of resistant to carbapenems Gram-negative bacteria (CR GNB) has severely challenged antimicrobial therapy. Many CR GNB isolates are only susceptible to polymyxins; however, therapy with polymyxins and other potentially active antibiotics presents some drawbacks, which have discouraged their use in monotherapy. In this context, along with strong pre-clinical evidence of benefit in combining antimicrobials against CR GNB, the clinical use of combination therapy has been raised as an interesting strategy to overcome these potential limitations of a single agent. Polymyxins, tigecycline and even carbapenems are usually the cornerstone agents in combination schemes. Optimization of the probability to attain the pharmacokinetic/pharmacodynamic targets by both cornerstone drug and adjuvant drug is of paramount importance to achieve better clinical and microbiological outcomes. Clinical evidence of the major drugs utilized in combination schemes and how they should be prescribed considering pharmacokinetic/pharmacodynamic characteristics against CR GNB will be reviewed in this article.

Resistencia a carbapenemes en aislamientos de pseudomonas aeruginosa: un ejemplo de interacción entre distintos mecanismos / Carbapenem resistance in pseudomonas aeruginosa isolates: an example of interaction between different mechanisms

Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes acarbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en suexpresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia.Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.

Objective. To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of othermechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. Methods. Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibitedequal expression of the IMP-13 enzyme, but only five of them were carbapenemresistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identifiedusing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. Results. The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presentedmutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and‑resistant isolates. The contribution of the combined presence of IMP-13 and reducedOprD to increased resistance was examined. Conclusions. Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistancemechanisms in order to enhance carbapenem resistance.

[Carbapenem resistance in Pseudomonas aeruginosa isolates: an example of interaction between different mechanisms]. / Resistencia a carbapenemes en aislamientos de Pseudomonas aeruginosa: un ejemplo de interacción entre distintos mecanismos.

OBJECTIVE: To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of other mechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. METHODS: Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibited equal expression of the IMP-13 enzyme, but only five of them were carbapenem-resistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. RESULTS: The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presented mutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and -resistant isolates. The contribution of the combined presence of IMP-13 and reduced OprD to increased resistance was examined. CONCLUSIONS: Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistance mechanisms in order to enhance carbapenem resistance.

Microbiological and epidemiological characterization of imipenem-resistant Pseudomonas aeruginosa strains from a Brazilian tertiary hospital: report from the SENTRY Antimicrobial Surveillance Program.

OBJECTIVES: To evaluate the antimicrobial susceptibility profile, the genetic similarity, and the mechanisms of carbapenem resistance among imipenem-resistant Pseudomonas aeruginosa isolates collected from a Brazilian tertiary teaching hospital. METHODS: Seventy-eight consecutive samples of P. aeruginosa were evaluated during 2000 and 2001. The antimicrobial susceptibility was evaluated by reference broth microdilution methods and the imipenem-resistant isolates were screened for metallo-beta-lactamase (MbetaL) production throughout disc approximation test and MbetaL Etest strips and isolates with positive screen test result were submitted to PCR assays using primers blaIMP-1, bla VIM-1, blaVIM-2 e blaSPM-1. The genetic similarity of MbetaL-producing strains was evaluated by automated ribotyping for epidemiological typing purpose. RESULTS: Resistance rates were high to the majority of antimicrobial agents tested except polymyxin B, which inhibited all samples at the Clinical and Laboratory Standards Institute breakpoint (< or = 2 microg/ml). Twenty-nine (37.2%) isolates were resistant to imipenem and these isolates showed great genomic variability. MbetaL production was demonstrated in two imipenem-resistant isolates, which were detected using blaSPM-1 and blaIMP-2-specific primers. Sequence analysis revealed the presence of blaSPM-1 and a novel blaIMP-type gene, blaIMP-16. CONCLUSION: The results of this study showed high resistance rates to the majority of antimicrobial agents among P. aeruginosa samples. High imipenem resistance rates were probably due to continuous selection of resistant mutants. The production of MbetaL did not represent a frequent mechanism of carbapenem resistance in this medical center; but a novel MbetaL was identified. Continued antimicrobial surveillance and infection control measures should be emphasized to minimize the emergence and dissemination of antimicrobial resistance.

[Guides for rational use of B-lactam antibiotics:resistance mechanism and clinical interpretation]. / Guías para el uso racional de antibióticos beta-lactámicos: mecanismos de resistencia y su interpretación clínica.

beta-lactams are the antibiotic compounds most widely used against hospital and community acquired infections. However, resistance has emerged in both Gram-positive and Gram-negative bacteria, limiting their therapeutic efficacy. The choice of appropriate treatment depends on analysis of susceptibility data that indicates a specific mechanism of resistance. Correct interpretation of susceptibility tests permits a rational approach to the resistance problem and selection of alternatives for treatment. The laboratory must first be able to identify accurately microorganisms to the species level and then test a minimum of relevant antimicrobials. beta-lactam resistance in Enterobacteriaceae is mainly due to the production of plasmid or chromosomal encoded beta-lactamases. In Gram-negative non-fermenting bacteria, impermeability and efflux are relatively more important to the treatment selected. In Gram-positive bacteria, resistance mechanisms can involve changes in penicillin-binding proteins (PBPs), production of new PBPs or synthesis of beta-lactamases. The range of therapeutic options must be based on the current status of local resistance mechanisms.

Extended-spectrum beta-lactamase producing enterobacteriaceae in a tertiary care hospital in Trinidad and Tobago.

Extended-spectrum beta-lactamase (ESBL) mediated resistance to third generation cephalosporins, amongst the family Enterobacteriaceae, is emerging worldwide. This is the Caribbean's first survey on ESBL production, and was conducted during two six-month periods in 1998 and 2001, in a tertiary health institution in Trinidad and Tobago. Consecutive ampicillin resistant isolates of the family Enterobacteriaceae from in-patients were screened for resistance to third generation cephalosporins, and for ESBL production. The proportion of isolates found to be ESBL producers was similar in both samples (40 of 560 and 23 of 361). Overall, ESBL production was more frequent in enterobacter, citrobacter and proteus (and related organisms) than in Klebsiella and Escherichia (11.2% and 4.6%, respectively, p < 0.001). In the 1998 sample, this proportion (9.8% versus 5.8%) was significant (p < 0.05), but the difference was more marked in the 2001 sample (13.6% versus 2.9%, p < 0.001). Continued distribution of these resistant bacterial strains is of concern. In the Caribbean region, more laboratory surveillance and increased infection control vigilance are recommended, with focus on specific genera in the family.

Extended-spectrum beta-lactamase producing enterobacteriaceae in a tertiary care hospital in Trinidad and Tobago

Extended-spectrum beta-lactamase (ESBL) mediated resistance to third generation cephalosporins, amongst the family Enterobacteriaceae, is emerging worldwide. This is the Caribbean's first survey on ESBL production, and was conducted during two six-month periods in 1998 and 2001, in a tertiary health institution in Trinidad and Tobago. Consecutive ampicillin resistant isolates of the family Enterobacteriaceae from in-patients were screened for resistance to third generation cephalosporins, and for ESBL production. The proportion of isolates found to be ESBL producers was similar in both samples (40 of 560 and 23 of 361). Overall, ESBL production was more frequent in enterobacter, citrobacter and proteus (and related organisms) than in Klebsiella and Escherichia (11.2 and 4.6, respectively, p < 0.001). In the 1998 sample, this proportion (9.8 versus 5.8) was significant (p < 0.05), but the difference was more marked in the 2001 sample (13.6 versus 2.9, p < 0.001). Continued distribution of these resistant bacterial strains is of concern. In the Caribbean region, more laboratory surveillance and increased infection control vigilance are recommended, with focus on specific genera in the family

All detectable high-molecular-mass penicillin-binding proteins are modified in a high-level beta-lactam-resistant clinical isolate of Streptococcus mitis.

All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the beta-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 microg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in a pbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

Multicenter evaluation of antimicrobial resistance to six broad-spectrum beta-lactams in Colombia using the Etest method. The Colombian Antimicrobial Resistance Study Group.

The need for comprehensive and quantitative accurate antimicrobial resistance surveillance systems has become acute as a guide to problem recognition and to focus local interventions. A multilaboratory (10 medical centers) Colombia surveillance project was initiated in early 1997 to monitor the potency and spectrum of six (cefepime, cefotaxime, ceftazidime, cefoperazone/sulbactam, aztreonam, and imipenem) broad-spectrum antimicrobial agents tested against 100 organisms per participant center (802 strains). Ten groups of organisms were tested by a reference-quality method (Etest; AB BIODISK, Solna, Sweden) with results validated by concurrent quality control and additional challenge strain analysis. Results from nine qualifying medical centers were tabulated, and 95.7 to 96.8% of quality assurance tests were within expected ranges. Only cefepime (90.1-100.0% susceptible) and imipenem (96.3-100.0%) were active against all Enterobacteriaceae at > 90% of susceptible isolates using the breakpoint concentrations recommended by the National Committee for Clinical Laboratory Standards. Among ceftazidime- (or cefotaxime- or aztreonam-) resistant Enterobacter spp. and Citrobacter freundii, cefepime remained active, but not cefoperazone with sulbactam. Escherichia coli and Klebsiella spp. strains having resistance phenotypes consistent with extended spectrum beta-lactamase production were discovered in approximately 5 to 10% of isolates. All tested drugs except ceftazidime (31.8-57.7% susceptible) were active against > 94% of oxacillin-susceptible staphylococci. Similar rates of resistance (9.1-14.8%) were observed in Pseudomonas aeruginosa for five of six drugs (not cefotaxime; 15.9% of strains were susceptible). Acinetobacter spp. isolates were most susceptible to imipenem (95.8%), cefepime (86.1%), and cefoperazone/sulbactam (83.3%). Overall for the 1997 order of antimicrobial spectrums for these tested compounds was: imipenem (96.6%) > cefepime (93.6%) > cefoperazone/sulbactam (90.5%) > cefotaxime (74.9%) > aztreonam (74.3% for Gram-negative bacilli only) > ceftazidime (73.2%). These data should be used to guide empiric regimens in Colombia, and additionally will provide a resistance statistical baseline to which future studies in this nation can be compared.

Estudio sobre cepas productoras de beta lactamasas plasmídicas de espectro extendido en Argentina / Plasmid mediated extended spectrum beta-lactamase strains isolated in Argentina

La inactivación de cefalosporinas de tercera generación (C3G) por beta lactamasas de enterobacterias era hasta hace poco responsabilidad exclusiva de cepas productoras de cefalosporinasas cromosómicas. Actualmente, se han descripto nuevas beta lactamasas plasmídicas, transferibles, de espectro ampliado (BLEA) que hidrolizan C3G. Para determinar la incidencia de enterobacterias productoras de BLEA se estudiaron las cepas aisladas de 824 pacientes de siete centros asistenciales de Buenos Aires. Se seleccionaron por: 1) obtención de un halo de <26mm, por el método de discos frente a cefotaxima (CTX) o ceftacidima (CAZ) y 2) aumento del halo a >30mm al emplear discos de CTX o CAZ adicionados de clavulanato (30:10). Reunieron estas condiciones 44 Klebsiella spp y 2 E. coli. Estas últimas resultaron ser simultaneamente hiperproductoras de beta lactamasa cromosónica y de TEM-1. Todas las cepas de Klebsiella fueron productoras de BLEA en base a: perfil de resistencia por CIM, punto isoeléctrico (pI) de las beta lactamasas, acción de inhibidores de beta lactamasas y transferencia de la resistencia a C3G. Las cepas productoras de BLEA, se aislaron en el 14,6 por ciento de pacientes internados en Unidades de Cuidados Intensivos, 3,6 por ciento en otras áreas y 1,2 por ciento de pacientes externos. Un 56,5 por ciento provinieron de infecciones urinarias, 17,3 por ciento de bacteriemias, 15,2 por ciento de infecciones respiratorias y 11 por ciento de otras localizaciones. Los fracasos terapéuticos con C3G predominaron entre las infecciones extraurinarias. De acuerdo a la CIM todas las cepas fueron resistentes a las penicilinas, excepto a temocilina; a las cefalosporinas de primera y segunda generación, excepto a la cefoxitina. Para las C3G todas presentaron CIM entre 50 y 100 veces más elevadas que las habituales en Klebsiella spp. no productoras de BLEA. Cefoperazona y CAZ fueron las más perjudicadas mientras que ceftizoxima y cefpiroma fueron las más estables. Aztreonama fue más afectada que carumonama. Todas fueron sensibles a imipenem y moxalactama y también a ciprofloxacina, excepto una cepa. Todas las cepas presentaron bandas a pI correspondientes a derivados SHV y todas, salvo una, a pI:5,4 correspondiente a TEM-1...

Estudio sobre cepas productoras de beta lactamasas plasmídicas de espectro extendido en Argentina / Plasmid mediated extended spectrum beta-lactamase strains isolated in Argentina

La inactivación de cefalosporinas de tercera generación (C3G) por beta lactamasas de enterobacterias era hasta hace poco responsabilidad exclusiva de cepas productoras de cefalosporinasas cromosómicas. Actualmente, se han descripto nuevas beta lactamasas plasmídicas, transferibles, de espectro ampliado (BLEA) que hidrolizan C3G. Para determinar la incidencia de enterobacterias productoras de BLEA se estudiaron las cepas aisladas de 824 pacientes de siete centros asistenciales de Buenos Aires. Se seleccionaron por: 1) obtención de un halo de <26mm, por el método de discos frente a cefotaxima (CTX) o ceftacidima (CAZ) y 2) aumento del halo a >30mm al emplear discos de CTX o CAZ adicionados de clavulanato (30:10). Reunieron estas condiciones 44 Klebsiella spp y 2 E. coli. Estas últimas resultaron ser simultaneamente hiperproductoras de beta lactamasa cromosónica y de TEM-1. Todas las cepas de Klebsiella fueron productoras de BLEA en base a: perfil de resistencia por CIM, punto isoeléctrico (pI) de las beta lactamasas, acción de inhibidores de beta lactamasas y transferencia de la resistencia a C3G. Las cepas productoras de BLEA, se aislaron en el 14,6 por ciento de pacientes internados en Unidades de Cuidados Intensivos, 3,6 por ciento en otras áreas y 1,2 por ciento de pacientes externos. Un 56,5 por ciento provinieron de infecciones urinarias, 17,3 por ciento de bacteriemias, 15,2 por ciento de infecciones respiratorias y 11 por ciento de otras localizaciones. Los fracasos terapéuticos con C3G predominaron entre las infecciones extraurinarias. De acuerdo a la CIM todas las cepas fueron resistentes a las penicilinas, excepto a temocilina; a las cefalosporinas de primera y segunda generación, excepto a la cefoxitina. Para las C3G todas presentaron CIM entre 50 y 100 veces más elevadas que las habituales en Klebsiella spp. no productoras de BLEA. Cefoperazona y CAZ fueron las más perjudicadas mientras que ceftizoxima y cefpiroma fueron las más estables. Aztreonama fue más afectada que carumonama. Todas fueron sensibles a imipenem y moxalactama y también a ciprofloxacina, excepto una cepa. Todas las cepas presentaron bandas a pI correspondientes a derivados SHV y todas, salvo una, a pI:5,4 correspondiente a TEM-1...(AU)