Distribución y caracterización molecular de betalactamasas en bacterias Gram negativas en Colombia, 2001-2016 / Distribution and molecular characterization of beta-lactamases in Gram-negative bacteria in Colombia, 2001-2016

Resumen Las betalactamasas, enzimas con capacidad hidrolítica frente a los antibióticos betalactámicos, son responsables del principal mecanismo de resistencia en bacterias Gram negativas; las de mayor impacto clínico y epidemiológico en los hospitales, son las betalactamasas de espectro extendido (BLEE), las de tipo AmpC y las carbapenemasas. El incremento en su frecuencia y su diseminación a nivel mundial ha limitado cada vez más las opciones terapéuticas tanto en infecciones adquiridas en los hospitales como las que se generan en la comunidad. En Colombia, las redes de vigilancia y los grupos de investigación iniciaron su estudio desde finales de los años 90 y, así, se logró la caracterización molecular de las diferentes variantes; además, se reportó una gran prevalencia y diseminación en los hospitales de mediana y alta complejidad, y se describió el impacto clínico de las infecciones que causan. Dichos estudios han evidenciado el alto grado de endemia de algunas de estas betalactamasas y, en consecuencia, la necesidad de una inmediata implementación de programas para inducir el uso prudente de los antibióticos y de medidas de vigilancia, que permitan controlar y prevenir su diseminación, con el fin de disminuir la morbimortalidad en los pacientes y preservar las opciones terapéuticas disponibles en la actualidad. En esta revisión, se recopiló la información sobre las variantes, la distribución geográfica y la caracterización molecular de las betalactamasas en Colombia, así como los estudios llevados a cabo desde finales de la década de 90 hasta el 2016, lo cual permitió tener un panorama de las betalactamasas que circulan en diferentes regiones, su incremento en el tiempo y sus implicaciones clínicas.

Abstract Beta-lactamases are enzymes with hydrolytic activity over beta-lactam antibiotics and they are the main resistance mechanism in Gram-negative bacteria. Extended-spectrum beta-lactamases (ESBL), AmpC, and carbapenemases have the greatest clinical and epidemiological impact in hospital settings. The increasing frequency and worldwide spread of these enzymes have limited the therapeutic options in hospital-acquired infections and those originating in the community. In Colombia, surveillance networks and research groups began studying them in the late 90s. Different variants of these enzymes have been molecularly characterized and their high prevalence and dissemination in medium and high complexity hospitals, along with a high clinical impact, have been reported. Furthermore, many studies in Colombia have evidenced high endemicity for some of these beta-lactamases, which requires an urgent implementation of antimicrobial stewardship programs in order to preserve the few therapeutic options and infection control strategies to prevent and limit their dissemination. In this publication, we carried out a review of the different enzyme variants, geographic distribution, and molecular characterization of these beta-lactamases in Colombia. Additionally, we describe the available information in the literature regarding studies conducted between the late 1990s and 2016, which provide an overview of the beta-lactamases circulating in different regions of Colombia, their increase over time, and their clinical implications.

Genomic analysis of bifunctional Class C-Class D ß-lactamases in environmental bacteria

β-lactamases, which are found in several bacterial species and environments, are the main cause of resistance to β-lactams in Gram-negative bacteria. In 2009, a protein (LRA-13) with two β-lactamase domains (one class C domain and one class D domain) was experimentally characterised, and an extended action spectrum against β-lactams consistent with two functional domains was found. Here, we present the results of searches in the non-redundant NCBI protein database that revealed the existence of a group of homologous bifunctional β-lactamases in the genomes of environmental bacteria. These findings suggest that bifunctional β-lactamases are widespread in nature; these findings also raise concern that bifunctional β-lactamases may be transferred to bacteria of clinical importance through lateral gene transfer mechanisms.

Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples

Abstract The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false–positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

Phenol degradation and genotypic analysis of dioxygenase genes in bacteria isolated from sediments

Abstract The aerobic degradation of aromatic compounds by bacteria is performed by dioxygenases. To show some characteristic patterns of the dioxygenase genotype and its degradation specificities, twenty-nine gram-negative bacterial cultures were obtained from sediment contaminated with phenolic compounds in Wuhan, China. The isolates were phylogenetically diverse and belonged to 10 genera. All 29 gram-negative bacteria were able to utilize phenol, m-dihydroxybenzene and 2-hydroxybenzoic acid as the sole carbon sources, and members of the three primary genera Pseudomonas, Acinetobacter and Alcaligenes were able to grow in the presence of multiple monoaromatic compounds. PCR and DNA sequence analysis were used to detect dioxygenase genes coding for catechol 1,2-dioxygenase, catechol 2,3-dioxygenase and protocatechuate 3,4-dioxygenase. The results showed that there are 4 genotypes; most strains are either PNP (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is positive) or PNN (catechol 1,2-dioxygenase gene is positive, catechol 2,3-dioxygenase gene is negative, protocatechuate 3,4-dioxygenase gene is negative). The strains with two dioxygenase genes can usually grow on many more aromatic compounds than strains with one dioxygenase gene. Degradation experiments using a mixed culture representing four bacterial genotypes resulted in the rapid degradation of phenol. Determinations of substrate utilization and phenol degradation revealed their affiliations through dioxygenase genotype data.

Prevalence and antimicrobial susceptibility of non-fermenting Gram-negative bacilli isolated from clinical samples at a tertiary care hospital

Abstract INTRODUCTION: We compared the prevalence and antimicrobial susceptibility of non-fermenting gram-negative bacilli (NFGNB) isolated from clinical samples at a Brazilian tertiary care hospital in 2008 and 2013. METHODS: Collected data included patient's name, age, sex, inpatient unit, laboratory record number, type of biological material, culture test result, and antimicrobial susceptibility of isolated strains. RESULTS: Out of 19,112 culture tests analyzed, 926 (4.8%) were positive for NFGNB. Among these, 45.2% were metallo-beta-lactamase (MBL) producing strains. CONCLUSION: Between 2008 and 2013, the number of MBL-producing NFGNB isolates increased by 21.5%, which was accompanied by a consequent reduction in susceptibility to antimicrobials.

NDM-1 (New Delhi metallo beta lactamase-1) producing Gram-negative bacilli: Emergence & clinical implications.

Backgound & objectives: resistance to carbapenems in Gram-negative bacteria conferred by NDM-1 is a global health problem. We investigated the occurrence of NDM-1 in clinical isolates of gram-negative bacilli in a tertiary care hospital in Kashmir valley, India. Methods: Gram-negative bacilli from different clinical isolates were included in the study. Antimicrobial susceptibility was performed by Kirby Bauer disk diffusion method and interpreted using Clinical Laboratory Standards Institute (CLSI) guidelines. Isolates resistant to carbapenems were subjected to different phenotypic test such as modified hodge test (MHT), boronic acid and oxacillin based MHT (bA-MHT and OXA-MHT), combined disk test and minimum inhibitory concentration (MIC) with imipenem and imipenem -EDTA for determination of class B metallo enzymes. Presence of blaNDM-1 gene was established by PCR and confirmed by sequencing. Results: Of the total 1625 gram-negative isolates received, 100 were resistant to imipenem. Of the 100 isolates, 55 (55%) were positive by modified Hodge test indicating carbapenemase production. Of the 100 isolates tested by MHT, BA-MHT and OXA-MHT, 29 (29%) isolates belonged to Class A and 15 (15%) to Class B, while 56 (56%) isolates were negative. Of the 15 class B metallo beta lactamase producers, nine carried the blaNDM-1 gene. NDM-1 was found among escherichia coli (2 isolates), Klebsiella pneumoniae (2 isolates), Citrobacter freundii (3 isolates), Acinetobacter spp (1 isolate), and one isolate of Pseudomonas aeruginosa. Isolates were resistant to all antibiotic tested except polymyxin B and tigecycline. Interpretation & conclusions: Our study showed the presence of clinical isolates expressing NDM-1 in Srinagar, Jammu & Kashmir, India. These isolates harbour plasmid mediated multiple drug resistant determinants and can disseminate easily across several unrelated genera. To halt their spread, early identification of these isolates is mandatory.

Detection of multi drug resistant bacteria in major hospitals in Kano, North-West, Nigeria

Two major hospitals in Kano, North West Nigeria have recorded increasing resistance of clinical pathogens to broad spectrum β lactams, mediated by extended spectrum β- lactamase (ESβL) and non ESBLs. A study was therefore undertaken to determine the occurrence and prevalence of plasmid and chromosomal mediated AmpC βL and carbapenemase in addition to already known ESBL due to increasing resistance of pathogens from the two hospitals to carbapenems, cephamycins and flouroquinolones. Antibiogram tests and ESBL, AmpC and carbapenemase production tests were performed on all the isolates. AmpC and carbapenemase producers were further screened for AmpC inducibility and metallo beta lactamase production respectively. Majority of the isolates (> 80%) were resistant to both β-lactam and non β-lactam antibiotics. Reduced susceptibility to levofloxacin, nitrofurantoin, nalidixic acid and ofloxacin among the isolates were observed with the exception of P. aeruginosa which is totally resistant to imipenem and levofloxacin. An overall prevalence of 14.4%, 11.9% and 11.9.3% for ESβL, AmpC and carbapenemase was observed respectively. About 7.9% of the AmpC producers can over expressed the chromosomally mediated AmpC and 85.8% of the carbapenemase producers require metal for their action. Co-production of either of two and/or all of the enzymes was observed in E. coli, P. mirabilis and P. aeruginosa. Antibiotic resistance among isolates from the two hospitals is increasing and the major cause of this resistance in the pathogens studied are production of AmpC, carbapenemase (especially Metallo β- lactamase) in addition to already known ESBL enzymes by the pathogens. Some of the isolates also possess the capacity to elaborate two or more of the enzymes concurrently, which would renders them resistant to a multitude of antibiotics.

Prevalencia de bacterias Gram negativas portadoras del gen bla KPC en hospitales de Colombia / Prevalence of Gram-negative bacteria harboring bla KPC gene in Colombian hospitals

Introducción. Las enzimas carbapenemasas de tipo KPC tienen gran capacidad de diseminación, son causantes de epidemias y se asocian a mayor mortalidad y estancia hospitalaria. En Colombia se han venido reportando cada vez más desde 2007, pero se desconoce la prevalencia hospitalaria. Objetivo. Estimar la prevalencia hospitalaria del gen bla KPC . Materiales y métodos. Se evaluó la presencia del gen bla KPC y su ´clonalidad´ en aislamientos de enterobacterias y Pseudomonas aeruginosa de pacientes hospitalizados. Resultados. De los 424 aislamientos evaluados durante el periodo de estudio, 273 cumplieron con criterios de elegibilidad, 31,1 % fue positivo para el gen bla KPC y, al ajustar por ´clonalidad´, la positividad fue de 12,8 %. El gen bla KPC se encontró con mayor frecuencia en Klebsiella pneumoniae seguido de P. aeruginosa y otras enterobacterias. A pesar de que la unidad de cuidados intensivos aportó el mayor número de aislamientos, no se encontró un patrón más prevalente del gen bla KPC en las ellas que en las otras salas. El aparato respiratorio fue el sitio anatómico de origen con la mayor prevalencia . No se presentó estacionalidad en la frecuencia de los aislamientos portadores del gen bla KPC. Conclusión. Este estudio reveló la alta prevalencia del gen bla KPC en diferentes microorganismos aislados en varias instituciones hospitalarias del país. La extraordinaria capacidad de propagación del gen bla KPC , las dificultades del diagnóstico y la limitada disponibilidad de antibióticos plantean la apremiante necesidad de fortalecer los sistemas de vigilancia epidemiológica y ajustar oportunamente las políticas institucionales de uso racional de antibióticos con el fin de contener su diseminación a otras instituciones de salud del país.

Introduction: KPC enzymes are carbapenemases with a great capability to disseminate and to cause epidemics. They are frequently associated with higher mortality rates and prolonged hospital stay. In Colombia, they have been progressively reported since 2007; however, its prevalence in hospitals is not known. Objective: To estimate the prevalence of bla KPC gene in hospitals. Methods and materials: The presence of bla KPC gene and its clonality were evaluated in clinical isolates of Enterobacteriacea and Pseudomonas aeruginosa in hospitalized patients. Results: Of the 424 isolates tested during the study period, 273 met eligibility criteria, and 31.1% were positive for bla KPC gene; after clonality adjustment, positivity was 12.8%. The bla KPC gene was more frequent in Klebsiella pneumonia, followed by P. aeruginosa and other Enterobacteriacea . Although intensive care units (ICU) provided the majority of the isolates, the bla KPC pattern was not more prevalent in ICUs than in other wards. The respiratory tract was the anatomic source with the highest prevalence. No seasonality was observed associated with the frequency of isolation of microorganisms carrying bla KPC gene. Conclusion: This study revealed a high prevalence of bla KPC gene in microorganisms isolated from different hospitals in Colombia. The extraordinary ability of bla KPC gene to spread, the difficulties for its diagnosis and the limited antibiotics available for its treatment pose the urgent need to strengthen epidemiological surveillance systems, and to timely adjust institutional policies for rational use of antibiotics in order to limit its dissemination to other institutions in the country.

Evolución de la resistencia antimicrobiana de bacilos Gram negativos en unidades de cuidados intensivos en Colombia / Evolution of antimicrobial resistance in Gram negative bacilli from intensive care units in Colombia

Introducción. La evolución de la resistencia bacteriana constituye una amenaza para la salud pública mundial. Los sistemas de vigilancia epidemiológica han integrado técnicas de biología molecular para mejorar las estrategias de control. Objetivo. Describir los perfiles moleculares y fenotípicos de los bacilos Gram negativos en unidades de cuidados intensivos de 23 hospitales de Colombia entre 2009 y 2012. Materiales y métodos. Se diseñó un estudio descriptivo en 23 hospitales del Grupo para el Estudio de la Resistencia Nosocomial (sic.) en Colombia. Se analizaron 38.048 aislamientos usando WHONET durante el periodo descrito. Se describieron perfiles de resistencia para Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa y Acinetobacter baumannii. En 1.248 cepas se realizó reacción en cadena de la polimerasa (PCR) para detectar las carbapenemasas clínicamente más relevantes. Resultados. Escherichia coli fue el microorganismo más frecuente (promedio=14,8 %); la frecuencia de aislamientos de K. pneumoniae aumentó de 11 % en 2009 a 15 % en 2012 (p<0,001). La tendencia de los perfiles de multirresistencia aumentó en todas las especies estudiadas. De los aislamientos de K. pneumoniae evaluados, 68,4 % fue positivo para KPC ( Klebsiella pneumoniae Carbapenemase ), mientras que la VIM ( Verona Integron-encoded Metallo-betalactamase ) en P. aeruginosa se observó en 46,5 %. Conclusiones. Se observó un incremento en la tendencia de los microorganismos hacia la multirresistencia y una amplia distribución de las carbapenemasas. La articulación de la biología molecular con los sistemas de vigilancia permitió integrar el análisis del fenotipo con los mecanismos de resistencia involucrados en las bacterias estudiadas. Este análisis permitirá la elaboración de guías para el uso adecuado de antimicrobianos y contribuirá a la contención de estas bacterias multirresistentes en Colombia.

Introduction: The continuous evolution of antimicrobial resistance poses a major threat to public health worldwide. Molecular biology techniques have been integrated to epidemiological surveillance systems to improve the control strategies of this phenomenon. Objective: To describe the phenotypic and molecular profiles of the most important Gram negative bacilli from intensive care units in 23 Colombian hospitals during the study period 2009-2012. Materials and methods: A descriptive study was conducted in 23 hospitals belonging to the Colombian Nosocomial Resistance Study Group. A total of 38.048 bacterial isolates were analyzed using WHONET over a four-year period. The antimicrobial resistant profiles were described for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii . Polymerase chain reaction was performed in 1.248 strains to detect the most clinically relevant carbapenemases. Results: Escherichia coli was the most frequently isolated organism (mean=14.8%). Frequency of K. pneumoniae increased significantly from 11% in 2009 to 15% in 2012 (p<0.001). All screened isolates had rising trends of multidrug-resistant profiles. KPC ( Klebsiella pneumoniae carbapenemase) was detected in 68.4% of K. pneumoniae isolates while VIM (Verona integron-encoded metallo-betalactamase) was present in 46.5% of them. Conclusion: In this study, an increase in the trend of multidrug-resistant organisms and a wide distribution of carbapenemases was observed. The integration of molecular biology to surveillance systems allowed the compilation of this data, which will aid in the construction of guidelines on antimicrobial stewardship for prevention in Colombia.

La evolución de la resistencia bacteriana en México, 1973-2013 / Evolution of bacterial resistance to antibiotics in México, 1973-2013

Introducción. La resistencia bacteriana a los antibióticos es un problema de salud mundial. Las investigaciones relacionadas con este problema emergente son indispensables para reconocer y desarrollar programas para su vigilancia y control. Objetivo. Revisar y comentar las contribuciones de los investigadores mexicanos en el área de la resistencia bacteriana a los antibióticos. Materiales y métodos. Se realizó una búsqueda de la literatura científica relacionada con la resistencia bacteriana a los antibióticos producida por investigadores mexicanos y registrada en Medline-PubMed entre 1973 y julio de 2013. Resultados. En 66 publicaciones, las contribuciones de investigadores mexicanos incluyeron datos sobre la resistencia de agentes patógenos entéricos como Salmonella Typhi, múltiples contribuciones sobre la producción de betalactamasas de espectro extendido, de metalobetalactamasas y de carbapenemasas, los mecanismos de resistencia en Pseudomonas aeruginosa y la evolución de la resistencia en cocos Gram positivos como Streptococcus pneumoniae , Staphylococcus aureus y Enterococcus spp., entre otros. Conclusiones. Los datos publicados en los últimos 40 años son fuente adecuada para entender la evolución de la resistencia bacteriana a los antibióticos y desarrollar programas para su control.

Introduction: Bacterial resistance to antibiotics is a worldwide public health concern. Research priorities for the study and control of this emerging problem include country-wide surveillance. Objective: To review and comment on the contributions by Mexican investigators towards a greater understanding of the mechanisms of bacterial antibiotic resistance. Materials and methods: A comprehensive search of the medical literature on Medline/PubMed between 1973 and July 2013 was performed. Results: The contributions of Mexican investigators have included descriptions of resistance in enteric pathogens, such as Salmonella Typhi, publications on the production of extended spectrum beta-lactamases, metallo-beta-lactamases, and carbapenemases, resistance mechanisms of Pseudomonas aeruginosa , and the evolution of resistance in Gram-positive pathogens, including Streptococcus pneumoniae , Staphylococcus aureus , and Enterococcus spp. Conclusion: The Mexican literature on mechanisms of bacterial resistance is relevant for the development of plans to control the antibiotic resistance crisis.

Antibacterial activity of the Antarctic bacterium Janthinobacterium sp: SMN 33. 6 against multi-resistant Gram-negative bacteria

Background The increment of resistant strains to commonly used antibiotics in clinical practices places in evidence the urgent need to search for new compounds with antibacterial activity. The adaptations that Antarctic microorganisms have developed, due to the extreme environment that they inhabit, promote them as a potential new source of active compounds for the control of microorganisms causing infections associated with health care. The aim of this study was to evaluate the antibacterial activity of an ethanol extract of the Antarctic bacterium Janthinobacterium sp., strain SMN 33.6, against nosocomial multi-resistant Gram-negative bacteria. Results Inhibitory activity against human Gram-negative bacterial pathogens, with concentrations that varied between 0.5 and 16 µg ml- 1, was demonstrated. Conclusions The ethanolic extract of Janthinobacterium sp. SMN 33.6 possesses antibacterial activity against a chromosomal AmpC beta-lactamase-producing strain of Serratia marcescens, an extended-spectrum beta-lactamase-producing Escherichia coli and also against carbapenemase-producing strains of Acinetobacter baumannii and Pseudomonas aeruginosa. This becomes a potential and interesting biotechnological tool for the control of bacteria with multi-resistance to commonly used antibiotics.

Actividad de cobre sobre bacilos gramnegativos multi-resistentes aislados en hospitales chilenos / Copper activity against multiresistant Gram negative bacilli isolated from Chilean hospitals

Introduction: Multiresistant nosocomial pathogens, especially Gram-negative bacilli (GNB), are a serious problem for public health systems worldwide. Due to their antimicrobial properties, copper alloys have been suggested as an alternative for the control of bacterial burden in surfaces in hospital environment. However, antibiotic multiresistance and copper resistance could be associated in GNB, and there is evidence that both kind of resistance genes (antibiotic and copper) can be located on the same genetic structures. For this reason antibiotic-multiresistant strains could survive in the presence of copper, selecting for bacterial phenotypes resistant to both antibacterial agents. Aim: To evaluate antibacterial activity of copper against nosocomial extended-spectrum β-lactamases (ESBL) (+) and ESBL (-) GNB, and carbapenems resistant or susceptible strains. Material and Method: This study included 390 strains of GNB isolated from Chilean hospitals: Acinetobacter baumannii and Pseudomonas aeruginosa resistant (CAR R) and susceptible (CAR S) to carbapenem antibiotics, and Klebsiella pneumoniae and Escherichia coli producers and non-producers of ESBL. Susceptibility levels to cupric sulphate were determined by agar dilution method and statistical analysis were used to determine the significance of the differences in the copper tolerance levels between the strains groups. Results: Statistically superior copper tolerance levels were found in the CAR R and ESBL producing strains of A. baumannii and K. pneumoniae, in relation with the CAR S and ESBL not-producing strains. Conclusion: A relation between a diminished susceptibility to ionic copper and to recent generation antimicrobial agents was observed in K. pneumoniae y A. baumannii strains.

Introducción: Los patógenos intrahospitalarios multi-resistentes constituyen un grave problema mundial de salud pública, especialmente los bacilos gramnegativos (BGN). El uso de cobre como antimicrobiano de superficie en hospitales se postula como una alternativa para el control de microorganismos en estos ambientes. Sin embargo, la multi-resistencia a antimicrobianos en BGN hospitalarios puede asociarse con la tolerancia a cobre, ya que existe evidencia que genes que codifican tolerancia a este metal pueden encontrarse en elementos genéticos que confieren resistencia a antimicrobianos. Por esta razón, cepas multi-resistentes a antimicrobianos podrían sobrevivir en presencia de cobre, seleccionando bacterias resistentes a ambos agentes antibacterianos. Objetivo: Investigar la actividad de cobre sobre BGN hospitalarios productores y no productores de β-lactamasas de espectro extendido (BLEE), y resistentes o susceptibles a antimicrobianos carbapenémicos. Material y Métodos: Se estudió 390 cepas de BGN aisladas en hospitales chilenos: Acinetobacter baumannii y Pseudomonas aeruginosa resistentes (CAR R) y susceptibles (CAR S) a carbapenémicos y Klebsiella pneumoniae y Escherichia coli productoras y no productoras de BLEE. Se investigó los niveles de susceptibilidad a sulfato cúprico, mediante dilución seriada en agar y se evaluó la significancia estadística de la diferencia de estos niveles entre los distintos grupos de cepas. Resultados: Se encontraron niveles de tolerancia a cobre superiores en cepas de A. baumannii y K. pneumoniae, CAR R y productoras de BLEE respectivamente, con respecto a sus pares CAR S y no productoras de BLEE. Conclusión: Observamos una relación entre la disminución de la susceptibilidad a cobre iónico y a antimicrobianos de última generación en K. pneumoniae y A. baumannii.

Extended Spectrum beta-lactamases in pyogenic gram-negative bacilli

To determine the frequency, distribution and sensitivity pattern of extended spectrum beta-lactamases producing Gram-negative bacilli from pus samples. Cross Sectional Comparative Study Study, place and duration: This study was conducted at the Microbiology Department, Fauji Foundation Hospital, Rawalpindi over a period of two years from 04/ 2004 to 03 / 2006 Inclusion criteria: All pus samples were included. Exclusion criteria: Dried pus swabs were not included in the study. Multidrug resistance and ESBLs-production were studied in a total of 138 Enteric Gram-negative Rods [GNR] obtained from pus specimens by the help of double disc diffusion technique. Susceptibility testing was done to various groups of antimicrobials. Frequency of ESBLs-production was 56.5%. ESBLs-positivity was highest at 61-70 years of age group [24.3%], followed by 11-20 yrs [17.5%]. ESBLs-positivity in females versus males was 58.2%: 54.2% and highest in indoor patients [92.3%]. ESBLs-production was highest amongst Escherichia coli [55.1%] followed by Klebsiella pneumoniae, [29.5%], Pseudomonas aeruginosa [9.0%], Aeromonas spp [2.6%]. The antibiotics showing best activity against ESBLs-producing K pneumoniae and E coli were beta lactam beta -lactamase inhibitors [piperacillin /tazobactam 100% and 80%-] and carbapenems [imipenem 100% each and meropenem 75% and 100%] respectively. ESBLs producing GNRs had a more antibiotic-resistant profile than non- ESBLs producers. They were not only resistant to beta-lactams but showed cross-resistance towards a wide variety of commonly used other antimicrobials. ESBLs producers are more resistant as compared to non-ESBLs producers. Considering the high prevalence of ESBLs in Enteric Gram-negative rods, it is suggested that all such isolates should be tested for the production of ESBLs in the routine microbiology laboratory

Effect of lysozyme or nisin on survival of some bacteria treated with high pressure at subzero temperature

The aim of this work was to examine the inactivation of some Gram-positive and Gram-negative bacteria exposed to the pressure of 193 MPa at -20 ºC in the presence of lysozyme or nisin at concentration of 400 mg/ml. The highest effect of pressure at subzero temperature and lysozyme was found with pressure sensitive Pseudomonas fluorescens; viable cells of this strain were not detected in 1 ml of sample after combined treatment. The action of pressure at subzero temperature and lysozyme or nisin against Escherichia coli led to synergistic reduction by 0.7 or 1.6 log cycles, respectively, while it was practically insignificant for two Staphylococcus aureus strains. Viability loss of E. coli and S. aureus occurred during storage for 20 h of the samples at 37 and 5 ºC, which were previously pressurized with lysozyme or nisin. The synergistic effect of pressure and nisin at pH 5 against E. coli cells just after the pressure treatment was lower than that at pH 7, however, the extent of the lethal effect after storage was higher.

Betalactámicos con inhibidores de betalactamasas: Amoxicilina-sulbactam / Betalactam antibiotics combined with bectalactamases inhibitors: Amoxicillin-sulbactam

La producción de betalactamasas constituye uno de los principales mecanismos de resistencia bacteriana a los antibióticos betalactámicos. La utilización de inhibidores de betalactamasas en combinación con antibióticos betalactámicos permite la inactivación de determinadas betalactamasas producidas por gérmenes Gram positivos, Gram negativos, anaerobios, y aun por micobacterias. Los inhibidores de betalactamasas representan una alternativa terapéutica mejorada respecto del resto de los betalactámicos al asegurar, en la mayoría de los casos, un mayor espectro antimicrobiano comparado con el de sus análogos. La actividad enzimática de las betalactamasas está dirigida específicamente a la hidrólisis del anillo betalactámico, con producción de un compuesto sin actividad antibacteriana. De acuerdo con su posición genómica dentro de los microorganismos, las betalactamasas pueden ser cromosómicas o plasmídicas. Actualmente existen tres inhibidores de betalactamasas localmente disponibles: ácido clavulánico, sulbactam y tazobactam. De ellos, sólo el sulbactam posee actividad antimicrobiana intrínseca sobre las proteínas ligadoras de penicilina. La experiencia clínica acumulada durante más de 20 años confirma que las combinaciones de betalactámicos-inhibidores de betalactamasas son efectivas en el tratamiento empírico inicial de infecciones respiratorias, intraabdominales, urinarias y ginecológicas, incluidas las de origen polimicrobiano. En el caso particular de amoxicilina-sulbactam, la evidencia citada indica que esta combinación es efectiva para el tratamiento de absceso periamigdalino, otitis media, sinusitis, neumonía extrahospitalaria, exacerbación aguda de enfermedad pulmonar obstructiva crónica (EPOC), infección del tracto urinario e infecciones ginecoobstétricas. Por su espectro y propiedades farmacológicas, la combinación amoxicilina-sulbactam constituye una excelente opción también para el tratamiento de infecciones de piel y partes blandas e infecciones intraabdominales.

Betalactamases production is one of the main bacterial resistance mechanisms to betalactam antibiotics. The use of bectalactamases inhibitors combined with betalactam antibiotics allows the inactivation of certain betalactamases produced by Gram positive, Gram negative and anaerobic organisms, and even by mycobacteria. Betalactamases inhibitors are an improved therapeutic alternative compared with the other betalactam since, in most cases, they cover a wider antimicrobial spectrum than their analogues. Betalactamases enzimatic activity is specifically directed to the betalactam ring hydrolisis, producing a compound without antibacterial activity. According to their genomic position within microorganisms, betalactamases can be either chromosomic or plasmidic. Currently there are three betalactamases inhibitors locally available: clavulanic acid, sulbactam and tazobactam. Of them, only sulbactam has an intrinsic antimicrobial activity against penicillin binding proteins. The clinical experience from over 20 years confirms that the combination of betalactam antibiotics is effective in the empirical initial treatment of respiratory, intraabdominal, urinary tract and gynecologic infections, including those of polymicrobial origin. In the specific case of amoxicillin-sulbactam, experiences have shown the effectiveness of the combination in the treatment of peritonsillar abscess, otitis media, sinusitis, community acquired pneumonia, acute exacerbation of chronic obstructive pulmonar disease (COPD), urinary tract infection and obstetric/ gynecologic infections. The spectrum and pharmacologic properties of this combination makes it also an excellent option for the treatment of skin/soft tissue and intraabdominal infections.

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation. Rev. Biol. Trop. 55 (3-4): 777-786. Epub 2007 December, 28.

Se determinó la diversidad y la carga de bacterias heterotróficas, así como los hongos asociados al suelo del manglar de Suva, Islas Fiji, utilizando el método de conteo de placas, usado también para medir la capacidad de bacterias aisladas para producir enzimas hidrolíticas como amilasa, gelatinasa y lipasa. La carga bacteriana heterotrófica resultó ser considerablemente más alta que la carga funguicida. Hubo predominancia de bacterias "Gram-positivas" del género de Bacillus. Otros géneros encontrados fueron Staphylococcus, Micrococcus, Listeria y Vibrio. La eficacia de esta microflora en la degradación del polietileno comercial de bolsas hechas de polietileno de alta densidad (HDPE) y de baja densidad (LDPE) fue estudiada en el laboratorio por un periodo de ocho semanas. La biodegradación fue medida en términos de pérdida de peso, la cual indicó una disminución del 5 %. Después de ocho semanas en el suelo de un manglar, el polietileno clase 1 y clase 3 contenía fundamentalmente Bacillus, pero en el polietileno clase 2 predominó el género Staphylococcus. Mientras que la mayoría de bacterias aisladas fueron capaces de producir enzimas hidrolíticas como la amilasa y la gelatinasa, la actividad lipolítica fue muy baja. La clase 2 (HDPE) experimentó la mayor biodegradación.

Actividades enzimáticas en aislamientos bacterianos de tractos digestivos de larvas y del contenido de pupas de Automeris zugana y Rothschildia lebeau (Lepidoptera: Saturniidae)

Enzymatic activities of bacteria isolated from the digestive tract of caterpillars and the pupal content of Automeris zugana and Rothschildia lebeau (Lepidoptera: Saturniidae). The enzymatic activities of bacteria isolated from the digestive tracts of caterpillars and the pupal contents of Automeris zugana and Rothschildia lebeau was studied. This digestive tract represents an extreme microenvironment due to its high pH and presence of antimicrobial substances secreted by the insect or derived from ingested plant tissue. At the same time, it contains large amounts of nutrient-rich food, for which microbes may compete among themselves and with the caterpillar. There is little information about the microbiota associated with tropical caterpillar guts, although bacteria from different genera have been isolated from gut and pupae samples. The study of the enzymatic activities generated by these organisms constitutes a starting point to understand their metabolic and physiological relationships with their hosts, and to find enzymes that have potential biotechnological applications. In this study we evaluated several enzymatic activities in two collections of bacteria isolated from caterpillar guts and pupae of the tropical lepidopteran species A. zugana and R. lebeau. Bacteria grown under aerobic conditions were tested for an array of enzymes, including gelatinases, caseinases, lipases, esterases, cellulases, xylanases, amylases and chitinases. Both collections displayed similar patterns of enzymatic activity. No isolate showed activity for all enzymatic tests, but as a whole, at least some bacteria in each collection were able to degrade each substrate tested. Isolates with the same taxonomic identification obtained from caterpillar guts and pupae had almost the same enzymatic activities. In both collections, it was possible to group bacterial isolates according to their enzyme activity pattern. In addition to a heterogeneous ensemble of isolates exhibiting two or less enzymatic activities, there were two groups with at least five activities that showed an apparent specialization for the substrates they were able to use. The first consisted exclusively of isolates of the family Enterobacteriaceae, which were positive for lipolytic and chitinolytic activities, but completely lacked amylasic, cellulolytic and xylanolytic activities. The second group, composed mainly of Gram-positive rods, exhibited the opposite pattern: they were positive for amylasic, cellulolytic and xylanolytic activities, lacked chitinolytic activity and had few isolates with lipolytic activity. This work forms the foundation for future research to explore the biotechnological potential of bacterial isolates from caterpillar guts. Rev. Biol. Trop. 55 (2): 401-415. Epub 2007 June, 29.

El tracto digestivo de orugas constituye un microambiente extremo, debido a su elevado pH y presencia de sustancias antimicrobianas secretadas por el insecto o derivadas del tejido vegetal ingerido. Al mismo tiempo, el intestino alberga gran cantidad de alimento, por el cual los microorganismos presentes podrían competir entre sí y con su hospedero. Existe poca información sobre la microbiota asociada con el intestino de orugas tropicales, aunque se ha demostrado la presencia de bacterias de diversos géneros tanto en el intestino como en el interior de pupas. El estudio de las actividades enzimáticas de estos microorganismos constituye un punto de partida en la comprensión de la posible relación metabólica y fisiológica que establecen con sus hospederos, a la vez que permite investigar enzimas con potenciales aplicaciones biotecnológicas. En este trabajo se evaluó la presencia de actividades gelatinolítica, caseinolítica, esterásica, lipolítica, quitinolítica, amilásica, celulolítica y xilanolítica en dos colecciones de aislamientos bacterianos provenientes de tractos digestivos de orugas y de pupas de los lepidópteros Automeris zugana y Rothschildia lebeau. Se utilizaron ensayos bioquímicos tradicionales para detectar enzimas secretadas en condiciones aerobias, en las que ambas colecciones exhibieron un comportamiento enzimático similar. Ningún aislamiento produjo un resultado positivo en todas las pruebas, pero como conjunto ambas colecciones fueron capaces de utilizar todos los sustratos evaluados. Los aislamientos obtenidos de pupas presentaron prácticamente las mismas actividades que sus homólogos provenientes de intestinos. En ambas colecciones fue posible agrupar los aislamientos de acuerdo con su patrón de producción de enzimas. Además de un conjunto heterogéneo de aislamientos poco activos (dos o menos actividades), se destacan dos grupos muy activos (al menos cinco actividades), que manifiestan una aparente especialización en los sustratos que utilizan. El primero de ellos está constituido exclusivamente por miembros de la familia Enterobacteriaceae, los cuales exhibieron un alto porcentaje de positividad en actividades lipolítica y quitinolítica, pero no demostraron la expresión de las actividades amilásica, celulolítica ni xilanolítica. El segundo grupo, formado en su gran mayoría por bacilos Gram-positivos, presenta la situación opuesta: alta positividad en actividades amilásica, celulolítica y xilanolítica, no detección de actividad quitinolítica y pocos aislamientos con actividad lipolítica. Este trabajo pretende ser la base de futuras investigaciones que exploren el potencial biotecnológico de aislamientos bacterianos provenientes del tracto digestivo de orugas.

Mecanismos enzimáticos de resistencia a antibióticos aminoglicósidos en bacilos gram negativos de hospitales chilenos / Enzymatic mechanisms of resistance to aminoglycoside antibiotics in gram negative bacilli from chilean hospitals

The presence of aminoglycoside modifying enzyme (AMEs) has been investigated by an agar diffusion method, in 344 strains of aminoglycoside-resistant Gram negative bacilli isolated in different Chilean hospitals. Most of the strains exhibited a combination of enzymatic mechanism of resistance, but 2 acetylatting (AAC(3)II and AAC(6')I) and one phosphorylating (APH(3')I) enzymes were mechanism detected in the strains. A significant increase in the frequency of strains producing AAC(6')I), possibly due to wide use of amikacin, has been found when results were compared with those of a report published in 1985