First characterization of K. pneumoniae ST11 clinical isolates harboring blaKPC-3 in Latin America / Primera caracterización de aislamientos clínicos de K. pneumoniae ST11 portadoresde blaKPC-3en América Latina

Abstract Antimicrobial resistance due to carbapenemase production in Enterobacteriaceaeclinical isolates is a global threat. Klebsiella pneumoniae harboring the blaKPCgene is one ofthe major concerns in hospital settings in Latin America.The aim of this study was to characterize the antibiotic resistance mechanisms and to typifyfour carbapenem-resistant K. pneumoniae clinical isolates from the city of Manizales, Colombia.We identified blaKPC-3in all four isolates by polymerase chain reaction and subsequentsequencing. The plasmid-mediated quinolone resistance genes qnrB19-like and aac(6)Ib-cr;fosfomycin resistance gene fosA and an insertion sequence IS5-like in mgrB (colistin resistance)were also detected. Sequence types ST11 with capsular type wzi75, and ST258 with wzi154,were characterized. The blaKPC-3gene was mobilized in a 100-kb IncFIB conjugative plasmidwith vagCD toxin-antitoxin system.This work reports multiple resistance genes in blaKPC-producing K. pneumoniae and the firstoccurrence of ST11 clinical isolates harboring blaKPC-3in Latin America.

Resumen La resistencia a antibióticos mediada por la producción de carbapenemasas en aislamientos clínicos de Enterobacteriaceae es una amenaza mundial. Klebsiella pneumoniae portador de blaKPC es uno de los mayores problemas a nivel hospitalario en Latinoamérica. El objetivo de este estudio fue caracterizar los mecanismos de resistencia antibiótica y tipificar cuatro aislamientos clínicos de K. pneumoniae resistentes a carbapenems obtenidos en la ciudad de Manizales, Colombia. Se identificó blaKPC-3 en todos los aislamientos mediante reacción en cadena de polimerasa y secuenciación. También se detectaron los genes de resistencia transferible a quinolonas qnrB19-like y aac(6')Ib-cr y a fosfomicina fosA, y la secuencia de inserción /S5-like en mgrB (asociada a la resistencia a colistina). Se caracterizaron los secuenciotipos ST11 (cápsula wzi75) y ST258 (cápsula wzi154). Se comprobó que blaKPC-3 fue movilizado por un plásmido conjugativo IncFIB-vagCD de 100kb. En este trabajo se reportan múltiples genes de resistencia en K. pneumoniae productor de blaKPC y se describen por primera vez aislamientos clínicos ST11 productores de blaKPC-3 en Latinoamérica.

Prevalence of Klebsiella pneumoniae carbapenemase - and New Delhi metallo-beta-lactamase-positive K. pneumoniae in Sergipe, Brazil, and combination therapy as a potential treatment option

Abstract INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae infection lacks treatment options and is associated with prolonged hospital stays and high mortality rates. The production of carbapenemases is one of the most important factors responsible for this multi-resistance phenomenon. METHODS: In the present study, we analyzed the presence of genes encoding carbapenemases in K. pneumoniae isolates circulating in one of the public hospitals in the city of Aracaju, Sergipe, Brazil. We also determined the best combination of drugs that display in vitro antimicrobial synergy. First, 147 carbapenem-resistant K. pneumoniae isolates were validated for the presence of blaKPC, bla GES, bla NDM, bla SPM, bla IMP, bla VIM, and bla OXA-48 genes using multiplex polymerase chain reaction. Thereafter, using two isolates (97 and 102), the role of double and triple combinational drug therapy as a treatment option was analyzed. RESULTS: Seventy-four (50.3%) isolates were positive for bla NDM, eight (5.4%) for bla KPC, and one (1.2%) for both bla NDM and bla KPC. In the synergy tests, double combinations were better than triple combinations. Polymyxin B and amikacin for isolate 97 and polymyxin B coupled with meropenem for isolate 102 showed the best response. CONCLUSIONS: Clinicians in normal practice use multiple drugs to treat infections caused by multi-resistant microorganism; however, in most cases, the benefit of the combinations is unknown. In vitro synergistic tests, such as those described herein, are important as they might help select an appropriate multi-drug antibiotic therapy and a correct dosage, ultimately reducing toxicities and the development of antibiotic resistance.

Identification of plasmid IncQ1 and NTEKPC-IId harboring bla KPC-2 in isolates from Klebsiella pneumoniae infections in patients from Recife-PE, Brazil

Abstract INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.

Successful medical drainage and surgical treatment for vertebral osteomyelitis and bilateral psoas abscess with gas formation caused by klebsiella pneumoniae in a diabetic patient

SUMMARY OBJECTIVE: We describe the case of a diabetic patient who developed vertebral osteomyelitis and bilateral psoas abscess with gas formation due to klebsiella pneumoniae. METHODS: A 64-year-old woman with a 4-year history of type-2 diabetes mellitus was admitted to the Emergency Department. The subject had a 2-day history of high-grade fever associated with chills and a 5-hour history of consciousness. She received empirical treatment with febrifuge, after which her fever decreased. RESULTS: Her fever recurred after an interval of three hours. A computed tomography scan of the abdomen revealed vertebral osteomyelitis and bilateral psoas muscle abscess with gas formation. Blood culture and purulent fluid described the growth of the Klebsiella pneumoniae. The patient received antibiotic therapy and bilateral drainage therapy after the drainage catheter was placed into the abscess cavity by CT-guidance. Due to the serious damage to the vertebral column and permanent pain, the patient underwent minimally invasive internal spinal fixation and recovered successfully. CONCLUSION: A case of vertebral osteomyelitis and bilateral psoas abscess with gas formation caused by Klebsiella pneumoniae in a diabetic patient. Antibiotic therapy, drainage, and minimally invasive internal spinal fixation were performed, which enabled a good outcome.

RESUMO OBJETIVO: Descrever o caso de uma paciente diabética que desenvolveu osteomielite vertebral e abcesso bilateral do psoas com formação de gás causada por klebsiella pneumoniae. MÉTODOS: Uma mulher de 64 anos de idade, com 4 anos de histórico de diabetes mellitus tipo 2, foi admitida no Serviço de Emergência. A paciente apresentava um quadro de dias de febre alta acompanhada de calafrios e um histórico de 5 horas de consciência. Ela recebeu tratamento empírico com antitérmico, após o qual a febre diminuiu. RESULTADOS: A febre retornou após um intervalo de três horas. Uma tomografia computadorizada do abdome revelou osteomielite vertebral e abcesso bilateral do músculo psoas com formação de gás. A cultura do sangue e o fluido purulento revelaram o crescimento de Klebsiella pneumoniae. A paciente recebeu antibióticos e terapia de drenagem bilateral após o cateter de drenagem ser posicionado na cavidade do abscesso com auxílio de TC. Devido a sérios danos à coluna vertebral e a dor permanente, a paciente foi submetida à fixação vertebral interna minimamente invasiva e recuperou-se com sucesso. CONCLUSÃO: Um caso de osteomielite vertebral e abscesso do psoas bilateral com a formação de gás causada por Klebsiella pneumoniae em uma paciente diabética. Antibioticoterapia, drenagem e fixação vertebral interna minimamente invasiva foram realizadas, o que permitiu um bom resultado.

Características clínico-epidemiológicas y patrones de prescripción para quemaduras en tres hospitales de Lima, Perú / Clinical-epidemiological characteristics and prescribing patterns for burns in three hospitals in Lima, Peru

RESUMEN Con el objetivo de describir las características clínico-epidemiológicas y los patrones de prescripción médica de pacientes con quemaduras de primer y segundo grado que acudieron a tres hospitales de referencia de Lima, se realizó un estudio transversal donde se recogieron datos demográficos, antecedentes médicos, evaluación clínica y tratamiento recibido en 561 participantes. El uso de antibióticos y de agentes humectantes se dio en 64,7% y 4,2% en los centros de atención inmediata; y en 41,7% y 44,7% en los servicios de atención especializada en quemaduras. La sulfadiazina argéntica fue el antibiótico tópico más utilizado en los servicios de atención inmediata, en comparación con los servicios de quemados (80,2% vs 34,5%). El manejo de quemaduras fue más exhaustivo en los servicios de quemados que en los de atención inmediata. Asimismo, más de un cuarto de los pacientes que acudieron por emergencia lo hicieron luego de 24 horas de ocurrida la quemadura.

ABSTRACT In order to describe the clinical-epidemiological characteristics and medical prescription patterns of patients with first- and second-degree burns who visited three reference hospitals in Lima, a cross-sectional study was carried out to collect data on demographics, medical history, clinical evaluation, and treatment received by 561 participants. The use of antibiotics and moisturizing agents was 64.7% and 4.2% in immediate care centers; and 41.7% and 44.7% in specialized burn-care services. Argenic sulfadiazine was the most commonly used topical antibiotic in immediate care services compared to burned units (80.2% vs. 34.5%). Burn management was more comprehensive in burn services than in immediate care. Also, more than a quarter of the patients who sought emergency care did so within 24 hours of the burn.

Economic burden of inpatients infected with Klebsiella pneumoniae carbapenemase / Impacto econômico do tratamento de pacientes infectados com Klebsiella pneumoniae carbapenemase

ABSTRACT Objective: To estimate the direct medical costs of drug therapy of Klebsiella pneumoniae carbapenemase (KPC) infection patients in hospital-based context. Methods: A cost-of-illness study conducted with a prospective cohort design with hospitalized adults infected by KPC. Data collection was performed using an instrument composed of sociodemographic data, clinical and prescription medication. Estimates of the direct costs associated to each treatment were derived from the payer's perspective, in the case of federal public hospitals from Brazil, and included only drug costs. These costs were based on the average price available at the Brazilian Price Database Health. No discount rate was used for the cost of drugs. The costs are calculate in American Dollar (US$). Results: A total of 120 inpatients participated of this study. The total drug cost of these inpatients was US$ 367,680.85. The systemic antimicrobial group was responsible for 59.5% of total costs. The direct drug cost per patients infected by KPC was conservatively estimated at nearly US$ 4,100.00, and about of 60% of costs occurred during the period of infection. Conclusion: The findings of our study indicate a thoughtful economic hazard posed by KPC that all healthcare sectors have to face. The increasing worldwide incidence of these bacteria represents a growing burden that most health systems are unable to deal with. There is an imperative need to develop protocols and new antimicrobials to treatment of KPC, aiming to rearrange resources to increase the effectiveness of healthcare services.

RESUMO Objetivo: Estimar os custos médicos diretos da terapia medicamentosa de pacientes com infecção por carbapenemase por Klebsiella pneumoniae carbapenemase (KPC) em contexto hospitalar. Métodos: Estudo de custo de doença realizado com desenho de coorte prospectiva, com adultos hospitalizados infectados por KPC. A coleta de dados foi realizada usando instrumento composto por dados sociodemográficos, medicamentos clínicos e prescritos. As estimativas dos custos diretos associados a cada tratamento foram derivadas da perspectiva dos pagadores, no caso dos hospitais públicos federais do Brasil, e incluíram apenas custos de medicamentos, os quais basearam-se no preço médio disponível na Price Database Health do Brasil. Nenhuma taxa de desconto foi utilizada para o custo dos medicamentos. Os custos foram calculados em dólares norte-americanos (US$). Resultados: Um total de 120 pacientes hospitalizados participou do estudo. O custo total da droga desses pacientes internados foi de US$ 367,680.85. O grupo antimicrobianos de uso sistêmico foi responsável por 59,5% dos custos totais. O custo direto estimado de forma conservadora, por paciente, foi de aproximadamente US$ 4,100.00, e cerca de 60% destes se deram durante o período de infecção. Conclusão: Os achados deste estudo apontam um risco econômico importante relacionado a KPC, o qual todos os setores de saúde terão que enfrentar. A incidência mundial em elevação destas bactérias representa carga crescente, e a maioria dos sistemas de saúde é incapaz de resolvê-la. Há necessidade imperativa de se desenvolverem protocolos e novos antimicrobianos para o tratamento de KPC, com o objetivo de reorganizar os recursos para aumentar a efetividade dos serviços de saúde.

Performance of "RESIST-3 O.K.N. K-SeT" immunochromatographic assay for the detection of OXA-48 like, KPC, and NDM carbapenemases in Klebsiella pneumoniae in Turkey

ABSTRACT In this study, the performance of the "RESIST-3 O.K.N. K-SeT" (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria (n = 22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for bla KPC, bla IMP, bla VIM, bla NDM, and bla OXA-48. The rates of bla NDM, bla OXA-48, and bla KPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both bla NDM and bla OXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132 K. pneumoniae. The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae carrying bla KPC, bla NDM, and/or bla OXA-48. On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring bla IMP and bla VIM. We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.

Klebsiella variicola and Klebsiella quasipneumoniae with capacity to adapt to clinical and plant settings / Klebsiella variicola y Klebsiella quasipneumoniae con capacidad para adaptarse al ambiente clínico y a las plantas

Abstract: Objective: To compare the genetic determinants involved in plant colonization or virulence in the reported genomes of K. variicola, K. quasipneumoniae and K. pneumoniae. Materials and methods: In silico comparisons and Jaccard analysis of genomic data were used. Fimbrial genes were detected by PCR. Biological assays were performed with plant and clinical isolates. Results: Plant colonization genes such as cellulases, catalases and hemagglutinins were mainly present in K. variicola genomes. Chromosomal β-lactamases were characteristic of this species and had been previously misclassified. K. variicola and K. pneumoniae isolates produced plant hormones. Conclusions: A mosaic distribution of different virulence- and plant-associated genes was found in K. variicola and in K. quasipneumoniae genomes. Some plant colonizing genes were found mainly in K. variicola genomes. The term plantanosis is proposed for plant-borne human infections.

Resumen: Objetivo: Comparar genes de colonización de plantas o de virulencia en los genomas reportados de K. variicola, K. quasipneumoniae y K. pneumoniae. Material y métodos: Se utilizaron análisis in silico y de Jaccard. Por PCR se detectaron genes de fimbrias. Se realizaron ensayos biológicos con aislados de plantas y clínicos. Resultados: Los genes de colonización de plantas como celulasas, catalasas y hemaglutininas se encontraron principalmente en genomas de K. variicola. Las β-lactamasas cromosómicas son características de la especie y en algunos casos estaban mal clasificadas. K. variicola y K. pneumoniae producen hormonas vegetales. Conclusiones: Se encontró una distribución en mosaico de los genes de asociación con plantas y de virulencia en K. variicola y K. quasipneumoniae. Principalmente en K. variicola se encontraron algunos genes involucrados en la colonización de plantas. Se propone el término plantanosis para las infecciones humanas de origen vegetal.

Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans / Clasificación errónea de aislados de Klebsiella variicola y Klebsiella quasipneumoniae obtenidos de plantas, animales y humanos

Abstract: Objective: Due to the fact that K. variicola, K. quasipneumoniae and K. pneumoniae are closely related bacterial species, misclassification can occur due to mistakes either in normal biochemical tests or during submission to public databases. The objective of this work was to identify K. variicola and K. quasipneumoniae genomes misclassified in GenBank database. Materials and methods: Both rpoB phylogenies and average nucleotide identity (ANI) were used to identify a significant number of misclassified Klebsiella spp. genomes. Results: Here we report an update of K. variicola and K. Quasipneumoniae genomes correctly classified and a list of isolated genomes obtained from humans, plants, animals and insects, described originally as K. pneumoniae or K. variicola, but known now to be misclassified. Conclusions: This work contributes to recognize the extensive presence of K. variicola and K. quasipneumoniae isolates in diverse sites and samples.

Resumen: Objetivo: Identificar genomas mal clasificados de K. variicola, y K. quasipneumoniae en la base de datos del GenBank. Material y métodos: En el presente estudio se usaron tanto análisis filogenéticos usando rpoB como la identidad media de nucleótidos (ANI, por sus siglas en ingles) para identificar un número significativo de genomas del género Klebsiella. Resultados: Se reportó una actualización de genomas de K. variicola y K. quasipneumoniae correctamente clasificados y una lista de aquellos aislamientos obtenidos de seres humanos, plantas, animales e insectos, descritos originalmente como K. pneumoniae o K. variicola pero ahora se conoce que están mal clasificados. Conclusiones: Este trabajo contribuye a la presencia extensiva de aislamientos de K. variicola y K. quasipneumoniae en diversos sitios y muestras.

Characterization of Klebsiella pneumoniae associated with cattle infections in southwest China using multi-locus sequence typing (MLST), antibiotic resistance and virulence-associated gene profile analysis

Abstract Klebsiella pneumoniae is important human and animal pathogen that causes a wide spectrum of infections. In this study, isolates from cattle nasal swabs samples were identified by 16S rRNA, and to evaluate the antimicrobial susceptibility, virulence gene carrying levels, and multilocus sequence typing of K. pneumoniae isolates. 33 isolates of K. pneumoniae were isolated and identified in 213 nasal swabs samples, of which 12 were hypervirulent K. pneumoniae strains. Extended Spectrum Beta-Lactamases genes were found in 93.4% of the strains. Of which, TEM was the most prevalent (93.4%), followed by CTX-M and SHV were 57.6% and 39.4%, respectively. A main mutation pattern of quinoloneresistance-determining region, Thr83-Ieu and Asp87-Asn in gyrA and Ser87-Ile in parC, was detected in 33 K. pneumoniae isolates. All the isolates harbored at least two virulence factor genes, with ureA (97.0%) and wabG (91.0%) exhibiting high carriage rates in 33 K. pneumoniae isolates. MLST revealed 7 sequence types, of which 3 STs (2541, 2581 and 2844) were newly assigned. Using eBURST, ST2844 and ST2541 were assigned to new clonal complex 2844. Our study provides evidence and biological characteristics of K. pneumoniae isolates from cattle upper respiratory tract in Southwest China.

Factores predictores de mortalidad intrahospitalaria en pacientes adultos con infecciones por Klebsiella pneumoniae resistente a carbapenémicos y colistín: un estudio de cohorte retrospectivo / Risk factors for in-hospital mortality among adult patients infected with colistin-resistant carbapenemase producing Klebsiella pneumoniae: a retrospective cohort study

Resumen Introducción: La emergencia de Klebsiella productora de carbapenemasas resistente a colistín representa un desafío clínico y un problema emergente. Objetivo: Evaluar la mortalidad intrahospitalaria y sus potenciales factores de riesgo en pacientes internados con infecciones clínicas por Klebsiella pneumoniae productora de carbapenemasas (KPC) resistente a colistín. Material y Método: Realizamos un estudio de cohorte retrospectivo, incluyendo pacientes adultos admitidos a un hospital universitario de tercer nivel en Buenos Aires, infectados por KPC resistente a colistín. El evento primario considerado fue la mortalidad intrahospitalaria. Se utilizaron modelos generalizados lineales para evaluar potenciales predictores de dicho evento. Resultados: En total, se identificaron 18 pacientes hospitalizados que presentaron una infección clínica por esta bacteria durante el año 2016 y que fueron incluidos en el análisis final. La mortalidad intrahospitalaria en esta cohorte fue de 38,9%. La presencia de bacteriemia, la injuria renal aguda al momento del diagnóstico y la presencia de shock séptico se asociaron a la ocurrencia del evento primario. Conclusión: El desarrollo de infecciones clínicamente relevantes por KPC resistente a colistín en pacientes internados es frecuente y presenta una elevada mortalidad. En nuestra cohorte, la presencia de shock e injuria renal aguda al momento del diagnóstico se asociaron a un incrementado riesgo de mortalidad intrahospitalaria. Futuras investigaciones deberían corroborar estos hallazgos e investigar factores adicionales que permitan identificar tempranamente a aquellos pacientes que presentarán eventos desfavorables.

ABSTRACT Background: The emergence of colistin resistant carbapenemase-producing Klebsiella represents a therapeutic challenge and a worldwide problem. Aim: To estimate the in-hospital mortality and identify the associated risk factors among patients with colistin-resistant carbapenemase-producing Klebsiella pneumoniae (KPC) that present with a clinical infection. Methods: We carried a retrospective cohort study, including adult patients infected with colistin-resistant KPC hospitalized at a tertiary teaching hospital in Buenos Aires, Argentina during the year 2016. The main outcome was in-hospital mortality. We used generalized lineal models to evaluate potential predictors of mortality. Results: 18 patients that developed a colistin-resistant KPC clinical infection were identified and included in the final analysis. In-hospital mortality in this cohort was 38.9%. The presence of bacteremia, acute renal injury at the time of diagnosis and septic shock were associated with the main outcome. Conclusions: Infections due to colistin-resistant KPC among in-hospital patients was frequent and was associated with high mortality rate. In our cohort, both shock and acute kidney injury were associated with a higher likelihood of poor outcomes. Further studies are warranted to evaluate the role of these and others risk factors so as to aid in the early detection of high risk patients.

Klebsiella pneumoniae productora de carbapenemasas en Perú: reporte de caso y discusión de la resistencia a los antimicrobianos / Carbapenemase producing Klebsiella pneumoniae in Peru: a case report and antimicrobial resistance discussion

Las carbapenemasas son uno de los mecanismos enzimáticos de resistencia antimicrobiana, que compromete la mayor parte de los antibióticos betalactámicos. Por lo general, su producción se debe al uso indiscriminado de antimicrobianos. A nivel mundial, la expansión de este mecanismo de resistencia es inminente y las medidas de control son limitadas. Con el objeto de discutir los problemas relacionados a este mecanismo emergente de resistencia, reportamos un caso de Klebsiella pneumoniae productora de carbapenemasas en Huancayo, la Región de la Sierra Central de Perú.

Carbapenemases are one of the major mechanisms of antimicrobial resistance, usually due to the indiscriminate use of antibiotics. The expansion of this mechanism of resistance at world level is imminent and control measures are limited. In the region of the Central Sierra of Peru - Huancayo, we report a case of carbapenemase-producing Klebsiella pneumoniae, with the purpose of discussing the problems related to this emerging mechanism of antibiotic resistance.

Antimicrobial activity evaluation and comparison of methods of susceptibility for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter spp. isolates

Abstract The production of KPC (Klebsiella pneumoniae carbapenemase) is the major mechanism of resistance to carbapenem agents in enterobacterias. In this context, forty KPC-producing Enterobacter spp. clinical isolates were studied. It was evaluated the activity of antimicrobial agents: polymyxin B, tigecycline, ertapenem, imipenem and meropenem, and was performed a comparison of the methodologies used to determine the susceptibility: broth microdilution, Etest® (bioMérieux), Vitek 2® automated system (bioMérieux) and disc diffusion. It was calculated the minimum inhibitory concentration (MIC) for each antimicrobial and polymyxin B showed the lowest concentrations for broth microdilution. Errors also were calculated among the techniques, tigecycline and ertapenem were the antibiotics with the largest and the lower number of discrepancies, respectively. Moreover, Vitek 2® automated system was the method most similar compared to the broth microdilution. Therefore, is important to evaluate the performance of new methods in comparison to the reference method, broth microdilution.

Detection and analysis of different interactions between resistance mechanisms and carbapenems in clinical isolates of Klebsiella pneumoniae

Abstract Carbapenems are considered last-line agents for the treatment of serious infections caused by Klebsiella pneumoniae, and this microorganism may exhibit resistance to β-lactam antibiotics due to different mechanisms of resistance. We evaluated 27 isolates of K. pneumoniae resistant to carbapenems recovered from inpatients at the University Hospital of Santa Maria-RS from July 2013 to August 2014. We carried out antimicrobial susceptibility, carbapenemase detection, testing for the presence of efflux pump by broth microdilution and loss of porin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Genetic similarity was evaluated by ERIC-PCR. High levels of resistance were verified by the minimum inhibitory concentration for the antimicrobials tested. The blaKPC gene was present in 89% of the clinical isolates. Blue-Carba and combined disk with AFB tests showed 100% concordance, while the combined disk test with EDTA showed a high number of false-positives (48%) compared with the gold-standard genotypic test. Four isolates showed a phenotypic resistance profile consistent with the overexpression of the efflux pump, and all clinical isolates had lost one or both porins. The ERIC-PCR dendrogram demonstrated the presence of nine clusters. The main mechanism of resistance to carbapenems found in the assessed isolates was the presence of the blaKPC gene.

Molecular detection of ß-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction

Abstract INTRODUCTION: Infections caused by β-lactamase-producing gram-negative bacteria, such as Klebsiella pneumoniae, are increasing globally with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the prevalence of antibiotic resistance genes (β-lactamase and integron genes) using multiplex PCR. METHODS One-hundred K. pneumoniae isolates were collected from different clinical samples. Antibiotic susceptibility testing was performed with thirteen different antibiotics. Multiplex-PCR was used to detect β-lactamase (bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC) and integron genes (int I, int II, and int III). RESULTS: The highest and lowest rate of resistance was exhibited against amikacin (93%) and imipenem (8%), respectively. The frequency of β-lactamase-positive K. pneumoniae was 37%, and the prevalence of the bla TEM, bla CTX-M, bla SHV , bla VEB, bla PER, bla GES, bla VIM, bla IMP, bla OXA, and bla KPC genes was 38%, 24%, 19%, 12%, 6%, 11%, 33%, 0%, 28%, and 23%, respectively. Of the 100 isolates, eight (8%) were positive for class I integrons; however, class II and III integrons were not detected in any of the strains. CONCLUSIONS: These results indicate co-carriage of a number of β-lactamase genes and antibiotic resistance integrons on the same plasmids harboring multi-drug resistance genes. It seems that these properties help to decrease treatment complications due to resistant bacterial infections by rapid detection, infection-control programs and prevention of transmission of drug resistance.

Epidemiología molecular de aislados clínicos de Klebsiella pneumoniae productores de carbapenemasas tipo KPC provenientes de dos hospitales públicos en los estados Carabobo y Zulia, Venezuela / Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolated from patients in two public hospitals in Carabobo and Zulia states, Venezuela

Klebsiella pneumoniae productora de carbapenemasas tipo KPC es uno de los principales agentes causante de infecciones nosocomiales a nivel mundial. En Venezuela se han identificado aislados de esta bacteria, sin embargo, se conoce poco sobre su dispersión. El objetivo de este estudio fue realizar la epidemiología molecular de aislados de K. pneumoniae productores de KPC provenientes de dos hospitales públicos ubicados en los estados Carabobo y Zulia. Se seleccionaron 32 aislados de K. pneumoniae clasificados fenotípicamente como productores de KPC, se les realizó la detección del gen bla KPC así como su ubicación en el transposón Tn4401 a través de PCR. El producto de PCR del gen blaKPC se secuenció para identificar los alelos circulantes. El análisis genotípico se realizó empleando las técnicas de amplificación por PCR de las secuencias repetidas extragénicas palindrómicas (rep-PCR) y la secuenciación de múltiples loci (MLST). Mediante ensayos de conjugación, se determinó si los genes bla KPC se encontraban en moléculas plasmídicas.Los resultados indican que los 32 aislados contenían la variante del gen bla KPC-2 asociada a la isoforma Tn4401 b y se distribuyeron en 9 secuencias tipo (ST), siendo una de ellas nueva. Los ensayos de conjugación indican que el 87,5% de los aislados tienen al gen bla KPC en plásmidos movilizables. En estos hospitales el gen bla KPC-2 se está dispersando a través de plásmidos que llevan al transposón Tn4401 b. Las ST más comunes pertenecen a los Complejos Clonales CC258 y CC147, que desempeñan un papel importante en la dispersión de la resistencia a carbapenemes a nivel mundial.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria ( K. pneumoniae carbapenemase ) are the most important causative agents of nosocomial infections worldwide. These isolates have been identified in Venezuela, but little is known about their local spread. The aim of this study was to perform molecular epidemiology of KPC-producing K. pneumoniae isolated from two public hospitals in the Carabobo and Zulia states of Venezuela. Thirty-two K. pneumoniaei solates, phenotypically classified as KPC producers were subjected to PCR to detect the presence of bla KPC genes and their location within transposon Tn4401 , and the bla KPC product was sequenced to identify the KPC allele. Genotypic analysis was performed using repeated extragenic palindromic PCR (rep-PCR) and Multi Locus Sequence Typing (MLST). Finally, a conjugation assay determined whether the bla KPC genes were carried on transferable plasmids. The results indicate that the 32 isolates contained the bla KPC-2 variant associated with isoform Tn4401 b, and were distributed in nine sequence types (ST), one of which was new. Conjugation assays indicate that 87.5% of the isolates contain the gene bla KPC on mobilizable plasmids. In these hospitals, the bla KPC-2 gene is spreading through the plasmids carrying the transposon Tn4401 b. The most common ST belongs to Clonal Complexes CC258 and CC147, which play an important role in the dispersion of resistance to carbapenems worldwide.