Restricción programada de cefalosporinas de tercera generación en contexto de un brote de bacilos gramnegativos productores de beta-lactamasas tipo AmpC en unidades críticas: una experiencia de la vida real / Third-generation cephalosporins programmed restriction in the context of an outbreak of AmpC beta-lactamase-producing gram-negative bacilli in critical units: a real-life experience

INTRODUCCIÓN: La restricción programada (RP) de antimicrobianos puede disminuir selectivamente la tasa de infecciones por determinados microorganismos. En este sentido, los bacilos gramnegativos productores de beta-lactamasas AmpC (BGN-blaAmpC) son seleccionados por el sobreuso de cefalosporinas de tercera generación (C3G). Estas bacterias, también adquieren genes y co-producen otras beta-lactamasas, como las de Nueva Delhi (BGN-blaNDM). OBJETIVOS: Disminuir la tasa de aislamiento de BGN-blaAmpC y BGN-blaNDM en cultivos de pacientes de la UCI luego de una RP de C3G en el marco de un brote nosocomial por estos microrganismos. MATERIALES Y MÉTODOS: Estudio cuasi-experimental, previo (P1= 12 meses) y posterior (P2= 12 meses) a una RP de C3G en un hospital de adultos, donde, en el contexto de brote mencionado, se aplicaron medidas de control de infecciones generales. El uso de antimicrobianos se expresó como "porcentaje de los días de tratamiento (%DDT)"/100 camas ocupadas al día (100-COD). Se compararon las tasas de aislamiento de BGN-blaAmpC y BGN-blaNDM en hemocultivos (HC), mini-lavados bronquio-alveolares (mB) y urocultivos (UC) en la UCI. RESULTADOS: En P2 el consumo de C3G fue 2,5% DDT/100-COD. Hubo un descenso en los aislamientos de BGN-blaAmpC en HC (RR 0,48 [0,2-0,9] p < 0,02) y mB (RR 0,52 [0,3-0,9] p < 0,02), así como también de BGN-blaNDM en HC (RR 8,1 [1,6-39,4] p < 0,00). Conclusiones: La RP de C3G se asoció con la reducción de los BGN-blaAmpC y BGN-blaNDM en HC, así como de los BGN-blaAmpC mB.

BACKGROUND: Programmed restriction (PR) of antimicrobials can selectively decrease the rate of infections by certain microorganisms. In this sense, AmpC beta-lactamase-producing gram-negative bacilli (GNB-blaAmpC) are selected for the overuse of third generation cephalosporins (3GC). These bacteria also acquire genes and co-produce other β-lactamases, such as New Delhi ones (GNB-blaNDM). AIM: To decrease the isolation rate of GNB- blaAmpC and GNB- blaNDM in cultures from ICU patients after a PR of 3GC. METHODS: Quasi-experimental study, before (P1= 12 months) and after (P2= 12 months) a PR of 3GC in an adults' hospital. The use of antibiotics was expressed as "percentage days of treatment (%DOT)" /100 beds occupied per day (100-BOD). The rates of GNB-blaAmpC and GNB-blaNDM were compared in blood cultures (BC), mini-bronchio alveolar lavages (mB) and urine cultures (UC) in the ICU. RESULTS: In P2, 3GC consumption was 2.5% DOT/100-COD. There was a decrease in GNB-blaAmpC from BC (RR 0.48 [0.2-0.9] p < 0.02) and mB (RR 0.52 [0.3-0.9] p < 0.02), as well as of GNB-blaNDM from BC (RR 8.1 [1.6-39.4] p < 0.00). Conclusions: PR of 3GC was linked to the reduction of GNB-blaAmpC and GNB-blaNDM in BC, as well as GNB-blaAmpC in mB from ICU patients.

Perfil fenotípico de resistência à colistina e tigeciclina em um hospital público no Brasil / Phenotypical profile of colistin and tigecycline resistance in a public hospital in Brazil / Perfil fenotípico de resistencia a la colistina y tigeciclina en un hospital público de Brasil

Justificativa e objetivos: Infecções Relacionadas à Assistência à Saúde (IRAS) causadas por bacilos Gram negativos multirresistentes (BGN-MDR) são consideradas um problema de saúde pública e um impacto nas taxas de mortalidade nas Unidades de Terapia Intensiva (UTI). O objetivo deste estudo foi verificar o perfil fenotípico de resistência à colistina e à tigeciclina, consideradas como último recurso terapêutico aos BGN-MDR. Métodos: Os dados foram coletados nas fichas de busca ativa do serviço de controle de infecções e prontuários médicos de pacientes internados em duas UTIs de um hospital público de Joinville, entre janeiro de 2016 e junho de 2017. Resultados: Ocorreram 256 IRAS por BGN, acometendo principalmente o gênero masculino (62%), com mediana de idade de 65 anos. Entre os BGN, 37% expressaram MDR; sendo as espécies mais frequentes: Klebsiella pneumoniae e (47%), Acinetobacter baumannii (23%) e Stenotrophomonas maltophilia (11%). A resistência de BGN-MDR à colistina e tigeciclina foi de 5% e de 12%, respectivamente; 5% dos isolados foram resistentes aos dois antibióticos. A taxa de óbito entre os pacientes com IRAS por BGN-MDR resistentes à colistina foi mais alta (60%) que aquelas à tigeciclina (45%). Conclusão: K. pneumoniae e A. baumannii produtores de carbapenemases, resistentes a colistina e tigeciclina prevaleceram entre os BGN-MDR, e estiveram associadas a maioria dos óbitos. Essas observações, junto com o alto uso de carbapenêmicos na terapia empírica, mostra a necessidade do uso racional de antimicrobianos.(AU)

Background and objectives: Healthcare-associated Infections (HAIs) caused by multidrug-resistant Gram-negative bacilli (GNB-MDR) are considered a public health problem and have an impact on mortality rates in Intensive Care Units (ICU). The aim of this study was to verify the phenotypic profile of resistance to colistin and tigecycline, considered as the last antimicrobial choice to treat BGNMDR infections. Methods: Data were collected on the active search records of the infection control service and medical records of patients admitted to two ICUs at a public hospital in Joinville between January 2016 and June 2017. Results: There were 256 HAIs caused by GNB, mainly affecting males (62%), with a median age of 65 years. Among GNBs, 37% expressed MDR; the most frequent species were: Klebsiella pneumoniae (47%), Acinetobacter baumannii (23%) and Stenotrophomonas maltophilia (11%). The resistance of GNB-MDR to colistin and tigecycline was 5% and 12%, respectively; 5% of the isolates were resistant to both antibiotics. The death rate among patients with HAIs caused by colistin-resistant GNB-MDR was higher (60%) than those to tigecycline (45%). Conclusion: Carbapenemase-producing K. pneumoniae and A. baumannii, resistant to colistin and tigecycline, prevailed among GNB-MDRs, and were associated with most deaths. These observations, coupled with the high use of carbapenems in empirical therapy, show the need for rational use of antimicrobials.(AU)

Justificación y objetivos: Las Infección nosocomial (IHs) causadas por bacilos Gram negativos multirresistentes (BGN-MDR) se consideran un problema de salud pública y un impacto en las tasas de mortalidad en las Unidades de Terapia Intensiva (UTI). El objetivo de este estudio fue verificar el perfil fenotípico de resistencia a la colistina ya la tigeciclina, consideradas como último recurso terapéutico a los BGN-MDR. Métodos: Los datos fueron recolectados en las fichas de búsqueda activa del servicio de control de infecciones y prontuarios médicos de pacientes internados en dos UTIs de un hospital público de Joinville, entre enero de 2016 y junio de 2017. Resultados: Ocurrieron 256 IHs por BGN, que afectan principalmente al género masculino (62%), con mediana de edad de 65 años. Entre los BGN, el 37% expresó MDR; siendo las especies más frecuentes: Klebsiella pneumoniae (47%), Acinetobacter baumannii (23%) y Stenotrophomonas maltophilia (11%). La resistencia de BGN-MDR a la colistina y tigeciclina fue del 5% y del 12%, respectivamente; 5% de los aislados fueron resistentes a los dos antibióticos. La tasa de muerte entre los pacientes con IH causadas por los BGN-MDR resistentes la colistina fue más alta (60%) que aquellas a tigeciclina (45%). Conclusión: K. pneumoniae y A. baumannii productoras de carbapenemases, resistentes la colistina y la tigeciclina, fueron más frecuentes entre los BGN-MDR y su asociación estuvo presente en la mayoría de las muertes. Estas observaciones, junto con el alto uso de carbapenems en la terapia empírica, muestran la necesidad de un uso racional de los antimicrobianos.(AU)

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach

ABSTRACT Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48 h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38 h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Perfiles genéticos bacterianos y análisis de brotes de las Enfermedades Transmitidas por Alimentos empleando Electroforesis de Campo Pulsado como herramienta para la vigilancia epidemiológica molecular / Bacterial genetic profiles and analysis of outbreaks of foodborne diseases using pulsed field gel electrophoresis as a tool for molecular epidemiological surveillance

Las Enfermedades de Transmisión Alimentaria (ETA) son un problema de salud pública con altos índices de morbilidad y mortalidad a nivel global. La vigilancia y estudio de brotes de las ETA a través de Electroforesis de Campo Pulsado (PFGE) constituye un soporte fundamental para la investigación epidemiológica. El objetivo del estudio es presentar la base de datos de perfiles genéticos bacterianos y analizar brotes de enfermedades transmitidas por alimentos empleando Electroforesis de Campo Pulsado. Estudio descriptivo observacional de carácter retrospectivo, muestreo por conveniencia en el que fueron estudiados 778 aislamientos bacterianos causantes de ETA. La Base de Datos Nacional (BDN) quedó conformada por los siguientes patógenos entéricos; Salmonella spp., Shigella sonnei, Vibrio cholerae, Campylobacter spp., Escherichia coli O157:H7 y Escherichia coli no O157 caracterizados por una diversidad de patrones únicos, clusters y brotes. La BDN de Salmonella spp., quedó representada por un total de 558 cepas con 248 PUN, de las cuales 22,6% (126 cepas) corresponden a Salmonella enterica ser. Typhimurium, 20,6% (115 cepas) a Salmonella enterica ser. Enteritidis, 9,1% (51 cepas) a Salmonella enterica ser. Newport, 1,6% (9 cepas) a Salmonella enterica ser. Muenchen, que al mismo tiempo son los serotipos que están asociados a brotes. Fueron confirmados un total de 13 brotes causados por Salmonella spp.; Shigella sonnei con 113 cepas estudiadas, 57 patrones únicos y 19 clusters detectados. Se identificaron 3 patrones PYJ16X01.0012, PYJ16X01.0034 y PYJ16X01.0014 como los predominantes. Vibrio cholerae con 18 cepas estudiadas, 9 patrones únicos y 4 clusters detectados. Se pudo establecer una relación genética del 100% entre cepas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa productora de toxinas ctxA y tcpA aislada del caso índice del brote de cólera. Campylobacter spp., con 62 cepas estudiadas, 42 patrones únicos y 10 clusters detectados. La BDN de E. coli productor de toxina shiga O157 y no O157, con 9 y 20 cepas de origen humano respectivamente, caracterizadas según sus factores de virulencia y subtipos. Se reconocieron 8 patrones electroforéticos PUN y 1 cluster para E. coli productor de toxina shiga O157, y 18 PUN y 1 clúster para E. coli productor de toxina shiga no O157.La disponibilidad de una Base de Datos Nacional de patógenos bacterianos transmitidos por alimentos constituye un importante avance para la salud pública, con un gran aporte en la vigilancia y epidemiología del país permitiendo la confirmación y detección de brotes discriminando aislamientos relacionados genéticamente y por consiguiente el estudio de relaciones clonales y probable origen(AU)

Foodborne diseases (FBD) are a problem of public health with high indexes of morbidity and mortality at global level. The surveillance and study of outbreaks of the FBD through pulsed field gel electrophoresis (PFGE) is a fundamental support for epidemiological research. The aim of the study is to present the database of bacterial genetic profiles and analyze outbreaks of FBD using PFGE. This was an observational descriptive retrospective study with convenience sampling in which 778 bacterial isolates causing FBD were studied. The National Database (NDB) was made up of the following enteric pathogens causing FBD: Salmonella spp., Shigella sonnei, Vibrio cholerae, Campylobacter spp., Escherichia coli O157: H7 and Escherichia coli no O157. Each of them was characterized by a diversity of unique patterns, clusters and outbreaks. The NDB of Salmonella spp. was represented by a total of 558 strains with 248 PUN, of which 22.6% (126 strains) correspond to Salmonella enterica ser. Typhimurium, 20.6% (115 strains) to Salmonella enterica ser. Enteritidis, 9.1% (51 strains) to Salmonella enterica ser. Newport, 1.6% (9 strains) to Salmonella enterica ser. Muenchen, which at the same time are the serotypes associated with outbreaks. A total of thirteen outbreaks caused by Salmonella spp., Shigella sonnei with 113 strains studied, 57 unique patterns and 19 clusters detected were confirmed. Three patterns PYJ16X01.0012, PYJ16X01.0034 and PYJ16X01.0014 were identified as the predominant. Vibrio cholerae with 18 strains studied, 9 unique patterns and 4 clusters were detected. A genetic relationship of 100% was established between strains of Vibrio cholerae O1 biotype El Tor serotype Ogawa toxin producer ctxA and tcpA isolated from the index case of the cholera outbreak. Campylobacter spp., with 62 strains studied, 42 unique patterns and 10 clusters were detected. The NDB of O157 and non-O157 Shiga toxin producing E. coli O157, with 9 and 20 strains of human origin respectively, were characterized according to their virulence factors and subtypes. We recognized 8 PUN electrophoretic patterns and 1 cluster for O157 Shiga toxin producing E. coli, and 18 PUN and 1 cluster for non-O157 Shiga toxin producing E. coli. The availability of a National Database of bacterial pathogens transmitted by food constitutes an important advance for public health, with a great contribution to the surveillance and epidemiology of the country allowing the confirmation and detection of outbreaks discriminating genetically related isolates and therefore, the study of clonal relationships and probable origin(AU)

A practical molecular identification of nonfermenting Gram-negative bacteria from cystic fibrosis

Abstract Identification of nonfermenting Gram-negative bacteria (NFGNB) of cystic fibrosis patients is hard and misidentification could affect clinical outcome. This study aimed to propose a scheme using polymerase chain reaction to identify NFGNB. This scheme leads to reliable identification within 3 days in an economically viable manner when compared to other methods.

Genes qnr en Enterobacteriaceae aisladas en un hospital de Venezuela / qnr genes in Enterobacteriaceae isolated from at a hospital in Venezuela

Resumen Introducción: La resistencia de enterobacterias a quinolonas se ha difundido por el mundo, fenómeno presente también en Venezuela. El mecanismo de esta resistencia pudiera estar mediado por genes incluidos en el cromosoma bacteriano o transmitirse en el interior de plásmidos. Objetivo: Evaluar la resistencia a quino-lonas, codificada por genes qnr, presentes en cepas de enterobacterias, aisladas en el Hospital Universitario de Cumaná, Venezuela. Métodos: A las cepas obtenidas se les realizaron pruebas de susceptibilidad antimicrobiana a quinolonas, β-lactámicos y aminoglucósidos. La presencia del gen qnr se determinó por RPC. Las enterobacterias portadoras del gen qnr fueron sometidas al proceso de conjugación bacteriana para comprobar su capacidad de transferencia. A las transconjugantes obtenidas se les realizó pruebas de susceptibilidad antimicrobiana y RPC para comprobar la transferencia de los genes. Resultados: Se encontraron elevados porcentajes de resistencia antimicrobiana a quinolonas y betalactámicos. El 33,6% de las cepas eran portadoras del gen qnrB, y 0,9% del gen qnrA. Se obtuvieron 23 cepas transconjugantes; de éstas, 20 portaban el gen qnrB, no se observó la presencia de qnrA. Discusión: En conclusión, el elevado porcentaje de genes qnr encontrado en las enterobacterias aisladas, y comprobada la presencia de éstos en plásmidos transferibles, complica la aplicación de tratamientos basados en quinolonas y fluoroquinolonas, por lo que es recomendable el uso racional de estos antimicrobianos, y proponer la rotación de la terapia antimicrobiana, a fin de evitar la selección de cepas resistentes.

Background: Enterobacteria resistant to quinolones is increasing worldwide, including Venezuela. The mechanism for this resistance could be due to genes included in the chromosome or in transmissible plasmids. Aim: To evaluate the resistance to quinolones, coded by qnr genes present in enterobacteria species, isolated in the University Hospital of Cumana, Venezuela. Methods: Antimicrobial susceptibility tests to quinolones, beta-lactams and aminoglycosides were carried out to all the isolates. The presence of qnr genes were determined by PCR. The isolates carrying the qnr genes were used for bacterial conjugation tests to determine the presence of transferable plasmids. Antimicrobial susceptibility tests and PCR were carried out in the transconjugants to verify the transfer of the genes. Results: High levels of antimicrobial resistance to quinolones and beta-lactams were found among the isolates. We found that 33.6% of the isolates carry the qnrB gene and 0.9% qnr A gene. Of the 23 transconjugants, 20 showed to have qnrB gene, but none qnrA. Discussion: We concluded that the high frequency of qnr genes found in the enterobacteria isolates and their presence on transferable plasmids, complicate the use of quinolones for the treatment of bacterial infections, thus, a treatment plan should be designed with the rational use and the rotation of different types of antimicrobials, in order to avoid the selection of increasingly resistant strains.

Resultados de la vigilancia nacional de la resistencia antimicrobiana de enterobacterias y bacilos Gram negativos no fermentadores en infecciones asociadas a la atención de salud, Colombia, 2012-2014 / Results of the national surveillance of antimicrobial resistance of Enterobacteriaceae and Gram negative bacilli in health care-associated infections in Colombia, 2012-2014

Resumen Introducción. En el tercer trimestre de 2012, comenzó a operar el Sistema Nacional de Vigilancia de Resistencia Antimicrobiana en las infecciones asociadas a la atención en salud, con el fin de recabar y analizar la información referente al problema en Colombia. Objetivo. Describir los perfiles de resistencia y los resultados de la vigilancia por el laboratorio con base en los datos recolectados en el Sistema. Materiales y métodos. Se hizo un estudio descriptivo y retrospectivo con base en la información del Sistema Nacional de Vigilancia en Salud Pública, Sivigila, 1 de septiembre de 2012 a 31 de diciembre de 2014, así como de las bases de datos Whonet con los datos notificados por las unidades primarias generadoras de datos y los resultados de la confirmación por el laboratorio de la caracterización fenotípica y genotípica de la resistencia a carbapenemasas en 1.642 aislamientos (927 de enterobacterias, 614 de Pseudomonas spp. y 101 de Acinetobacter spp.). Resultados. La resistencia de Escherichia coli a las cefalosporinas de tercera generación presentó un incremento significativo, alcanzando 26,3 % en unidades de cuidados intensivos y 22,5 % en otras áreas de hospitalización. La resistencia a ertapenem de Klebsiella pneumoniae registró un incremento y alcanzó 14,6 % en unidades de cuidados intensivos. La resistencia de Acinetobacter baumannii a los carbapenémicos superó el 50 % en dichas unidades, en tanto que en Pseudomonas aeruginosa se presentaron porcentajes más bajos (38,8 %). Las carbapenemasas más frecuentes en enterobacterias fueron la KPC (n=574), seguida de la NDM (n=57); en P. aeruginosa, la VIM (n=229) y la KPC (n=114), y en A. baumannii, la OXA-23 (n=87). Se detectaron varias combinaciones de carbapenemasas, siendo la de KPC y VIM la más frecuente en Pseudomonas spp., y en enterobacterias. Conclusión. La información obtenida a partir del Sistema Nacional de Vigilancia ha permitido conocer los perfiles y los mecanismos de resistencia a carbapenémicos de las cepas que están circulando en las instituciones de salud del país.

Abstract Introduction: The Colombian National Antimicrobial Resistance Monitoring System for the surveillance of healthcare-associated infections was set up to meet this problem in the third quarter of 2012. Objective: To describe resistance profiles and laboratory-based surveillance based on the information collected by the System. Materials and methods: We conducted a retrospective and descriptive study of the information notified to the Colombian Public Health Surveillance System (Sivigila), and in the Whonet databases covering the period from July 2012 to December 2014 provided by the primary data-generating units in the country, as well as laboratory surveillance results from 1,642 phenotypic and genotypic tests on carbapenemase isolates (927 from Enterobacteriaceae, 614 from Pseudomonas spp. and 101 from Acinetobacter spp.). Results: There was a significant increase in Escherichia coli resistance to third-generation cephalosporins (reaching 26.3% in ICUs and 22.5% in other hospital wards), and Klebsiella pneumoniae resistance to ertapenem also increased (reaching 14.6% in ICUs). Acinetobacter baumannii carbapenem resistance exceeded 50% in ICUs whereas Pseudomonas aeruginosa had lower carbapenem resistance (38.8%). KPC (n = 574) and NDM (n=57) were the most frequently occurring carbapenemases in Enterobacteriaceae, VIM (n=229) and KPC (n=114) in P. aeruginosa, and OXA-23 in A. baumannii (n=87); several carbapenemase combinations were identified, KPC + VIM being the most common in Pseudomonas spp. and Enterobacteriaceae. Conclusion: The data from the surveillance of healthcare-associated infections revealed significant carbapenem resistance profiles and antimicrobial resistance mechanisms circulating in Colombian healthcare institutions.

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.

Pressure ulcer as a reservoir of multiresistant Gram-negative bacilli: risk factors for colonization and development of bacteremia

Abstract The purpose of this study was to identify the risk factors that predispose patients who are hospitalized with pressure ulcers (PUs) colonized by Gram-negative bacilli (GNB) to develop bacteremia. In addition, we also detected main phenotypes of resistance in infected and uninfected PUs. A prospective cohort study was conducted at the Clinical Hospital of the Federal University of Uberlândia including patients with Stage II or greater PUs, colonized or not with GNB, from August 2009 to July 2010. Infected ulcers were defined based on clinical signs and on positive evaluation of smears of wound material translated by a ratio of polymorphonuclear cells to epithelial cells ≥2:1, after Giemsa staining. A total of 60 patients with Stage II PUs were included. Of these 83.3% had PUs colonized and/or infected. The frequency of polymicrobial colonization was 74%. Enterobacteriaceae and GNB non-fermenting bacteria were the most frequent isolates of PUs with 44.0% of multiresistant isolates. Among patients who had infected PUs, six developed bacteremia by the same microorganism with a 100% mortality rate. In addition, PUs in hospitalized patients were major reservoir of multiresistant GNB, also a high-risk population for the development of bacteremia with high mortality rates.

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.

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.

Bacteriological analysis of necrotic pulp and fistulae in primary teeth

Objectives: Primary teeth work as guides for the eruption of permanent dentition, contribute for the development of the jaws, chewing process, preparing food for digestion, and nutrient assimilation. Treatment of pulp necrosis in primary teeth is complex due to anatomical and physiological characteristics and high number of bacterial species present in endodontic infections. The bacterial presence alone or in association in necrotic pulp and fistula samples from primary teeth of boys and girls was evaluated. Material and Methods: Necrotic pulp (103) and fistula (7) samples from deciduous teeth with deep caries of 110 children were evaluated. Bacterial morphotypes and species from all clinical samples were determined. Results: A predominance of gram-positive cocci (81.8%) and gram-negative coccobacilli (49.1%) was observed. In 88 out of 103 pulp samples, a high prevalence of Enterococcus spp. (50%), Porphyromonas gingivalis (49%), Fusobacterium nucleatum (25%) and Prevotella nigrescens (11.4%) was observed. Porphyromonas gingivalis was detected in three out of seven fistula samples, Enterococcus spp. in two out of seven samples, and F. nucleatum, P. nigrescens and D. pneumosintes in one out of seven samples. Conclusions: Our results show that Enterococcus spp. and P. gingivalis were prevalent in necrotic pulp from deciduous teeth in boys from 2 to 5 years old, and that care of the oral cavity of children up to five years of age is important. .

Performance of Kiestra Total Laboratory Automation Combined with MS in Clinical Microbiology Practice

BACKGROUND: Microbiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens. METHODS: Early growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription. RESULTS: Pathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients. CONCLUSIONS: The requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner.

Atividade antimicrobiana da Terminalia arjuna Wight & Arn.: uma planta etnomedicinal contra patógenos causadores de infecções no ouvido / Antimicrobial activity of Terminalia arjuna Wight & Arn.: an ethnomedicinal plant against pathogens causing ear infection

Infecções de ouvido representam uma das doenças mais comuns do mundo. Diferentes agentes etiológicos são responsáveis por tais infecções. OBJETIVO: Avaliar o potencial antimicrobiano de extratos de folhas e da casca de Terminalia arjuna contra Staphylococcus aureus, Acinetobacter sp., Proteus mirabilis, Escherchia coli, Pseudomonas aeruginosa e Candida albicans - patógenos que causam infecções de ouvido e como esses extratos se comparam às medicações tópicas atualmente disponíveis. MATERIAIS E MÉTODOS: Metanol, etanol, acetona, extratos aquosos (quentes e frios) de folhas e casca da T. arjuna foram testados para avaliação de suas atividades antimicrobianas. RESULTADOS: Dos três solventes orgânicos avaliados, o extrato acetônico das folhas foi o que teve o melhor resultado contra S. aureus. O extrato orgânico da casca exibiu quase o mesmo grau de ação antimicrobiana entre todas as bactérias gram-negativas, exceto contra P. aeruginosa. Entretanto, o extrato aquoso da casca de T. arjuna exibiu boa atividade contra S. aureus. Nenhum dos extratos exibiu atividade antifúngica. CONCLUSÃO: O extrato orgânico obtido a partir da casca e folhas de T. arjuna pode ser usado para tratar infecções bacterianas de ouvido, especialmente aquelas causadas por S. aureus, que exibiu maiores zonas de inibição do que gotas herbáceas; entretanto, ainda necessitamos de estudos mais detalhados - como ensaios in vivo e avaliações de propriedades farmacocinéticas para avaliarmos sua utilidade terapêutica no tratamento de infecções de ouvido.

Ear infection is one of the common diseases occurring throughout the world. Different etiological agents are responsible for ear infections. AIM: To assess the antimicrobial potential of Terminalia arjuna leaves and bark extracts against Staphylococcus aureus, Acinetobacter sp., Proteus mirabilis, Escherchia coli, Pseudomonas aeruginosa and Candida albicans, pathogens causing ear infections and their comparison with locally available ear drops. MATERIALS AND METHODS: Methanol, ethanol, acetone, aqueous (hot and cold) extracts from the leaves and bark of T. arjuna were tested for their antimicrobial activity. RESULTS: Of the three organic solvents evaluated, acetonic leaf extract was found to be best against S. aureus. Organic bark extract showed almost equal inhibition of all tested Gram negative bacteria except P. aeruginosa. However, aqueous extract of T. arjuna bark exhibited good activity against S. aureus. All the extracts were unable to exhibit any antifungal activity. CONCLUSION: Organic extract obtained from the T. arjuna bark and leaves may be used to treat the bacterial ear pathogens especially S. aureus, which has shown greater inhibition zones than the herbal drops, however, we still need more detailed studies as in vivo testing and pharmacokinetics properties for their therapeutic utility in treating ear infections.

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.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Multiplex PCR for bla(CTX-M) & bla(SHV) in the extended spectrum beta lactamase (ESBL) producing gram-negative isolates.

BACKGROUND & OBJECTIVE: Phenotyping is commonly used for detection of extended spectrum beta lactamase (ESBL) production in gram-negative isolates. ESBLs are mainly coded for by three important genes, namely bla(TEM), bla(SHV) and bla(CTX-M). In this study we used a multiplex PCR as a rapid method to identify two common genes (bla(CTX-M) & bla(SHV)) responsible for extended spectrum beta lactamase production in members of Enterobacteriaceae family isolated from different clinical samples from a specialty hospital at Chennai. METHODS: A total of 260 non repetitive clinical isolates from 240 patients (some patients had more than one organism also), was selected for the study. Of these 33 were from sputum, 64 from urine, 46 from blood, 28 from pus aspirates, 58 from endotracheal secretions and 31 from other miscellaneous specimens. Phenotypic identification for ESBL production was confirmed by double disk synergy test (DDST) and phenotypic confirmatory double disk test (PCDDT) according to CLSI guidelines. Multiplex PCR for bla(CTX-M) and bla(SHV) was performed for the ESBL positive isolates. RESULTS: bla(SHV) like genes were found in 6 of 42 E.coli (14%), 7 of 46 Enterobacter species (15%), 28 of 62 Klebsiella species (45%) and bla(SHV) was not detected in any of the 50 isolates of non-fermenting gram-negative isolates. (Pseudomonas and Acinetobacter species) bla(CTX-M) like genes were found in 21 of 42 E. coli (50%), 13 of 46 Enterobacter species (28%), 25 of 62 (40%) Klebsiella species and 1 of 50 nonfermenting gram-negative bacilli (2%). INTERPRETATION & CONCLUSION: Our study demonstrated rapid detection of bla(SHV) and bla(CTX-M) in isolates belonging to Enterobacteriaceae and other non-fermenting clinical isolates using multiplex PCR. This genotypic method provided a rapid and efficient differentiation of ESBLs in the laboratory.

Antibacterial activities of multi drug resistant Myroides odoratimimus bacteria isolated from adult flesh flies (Diptera: Sarcophagidae) are independent of metallo beta-lactamase gene / Atividades antibacterianas de Myroides odoratimimus isolada de moscas varejeiras adultas (Diptera: Sarcophagidae) são independentes do gene metalo beta lactamase

Flesh flies (Diptera: Sarcophagidae) are well known cause of myiasis and their gut bacteria have never been studied for antimicrobial activity against bacteria. Antimicrobial studies of Myroides spp. are restricted to nosocomial strains. A Gram-negative bacterium, Myroides sp., was isolated from the gut of adult flesh flies (Sarcophaga sp.) and submitted to evaluation of nutritional parameters using Biolog GN, 16S rRNA gene sequencing, susceptibility to various antimicrobials by disc diffusion method and detection of metallo â-lactamase genes (TUS/MUS). The antagonistic effects were tested on Gram-negative and Gram-positive bacteria isolated from human clinical specimens, environmental samples and insect mid gut. Bacterial species included were Aeromonas hydrophila, A. culicicola, Morganella morganii subsp. sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp., Serratia sp., Kestersia sp., Ignatzschineria sp., Bacillus sp. The Myroides sp. strain was resistant to penicillin-G, erythromycin, streptomycin, amikacin, kanamycin, gentamycin, ampicillin, trimethoprim and tobramycin. These strain showed antibacterial action against all bacterial strains except W. confusa, Ignatzschineria sp., A. hydrophila and M. morganii subsp. sibonii. The multidrug resistance of the strain was similar to the resistance of clinical isolates, inhibiting growth of bacteria from clinical, environmental and insect gut samples. The metallo â-lactamase (TUS/MUS) genes were absent, and resistance due to these genes was ruled out, indicating involvement of other secretion machinery.

Moscas varejeiras (Diptera: Sarcophagidae) são causa conhecida de miíase e as bactérias de seus intestinos nunca foram estudadas quanto à atividade antibacteriana. Estudos antimicrobianos de Myroides spp restringem-se à cepas hospitalares. Uma bactéria Gram negativa, Myroides sp, foi isolada do intestino de moscas varejeiras adultas (Sarcophaga sp) e submetida à avaliação de parâmetros nutricionais pelo sistema BIOLOG GN, ao sequenciamento genético 16S rRNA, à sensibilidade a vários antimicrobianos pelo método de difusão de discos e à detecção dos genes de metalo beta lactamases (TUS/MUS). Os efeitos antagonistas foram testados contra bactérias Gram negativas e Gram positivas isoladas de material clínico humano, amostras ambientais e intestino do inseto. As espécies bacterianas incluíram Aeromonas hydrophila, A. culicicola, Morganella morganii subsp sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp, Serratia sp, Kestersia sp, Ignatzschineria sp e Bacillus sp. A cepa Myroides sp foi resistente à penicilina G, eritromicina, estreptomicina, amicacina, canamicina, gentamicina, ampicilina, trimetoprim e tobramicina. Esta cepa apresentou atividade antimicrobiana contra todas as cepas exceto W.confusa, Ignatzschineria sp, A. hydrophila e M. morgani subsp sibonii. A resistência múltipla da cepa foi semelhante à de isolados clínicos, inibindo bactérias das amostras clínicas, ambientais e do intestino do inseto. Os genes de metalo beta lactamases (TUS/MUS) estavam ausentes, excluindo-se a resistência mediada por esses genes, o que indica o envolvimento de um mecanismo alternativo de secreção.