A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual, SPR and NMR screening.

NDM-1 can hydrolyze nearly all available ß-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of 15N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.

Earliest identification of New Delhi metallo-ß-lactamase 1 (NDM-1) in Acinetobacter pittii in Brazil.

We report the occurrence in Brazil of the bla NDM-1 gene in Acinetobacter pittii, prior to the previously described first reports regarding the species Providencia rettgeri and Enterobacter hormaechei. Clinical isolates were investigated by polymerase chain reaction followed by bidirectional sequencing, and species was confirmed by 16S rDNA sequencing and matrix-assisted laser desorption-ionization time-of-flight spectrometry. A. pittii carrying bla NDM-1 was confirmed in a patient with no national or international travel history, or transfer from another hospital. The findings warn of the possibility of silent spread of bla NDM-1 to the community.

Earliest identification of New Delhi metallo-ß-lactamase 1 (NDM-1) in Acinetobacter pittii in Brazil

Abstract We report the occurrence in Brazil of the bla NDM-1 gene in Acinetobacter pittii, prior to the previously described first reports regarding the species Providencia rettgeri and Enterobacter hormaechei. Clinical isolates were investigated by polymerase chain reaction followed by bidirectional sequencing, and species was confirmed by 16S rDNA sequencing and matrix-assisted laser desorption-ionization time-of-flight spectrometry. A. pittii carrying bla NDM-1 was confirmed in a patient with no national or international travel history, or transfer from another hospital. The findings warn of the possibility of silent spread of bla NDM-1 to the community.

Antimicrobial susceptibility profiles of gram-negative bacteria causing infections collected across India during 2014-2016: Study for monitoring antimicrobial resistance trend report.

BACKGROUND: The emergence of antibiotic resistance among bacterial pathogens in the hospital and community has increased the concern to the health-care providers due to the limited treatment options. Surveillance of antimicrobial resistance (AMR) in frequently isolated bacterial pathogens causing severe infections is of great importance. The data generated will be useful for the clinicians to decide empiric therapy on the local epidemiological resistance profile of the antimicrobial agents. This study aims to monitor the distribution of bacterial pathogen and their susceptibility pattern to the commonly used antimicrobial agents. MATERIALS AND METHODS: This study includes Gram-negative bacilli collected from intra-abdominal, urinary tract and respiratory tract infections during 2014-2016. Isolates were collected from seven hospitals across India. All the study isolates were characterised up to species level, and minimum inhibitory concentration was determined for a wide range of antimicrobials included in the study panel. The test results were interpreted as per standard Clinical Laboratory Standards Institute guidelines. RESULTS: A total of 2731 isolates of gram-negative bacteria were tested during study period. The most frequently isolated pathogens were 44% of Escherichia coli (n = 1205) followed by 25% of Klebsiella pneumoniae (n = 676) and 11% of Pseudomonas aeruginosa (n = 308). Among the antimicrobials tested, carbapenems were the most active, followed by amikacin and piperacillin/tazobactam. The rate of extended-spectrum beta-lactamase (ESBL)-positive isolates were ranged from 66%-77% in E. coli to 61%-72% in K. pneumoniae, respectively. Overall, colistin retains its activity in > 90% of the isolates tested and appear promising. CONCLUSION: Increasing rates of ESBL producers have been noted, which is alarming. Further, carbapenem resistance was also gradually increasing, which needs much attention. Overall, this study data show that carbapenems, amikacin and colistin continue to be the best agents available to treat drug-resistant infections. Thus continuous monitoring of susceptibility profile of the clinically important Gram-negative pathogens is of great importance to guide effective antimicrobial therapy.

Escherichia coli y Klebsiella pneumoniae productores de betalactamasa de espectro extendido en infecciones de vías urinarias: evolución de la resistencia antibiótica y opciones terapéuticas / Extended-spectrum beta-lactamase-producing Escherichia coliand Klebsiella pneumoniae from urinary tract infections: Evolution of antimicrobial resistance and treatment options

Antecedentes y objetivos: Se analiza la presencia, a lo largo de los años en infecciones urinarias, de aislados de Escherichia coli y Klebsiella spp. productores de betalactamasa de espectro extendido (BLEE), y su sensibilidad a antibióticos. Material y método: Se realizó un estudio retrospectivo entre 2012 y 2016. La sensibilidad a ciprofloxacino, tobramicina, cefoxitina, fosfomicina, nitrofurantoína, cotrimoxazol y carbapenémicos se analizó mediante el sistema MicroScan®. Resultados: Se procesaron 95.399 muestras y se aislaron 9.772 E. coli, 1.784 Klebsiella pneumoniae y 248 Klebsiella oxytoca. Las cepas con BLEE fueron más frecuentes en mujeres, aunque disminuyó durante 2015 y 2016 (65,7-67,2%). La prevalencia de K. pneumoniae BLEE aumentó todos los años (28,1% en el 2016). La prevalencia media de E. coli BLEE fue del 10,5%, con escasas oscilaciones. Las mayores resistencias ocurrieron a ciprofloxacino y cotrimoxazol, 89,5 y 94,7% en 2015, respectivamente, y las menores a imipenem. Fosfomicina y nitrofurantoína fueron muy activos sobre E. coli BLEE. Conclusiones: E. coli y K. pneumoniae con BLEE fueron prevalentes y, sobre todo esta última, con una importante resistencia a ciprofloxacino y cotrimoxazol. La sensibilidad a imipenem fue elevada

Background and objectives: A study of the susceptibility to antimicrobials of the extended spectrum beta-lactamase phenotypes (ESBL) in Escherichia coli and Klebsiella spp. was performed to discover the evolution of this type of resistance from urinary tract infections. Material and method: A retrospective study was carried out between 2012 and 2016. Susceptibility to ciprofloxacin, tobramycin, cefoxitin, fosfomycin, nitrofurantoin, co-trimoxazole, and carbapenems was analyzed using MicroScan® system. Results: A total of 95,399 samples were processed and 9,772 E. coli, 1,784 Klebsiella pneumoniae and 248 Klebsiella oxytoca were isolated. ESBL strains were more frequent in women, although they decreased during 2015 and 2016 (65.7-67.2%). The prevalence of K. pneumoniae ESBL increased annually (28.1% in 2016). The average prevalence of E. coli ESBL was 10.5% with few oscillations. Higher resistance occurred to ciprofloxacin and cotrimoxazole, 89.5 and 94.7% in 2015, respectively, and there was lesser resistance to imipenem. Fosfomycin and nitrofurantoin were very active on E. coli ESBL. Conclusions: ESBL producing E. coli and K. pneumoniae were prevalent, especially the latter, with a significant resistance to ciprofloxacin and cotrimoxazole. Susceptibility to imipenem was high

Saudi plants as a source of potential ß-lactamase inhibitors.

This study was performed to assess the potential ß-lactamase inhibitory properties of nineteen crude Saudi plant extracts belonging to eight families against extended spectrum ß-lactamase (ESßL) strains of Klebsiella pneumoniae and other medically important pathogens. A total of 276 microbial isolates of pathogenic bacteria were used in this study; only 15 of them showed decreased sensitivity to one or several of ceftazidime, aztreonam, cefotaxime or ceftriaxone, which are deemed to be possible producers of ESßL. Antibacterial activities of plant extracts were carried out against ESßL positive isolates by the disc diffusion method. The potential ESßL suppressing activities of plant extracts and prepared fractions, (chloroform and methanol), with or without antibiotic were studied by disc diffusion method. Results revealed that selected plant extracts showed no antibacterial activity against tested strains; meanwhile, only Echinops viscosus, Pulicaria arabica, Tephrosia nubica, Chrozophora oblongifolia, and Clutia myricoides showed pronounced ESßL inhibitory activities. The extracts were quantified for phenolic compounds and their antioxidant properties. Bio-guided fractionation of the active extracts revealed that the chloroform fraction of C. myricoides possess a promising ESßL inhibitory activity. The separation and the structural elucidation of the active compounds from C. myricoides will offer beneficial leads for developing ß-lactamase inhibitors.

New Delhi metallo-ß-lactamase - type carbapenemases producing Escherichia coli isolates from hospitalized patients: A pilot study.

BACKGROUND & OBJECTIVES: Resistances to carbapenem group of antimicrobials among Escherichia coli due to production of carbapenemases, especially the New Delhi metallo-ß-lactamase (NDM) types, pose serious challenges in the treatment of infections in healthcare settings. This study was undertaken to detect NDM producing E. coli isolates from hospitalized patients with urinary tract infection (UTI). METHODS: A total of 30 non-repetitive isolates of E. coli from hospitalized patients with clinical suspicion of UTI were subjected to antimicrobial susceptibility testing. Screening for the production of extended-spectrum ß-lactamases (ESBL) was carried out by minimum inhibitory concentration (MIC) test strip ESBL followed by phenotypic confirmation by double-disc synergy test. Phenotypic confirmation of carbapenemase production was carried out by MIC test strip metallo-ß-lactamases. Molecular identification of the blaNDM gene was carried out by polymerase chain reaction (PCR) and sequencing of the amplified fragment. RESULTS: Seventeen of the 30 isolates were detected as ESBL producers, of which three were found to be carbapenemase producers. NDM genes were detected by PCR followed by gene sequencing in all three isolates positive for ESBL as well as carbapenemase. The amino acid sequence of the three isolates showed complete identity to the reference sequences of NDM-1, NDM-4 and NDM-8, respectively. INTERPRETATION & CONCLUSIONS: Our study showed the circulation of NDM variants among the clinical isolates of E. coli that were producers of ESBL as well as carbapenemase.

Aumento significativo de la resistencia a fosfomicina en cepas de Escherichia coli productoras de Beta-lactamasas de espectro extendido (BLEE) aisladas de urocultivos (2005-2009-2011) / Increasing prevalence of fosfomycin resistance in extended-spectrum-beta-lactamaseproducing Escherichia coli urinary isolates (2005-2009-2011)

Introducción. Escherichia coli es el principal uropatógeno. La aparición de cepas productoras de Beta-lactamasas de espectro extendido (BLEE), que con frecuencia presentan multirresistencia, deja pocas opciones terapéuticas, y es necesario realizar un seguimiento de su sensibilidad a lo largo del tiempo. En el presente trabajo se presentan los porcentajes de aislados urinarios de E.coli productores de BLEE durante 2005, 2009 y 2011 y se comparan los resultados de la determinación de su sensibilidad a antibióticos de diferentes grupos, fosfomicina entre ellos. Métodos. Se analizaron 5.053, 6.324 y 6.644 aislados urinarios de E. coli en 2005, 2009 y 2011 respectivamente. Se excluyeron duplicados. La sensibilidad se determinó por microdilución con el sistema Wider (Soria Melguizo S.A.) y se seleccionó el fenotipo que indicaba producción de BLEE (CLSI 2009). Resultados. El 3,9% de las cepas (198) resultó productor de BLEE en 2005, el 7,3% (463) en 2009 y el 8,7% (584) en 2011. Se detectó resistencia a carbapenemicos en 2009, aunque continúan con un 95% de sensibilidad. Entre los no-Betalactámicos, colistina fue el más activo, seguido de nitrofurantoina. Ciprofloxacino y sulfametoxazol-trimetoprim presentaron un 80% y 60% de resistencia, respectivamente. Se observó una tendencia al aumento de la resistencia en fosfomicina, desde 0% a 9,3 llegando al 14,4% en 2011. Conclusiones. Se observó una creciente prevalencia de cepas de E. coli productoras de BLEE aisladas de urocultivos, alcanzando el 8,7% en 2011. Los carbapenemicos siguen siendo los antibióticos más activos frente a este tipo de cepas. El aumento de resistencia a fosfomicina fue significativo(AU)

Introduction. Escherichia coli is the most important uropathogen. The appearance of extended- spectrum beta-lactamase (ESBL)-producing E.coli in urinary tract infections (UTI) constitutes an important therapeutic challenge that requires the study of its evolution throughout time in order to establish a suitable empirical treatment. Our aim was to determine the prevalence of ESBL-producing E. coli urinary isolates in 2005, 2009 and 2011. We also determined the antimicrobial coresistance to several agents, including fosfomycin. Methods. We analyzed 5053, 6324 and 6644 E. coli isolates obtained from urine cultures in 2005, 2009 and 2011 respectively. Duplicate isolates were excluded. Antimicrobial susceptibility was determined by the Wider microdilution system (Soria Melguizo S.A.) and the phenotypic pattern of resistance that indicated a BLEE-producing E.coli was selected (CLSI 2009). Results. 3.9% of strains (198) were ESBL producers in 2005, 7.3% (463) in 2009 and 8.7% (584) in 2011. Resistance to carbapenems was detected in 2009, they inhibited more than the 95% of strains in 2011. Among the non-beta-lactams, colistin was the most active antibiotic followed by nitrofurantoin. Ciprofloxacin and sulfamethoxazole-trimethoprim were not effective with 80% and 60% resistant isolates, respectively. An increasing resistance trend, from 0% to 9.3% in 2009 and 14.4% in 2011 was observed for fosfomycin. Conclusions. From 2005 our institution had an increasing prevalence of ESBL-producing E. coli rising to 8.7% in 2011. Carbapenems are still the most active agents. The increase of resistance was significant for fosfomycin(AU)

Characterization of purified New Delhi metallo-ß-lactamase-1.

New Delhi metallo-ß-lactmase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to almost all clinically used ß-lactam antibiotics, its presence within an easily transmissible plasmid bearing a number of other antibiotic resistance determinants, its carriage in a variety of enterobacteria, and its presence in both nosocomial and community-acquired infections. To improve our understanding of the molecular basis of this threat, NDM-1 was purified and characterized. Recombinant NDM-1 bearing its native leader sequence was expressed in Escherichia coli BL21 cells. The major processed form found to be released into culture media contains a 35-residue truncation at the N-terminus. This form of NDM-1 is monomeric and can be purified with 1.8 or 1.0 equiv of zinc ion, depending on the experimental conditions. Treatment of dizinc NDM-1 with EDTA results in complete removal of both zinc ions, but the relatively weaker chelator PAR chelates only 1 equiv of zinc ion from folded protein but 1.9 equiv of zinc ion from denatured protein, indicating different affinities for each metal binding site. UV-vis spectroscopy of the dicobalt metalloform along with molecular dynamics simulations of the dizinc metallo form indicates that the dinuclear metal cluster at the active site of NDM-1 is similar in structure to other class B1 metallo-ß-lactamases. Supplementation of excess zinc ions to monozinc NDM-1 has differential effects on enzyme activity with respect to three different classes of ß-lactam substrates tested, penems, cephems, and carbapenems, and likely reflects dissimilar contributions of the second equivalent of metal ion to the catalysis of the hydrolysis of these substrates. Fits to these concentration dependencies are used to approximate the K(d) value of the more weakly bound zinc ion (2 µM). NDM-1 achieved maximal activity with all substrates tested when supplemented with approximately 10 µM ZnSO(4), displaying k(cat)/K(M) values ranging from 1.4 × 10(6) to 2.0 × 10(7) M(-1) s(-1), and a slight preference for cephem substrates. This work provides a foundation for an improved understanding of the molecular basis of NDM-1-mediated antibiotic resistance and should allow more quantitative studies to develop targeted therapeutics.

Determinación de beta lactamasas de espectro expandido en el laboratorio: análisis comparativo de tres métodos / Determination of expanded spectrum beta lactamases in the laboratory: comparative analysis of three methods

58 cepas (16 escherichia coli y 42 klebsiella pneumoniae) aisladas de muestras clínicas entre septiembre 1999 y julio 2000 en las secciones de bacteriología del Laboratorio Metropolitano Hospital José Gregorio Hernández y Hospital de Clínicas Caracas, con patrón sugestivo de ser productoras de ß-lactamasa de espectro expandido (BLEE) sensibles a cefoxitin y resistentes a ceftazidime, cefotaxime, ceftriaxone y aztreonam) fueron evaluadas de forma prospectiva por los métodos del doble disco (DD), Etest (AB Biodisk) y ATB BLSE (bioMérieux) para la detección de BLEE, tomando como técnica de referencia la de DD, obteniéndose en general un 100 por ciento de correlación con la prueba de Etest y un 86,2 por ciento con la de ATB BLSE, estadísticamente no significativa (p>0,05). Se recomienda el uso de ambas técnicas para la confirmación de cepas productoras de BLEE en conjunto con el despistaje inicial realizado a través del análisis de los patrones de resistencia obtenido por las metodologías usadas rutinariamente en los laboratorios para la realización del antibiograma.

Failure of therapy in pseudomonas endocarditis: selection of resistant mutants.

Despite optimal use of available antibacterial agents, endocarditis due to Pseudomonas aeruginosa is commonly associated with poor response to medical treatment. Two patients are described in whom emergence of resistance to beta-lactam antibiotics was associated with clinical failure. A subpopulation of resistant mutants (10(-7)) was found within the initial, apparently sensitive population of bacteria. These resistant mutants were similar to posttherapy isolates in their increased production of beta-lactamase and in their identical pattern of resistance to beta-lactam antibiotics. Moreover, the only beta-lactamase produced was type Id, and this enhanced production proved to be constitutive. A relatively large inoculum (10(6) colony-forming units/g of tissue) of bacteria was found postoperatively in the heart valves of both patients. The failure to respond is postulated to be due to the selection of these producers of high levels of beta-lactamase in a large bacterial inoculum.