Presencia de genes fimH y afa en aislamientos urinarios de Escherichia coli productora de betalactamasas de espectro extendido en Lima, Perú / Presence of fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases producing Escherichia coli in Lima, Peru

RESUMEN Con el objetivo de determinar la presencia de los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de betalactamasas de espectro extendido (BLEE), se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño en Lima, Perú. Se incluyeron 75 aislamientos urinarios de Escherichia coli. La identificación de genes se realizó por reacción en cadena de la polimerasa. De los 75 aislamientos, 74 (98,7%) fueron positivos para el gen fimH y 6 (8,0%) fueron positivos para el gen afa. Se evidenció la presencia de los factores de virulencia producidos por los genes fimH y afa en aislamientos urinarios de Escherichia coli productoras de BLEE.

ABSTRACT Descriptive study conducted in order to determine the presence of the fimH and afa genes in urinary isolates of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli. Isolates from project TO-06/09 of the Instituto Nacional de Salud del Niño in Lima, Peru were used. A total of 75 urinary isolates of Escherichia coli were included. Gene identification was performed by polymerase chain reaction. From the 75 isolates, 74 (98.7%) were positive for the fimH gene and 6 (8.0%) were positive for the afa gene. Virulence factors produced by the fimH and afa genes were evident in urinary isolates of ESBL producing Escherichia coli.

Caracterización molecular y detección de genes blaCTX-M grupos 1 y 9 en Klebsiella pneumoniae resistentes a ceftazidima, en un hospital de San José de Cúcuta, Colombia / Molecular characterization and detection of genes blaCTX-M groups 1 and 9 in Klebsiella pneumoniae resistant to ceftazidime, in a hospital in San José de Cúcuta, Colombia

Resumen Introducción: La expresión de β-lactamasas CTX-M pertenecientes a los grupos 1 y 9 en Klebsiella pneumoniae produce grados altos de resistencia a ceftazidima, y presentan una amplia distribución mundial. Objetivo: Identificar y caracterizar los genes blaCTX-M-Grupo1 y blaCTX-M-Grupo9 en aislados de K. pneumoniae resistentes a ceftazidima en un hospital de San José de Cúcuta, Colombia. Material y Método: Se diseñaron partidores para la identificación de K. pneumoniae y los genes blaCTX-M mediante reacción de polimerasa en cadena (RPC). Posteriormente se realizó el análisis de la relación genética de estos aislados por medio de la RPC basada en secuencias repetitivas (RPC-REP). Resultados: Treinta y ocho por ciento de los 24 aislados identificados por RPC como K. pneumoniae presentaron los genes blaCTX-M-3, blaCTX-M-15 y blaCTX-M-32 (Grupo CTX-M-1) y 42% los genes blaCTX-M14, blaCTX-M-24 y blaCTX-M-27 (Grupo CTX-M-9). El análisis filogenético agrupó los aislados de K. pneumoniae en cuatro clusters, mostrando correlación en los clusters I, II y IV, al comparar los perfiles genéticos con el tipo de muestra y grupo de genes. Discusión: Se encontró una frecuencia similar de los genes blaCTX-M-Grupo1 y blaCTX-M-Grupo 9 en aislados de K. pneumoniae resistentes a ceftazidima. La correlación entre la RPC-REP con los grupos de CTX-M y el tipo de muestra reveló la presencia de tres patrones clonales.

Background: The expression of CTX-M β-lactamases belonging to groups 1 and 9 in Klebsiella pneumoniae produces high levels of resistance to ceftazidime, and they have a wide distribution worldwide. Aim: To identify and characterize the blaCTX-M-Group1 and blaCTX-M-Group9 genes in K. pneumoniae isolates resistant to ceftazidime in a hospital in San José de Cúcuta, Colombia. Material and Methods: Primers were designed for the identification of K. pneumoniae and blaCTX-M genes by PCR. Subsequently, the genetic relationship of these isolates was analyzed by REP-PCR. Results: A 38% of the 24 isolates identified by PCR as K. pneumoniae showed blaCTX-M-3. blaCTX-M-15 y blaCTX-M-32 genes (Group CTX-M-1) and 42% blaCTX-M14. blaCTX-M-24 y blaCTX-M-27 genes (Group CTX-M-9). The phylogenetic analysis grouped the K. pneumoniae isolates into 4 clusters, showing correlation in clusters I, II and IV, when comparing the genetic profiles with the type of sample and group of genes. Discussion: We found a similar frequency of blaCTX-M-Group 1 and blaCTX-M-Group 9 genes in isolates of K. pneumoniae resistant to ceftazidime. The correlation between the REP-PCR with the CTX-M groups and the type of sample revealed the presence of three clonal patterns.

High prevalence of CTX-M-1 group in ESBL-producing enterobacteriaceae infection in intensive care units in southern Chile

ABSTRACT Enterobacteria-producing extended-spectrum β-lactamases (ESBL) play an important role in healthcare infections, increasing hospitalization time, morbidity and mortality rates. Among several ESBLs that emerge from these pathogens, CTX-M-type enzymes had the most successful global spread in different epidemiological settings. Latin America presents high prevalence of CTX-M-2 in ESBL-producing enterobacterial infections with local emergence of the CTX-M-1 group. However, this high prevalence of the CTX-M-1 group has not yet been reported in Chile. The aim of this study was to identify ESBLs among enterobacteria isolated from clinical samples of critically ill patients from southern Chile. One-hundred thirty seven ESBL-producing bacteria were isolated from outpatients from all critical patient units from Hernán Henríquez Aravena Hospital. Phenotype characterization was performed by antibiogram, screening of ESBL, and determination of minimum inhibitory concentration (MIC). PCR was used for genetic confirmation of resistance. Molecular typing was performed by ERIC-PCR. ESBL-producing isolates were identified as Klebsiella pneumoniae (n = 115), Escherichia coli (n = 18), Proteus mirabilis (n = 3), and Enterobacter cloacae (n = 1), presenting multidrug resistance profiles. PCR amplification showed that the strains were positive for blaSHV (n = 111/81%), blaCTX-M-1 (n = 116/84.7%), blaTEM (n = 100/73%), blaCTX-M-2 (n = 28/20.4%), blaCTX-M-9 (0.7%), blaPER-1 (0.7%), and blaGES-10 (0.7%). The multiple production of ESBL was observed in 93% of isolates, suggesting high genetic mobility independent of the clonal relationship. The high frequency of the CTX-M-1 group and a high rate of ESBL co-production are changing the epidemiology of the ESBL profile in Chilean intensive care units. This epidemiology is a constant and increasing challenge, not only in Chile, but worldwide.

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

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

Caracterización molecular y patrón de susceptibilidad antimicrobiana de Escherichia coli productora de β -lactamasas de espectro extendido en infección del tracto urinario adquirida en la comunidad / Molecular characterization and antimicrobial susceptibility pattern of extended-spectrum β -lactamase-producing Escherichia coli as cause of community acquired urinary tract infection

Resumen Introducción Las infecciones del tracto urinario adquiridas en la comunidad (ITUac) causadas por cepas de Escherichia coli productoras de β-lactamasas de espectro extendido (BLEE), principalmente por cepas que contienen el gen blaCTX-M-15, es un fenómeno creciente a nivel mundial. Objetivo Determinar el patrón de susceptibilidad a antimicrobianos de cepas de E. coli productoras de BLEE causantes de ITUac y conocer su patrón molecular. Materiales y Métodos Se realizó un estudio descriptivo en Oaxaca, México, donde se incluyeron 288 cepas de E. coli aisladas de pacientes adultos con posible ITUac. Para obtener los patrones de susceptibilidad antimicrobiana se siguieron los criterios del CLSI y para obtener el análisis molecular se utilizó la técnica de RPC. Resultados Del total de cepas de E. coli aisladas, 31,3% fueron productoras de BLEE, presentando una menor susceptibilidad a antimicrobianos que las cepas no productoras de estas enzimas. El 95,6% de las cepas BLEE estudiadas fueron portadoras del gen blaCTX-M. Conclusiones Un tercio de las ITUac causadas por E. coli en nuestra población fueron causadas por cepas BLEE, mostrando un alto nivel de resistencia a los antimicrobianos comúnmente utilizados en su tratamiento y disminuyendo las opciones terapéuticas para tratamientos empíricos en esta población.

Background Community acquired urinary tract infections (CaUTI) caused by strains of extended-spectrum β-lactamases (ESBL) - producing Escherichia coli, mainly by strains carrying the blaCTX-M-15 gene, is a growing phenomenon worldwide. Aim To determine the antibiotic susceptibility pattern of ESBL-producing E. coli as cause of CaUTI and to identify their molecular pattern. Methods A descriptive study was performed in the city of Oaxaca, Mexico, from where 288 strains of CaUTI-producing strains of E. coli in adults with possible UTI were isolated. The CLSI criteria was followed to determine the antimicrobial susceptibility patterns, and their molecular characterization was performed by using PCR. Results 31.3% of E. coli strains isolated in our population were ESBL producers, which presented higher levels of antibiotic resistance than those of non-producers of these enzymes. 95.6% of the studied strains were carriers of the blaCTX-M gene. Conclusions One-third of the Ca-UTI caused by E. coli in our population are caused by ESBL-producing strains, which present high levels of resistance to the antibiotics widely used in our community. This situation considerably decreases the number of antibiotics available for an empiric treatment against these infections.

Incidencia de bacterias multi-resistentes en unidades de cuidados intensivos de hospitales chilenos / Incidence of multi-resistant bacteria in Intensive Care Units of Chilean hospitals

Resumen Introducción: La vigilancia de incidencia de bacterias multi-resistentes es un indicador que permite estimar mejor la magnitud de la resistencia bacteriana en los servicios hospitalarios. Objetivo: Evaluar la incidencia de bacterias multi-resistentes relevantes en unidades de cuidados intensivos del país y establecer las diferencias entre población adulta y pediátrica. Metodología: Se solicitó a los hospitales participantes información del número de aislados de siete bacterias multi-resistentes epidemiológicamente relevantes de unidades de cuidados intensivos (UCI) de adulto y pediátrico entre enero de 2014 y octubre de 2015, y el número de días-cama ocupados en dichas unidades en el mismo período. Con estos datos se calculó incidencia por 1.000 pacientes-día para cada unidad. Resultados: Se recibió información de 20 UCI adultos y 9 UCI pediátricas. En UCI adultos las bacterias de mayor incidencia fueron K. pneumoniae productora de BLEE [4,72 × 1.000 días cama (1,21-13,89)] y S. aureus resistente a oxacilina [3,85 (0,71-12,66)]. En pediatría la incidencia fue menor, destacando K. pneumoniae productora de BLEE [2,71 (0-7,11)] y P. aeruginosa resistente a carbapenémicos [1,61 (0,31-9,25)]. Conclusión: Se observan importantes diferencias entre los distintos hospitales en la incidencia de las bacterias estudiadas. La incidencia de bacterias multi-resistentes en UCI de adultos es significativamente mayor que en UCI pediátrica para la mayoría de las bacterias estudiadas.

Introduction: Incidence of multi-resistant bacteria is an indicator that permits better estimation of the magnitude of bacterial resistance in hospitals. Aim: To evaluate the incidence of relevant multi-drug resistant bacteria in intensive care units (ICUs) of Chile. Methods: Participating hospitals submitted information about the number of isolates from infected or colonized patients with 7 epidemiologically relevant multi-resistant bacteria in adult and pediatric ICUs between January 1, 2014 and October 31, 2015 and the number of bed days occupied in these units in the same period was requested. With these data incidence was calculated per 1,000 patient days for each unit. Results: Information from 20 adults and 9 pediatric ICUs was reviewed. In adult ICUs the bacteria with the highest incidence were K. pneumoniae ESBL [4.72 × 1,000 patient day (1.21-13.89)] and oxacillin -resistant S. aureus [3.85 (0.71-12.66)]. In the pediatric units the incidence was lower, highlighting K. pneumoniae ESBL [2.71 (0-7.11)] and carbapenem -resistant P. aeruginosa [1.61 (0.31-9.25)]. Conclusion: Important differences between hospitals in the incidence of these bacteria were observed. Incidence of multi-resistant bacteria in adult ICU was significantly higher than in pediatric ICU for most of the studied bacterias.

Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM) and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR). Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56%) were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.

Comparison of pulsed-field gel electrophoresis & repetitive sequence-based PCR methods for molecular epidemiological studies of Escherichia coli clinical isolates.

Background & objectives: PFGE, rep-PCR, and MLST are widely used to identify related bacterial isolates and determine epidemiologic associations during outbreaks. This study was performed to compare the ability of repetitive sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) to determine the genetic relationships among Escherichia coli isolates assigned to various sequence types (STs) by two multilocus sequence typing (MLST) schemes. Methods: A total of 41 extended-spectrum β-lactamase- (ESBL-) and/or AmpC β-lactamase-producing E. coli clinical isolates were included in this study. MLST experiments were performed following the Achtman’s MLST scheme and the Whittam’s MLST scheme, respectively. Rep-PCR experiments were performed using the DiversiLab system. PFGE experiments were also performed. Results: A comparison of the two MLST methods demonstrated that these two schemes yielded compatible results. PFGE correctly segregated E. coli isolates belonging to different STs as different types, but did not group E. coli isolates belonging to the same ST in the same group. Rep-PCR accurately grouped E. coli isolates belonging to the same ST together, but this method demonstrated limited ability to discriminate between E. coli isolates belonging to different STs. Interpretation & conclusions: These results suggest that PFGE would be more effective when investigating outbreaks in a limited space, such as a specialty hospital or an intensive care unit, whereas rep-PCR should be used for nationwide or worldwide epidemiology studies.

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.

ß-lactamases produced by amoxicillin-clavulanate-resistant enterobacteria isolated in Buenos Aires, Argentina: A new blaTEMgene / ß-lactamasas producidas por enterobacterias resistentes a amoxicilina-ácido clavulánico aisladas en Buenos Aires, Argentina: un nuevo gen blaTEM

La resistencia a la combinación de ß-lactámico/inhibidor de ß-lactamasa en enterobacterias es un problema creciente que no ha sido estudiado intensamente en Argentina. En el presente trabajo, 54/843 enterobacterias recolectadas en un hospital universitario de la ciudad de Buenos Aires fueron resistentes a ampicilina-sulbactama, pero se mantuvieron sensibles a las cefalosporinas de segunda y tercera generación. Se analizaron los mecanismos enzimáticos presentes en los aislamientos que también fueron resistentes a amoxicilina-ácido clavulánico (AMC) (18/54). La secuenciación reveló dos variantes diferentes de blaTEM-1, donde blaTEM-1b es el alelo más frecuentemente detectado (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis y 1 Raoultella terrigena), seguidos por blaTEM-1a(1 K. pneumoniae). La resistencia a AMC parece estar asociada principalmente con la hiperproducción de TEM-1 (sobre todo en E. coli) o con la coexpresión con ß-lactamasas tipo OXA-2 y/o SHV (K. pneumoniae y P. mirabilis). Se describió una nueva variante de blaTEM(TEM-163) en un aislamiento de E. coli que presentó una CIM frente a AMC de 16/8 µg/ml. La enzima TEM-163 contiene dos sustituciones de aminoácidos respecto de TEM-1, Arg275Gln y His289Leu. Teniendo en cuenta la alta actividad específica observada y la baja IC50 para el ácido clavulánico, el patrón de resistencia de este aislamiento parece obedecer a la hiperproducción de la nueva variante de la ß-lactamasa de amplio espectro, en lugar de vincularse con un comportamiento similar al de una TEM resistente a inhibidores (IRT).

Resistance to ß-lactam/ß-lactamase inhibitors in enterobacteria is a growing problem that has not been intensively studied in Argentina. In the present work, 54/843 enterobacteria collected in a teaching hospital of Buenos Aires city were ampicillin-sulbactam-resistant isolates remaining susceptible to second-and third-generation cephalosporins. The enzymatic mechanisms present in the isolates, which were also amoxicillin-clavulanic acid (AMC)-resistant (18/54) were herein analyzed. Sequencing revealed two different variants of blaTEM-1, being blaTEM-1b the most frequently detected allelle (10 Escherichia coli, 3 Klebsiella pneumoniae, 2 Proteus mirabilis and 1 Raoultella terrigena) followed by blaTEM-1a(1 K. pneumoniae). Amoxicillin-clavulanate resistance seems to be mainly associated with TEM-1 overproduction (mostly in E. coli) or co-expressed with OXA-2-like and/or SHV ß-lactamases (K. pneumoniae and P. mirabilis). A new blaTEMvariant (TEM-163) was described in an E. coli strain having an AMC MIC value of 16/8 µg/ml. TEM-163 contains Arg275Gln and His289Leu amino acid substitutions. On the basis of the high specific activity and low IC50 for clavulanic acid observed, the resistance pattern seems to be due to overproduction of the new variant of broad spectrumß-lactamase rather than to an inhibitor-resistant TEM (IRT)-like behavior.

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis / Detecção de beta-lactamase em Staphylococcus aureus e Staphylococcus coagulase negativa isolados de mastite bovina

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Os objetivos do presente estudo foram avaliar a presença/produção de beta-lactamases por ambos os métodos fenotípicos e genotípicos, verificar se os resultados são dependentes do tipo de bactéria (Staphylococcus aureus contra Staphylococcus coagulase negativa - CNS) e verificar a concordância entre os testes. Um total de 200 amostras bactérianas oriundas de 21 rebanhos distintos foram incluídos, sendo 100 CNS e 100 S. aureus. A presença/detecção de beta-lactamase foi realizada por diferentes testes (PCR, teste trevo (clover leaf test) - CLT, disco Nitrocefin e resistência in vitro à penicilina). Os resultados de todos os testes não foram dependentes do tipo de bactérias (CNS ou S. aureus). Vários isolados de S. aureus produtores de beta-lactamase eram de um mesmo rebanho. Testes fenotípicos excluindo resistência in vitro à penicilina mostraram uma forte associação medida pelo coeficiente kappa para ambas as espécies de bactérias. Nitrocefina e CLT são testes mais confiáveis para detectar a produção de beta-lactamase em estafilococos.

Detection of FUS-1 (OXA-85), a Class D Betalactamase from Fusobacterium nucleatum Subspecies Polymorphum in Nigeria.

Aims: Beta-lactamase production and subsequent resistance to β-lactam drugs has been a global concern in the treatment of Gram negative anaerobes. The aim of this study was to identify F. nucleatum strains producing Class D β-lactamase through the detection of FUS-1 (OXA-85) resistance gene. Place and Duration of Study: Department of Preventive Dentistry, Lagos University Teaching Hospital, Idi-Araba, between February 2010 and November 2010. Methodology: Twenty two oral clinical samples were obtained from patients with chronic periodontitis who admitted to previous use of amoxicillin. Antibacterial susceptibility of the bacterial isolates was determined by E-test on Brucella Blood agar. Amplification of the bacterial DNA was carried out by PCR using F. nucleatum species-specific primer, FUS-1 specific for blaFUS-1 and strain-specific primers for subspecies nucleatum,, fusiforme, polymorphum and vincentii. Results: From the 19 samples collected, F. nucleatum was isolated, and the identity of the isolates was confirmed by PCR. Four of the isolates produced similar bands with the control strain, 3 (15.7%) strains were able to produce amplication with FUS-1 primer specific for blaFUS-1 gene found in β-lactamase producing F. nucleatum subsp. polymorphum. Conclusion: This study shows the presence of class D β-lactamase producing F. nucleatum species in Nigeria.

Metalo-ß-lactamasas en aislamientos clínicos de pseudomonas aeruginosa en Lima, Perú / Metalo-ß-lactamases in clinical isolates of pseudomonas aeruginosa in Lima, Peru

Con el objetivo de detectar y caracterizar molecularmente las metalo-ß-lactamasas (MßL) en aislamientos clínicos de Pseudomonas aeruginosa, se realizó un estudio trasversal en seis hospitales de referencia de Lima (Perú) en agosto de 2011. Se evaluó 51 aislamientos de P. aeruginosa, resistentes a ceftazidima y con sensibilidad reducida a carbapenémicos. El ensayo fenotípico se realizó con el método de aproximación de discos con sustratos (ceftazidima, imipenem y meropenem) y con ácido etilendiaminotetraacético (EDTA). La detección de genes MßL se realizó mediante la técnica de reacción en cadena de polimerasa multiplex. A través del método fenotípico se detectaron MßL en el 15,7% de los aislamientos, en todos ellos la detección de genes mostró la presencia del gen blaIMP. La descripción del primer reporte de MßL en aislamientos de P. aeruginosa en el Perú debería alertar a los equipos de vigilancia epidemiológica intrahospitalaria para promover su control y prevenir su diseminación.

The aim of this study was to detect and characterize molecularly metallo-ß-lactamase (MßL) in clinical isolates of Pseudomonas aeruginosa. We carry out a cross sectional study in six publics hospital in Lima on August 2011. 51 isolates of P. aeruginosa resistant to ceftazidime and reduced susceptibility to carbapenemes were evaluated.The phenotypic assay was performed using the approximation method with substrate disks (ceftazidime, imipenem and meropenem) and ethylenediaminetetraacetic acid (EDTA). MßL gene detection was performed using the technique of polymerase chain reaction (PCR) multiplex. Through MßL detected phenotypic method in 15.7% of isolates. Detection of genes revealed the presence of the gene in the 8 isolates blaIMP. The first report of MßL in P. aeruginosa in Peru was described, this should alert the monitoring equipment in the institutions to promote control their spread.

Evaluation of different methods for the detection of KPC b-lactamase in Klebsiella pneumoniae and escherichia coli

The worldwide increase in the occurrence and dissemination of Klebsiella pneumoniae carbapenemase [KPC] among Gram-negative pathogens makes critical the early detection of these enzymes. The aim of this study is the evaluation of the most appropriate method for the detection and identification of KPC producing Klebsiella pneumonia and Escherichia coli. Modified Hodge test [MHT], Boronic acid lest [BAT] and polymerase chain reaction [PCR] were evaluated for the detection of KPC-possessing isolates. A total of 60 urine samples and 60 sputum samples were collected from 79 patients in Chest Intensive Care Unit and Trauma intensive Care Unit in Assiut University Hospital. Sixty four isolates [26 Klebsiella pneumonia and 38 E. coil] were identified by conventional methods. The antibiotic susceptibility testing of isolates showed that 19% were resistant to meropenem, 17% were resistant to imipenem and 95% were resistant to ertapenem. Thirty seven isolates were MHT positive while twenty three only of them were Boronic acid and PCR positive. The results indicate that the MHT lack sensitivity while Boronic acid test is an excellent inhibitor of KPCs. PCR is the standard method for the detection of the KPCs but it's highly expensive

Detection of bla SHV and bla CTX-M genes in ESBL producing Klebsiella pneumoniae isolated from Egyptian patients with suspected nosocomial infections

The correct identification of the genes involved in ESBL mediated resistance is necessary for the surveillance and epidemiological studies of their transmission in hospitals. The aim of the present study was to find the prevalence of ESBL producing Klebsiella pneumonia among K. pneumonia isolates separated from Egyptian patients with suspected nosocomial infections, to detect their drug resistance pattern and to look for bla SHV and bla CTX-M genes in such organisms. 138 K. pneumonia isolates from Egyptian patients with suspected nosocomial infections were screened for ESBL production by the pattern of antimicrobial susceptibilities. Phenotypic identification for ESBL production was confirmed by double disc synergy test, phenotypic confirmatory double disc test and by MicroScan panel system. bla SHV and bla CTX-M genes in ESBL producing K. pneumonia were detected using multiplex PCR. The prevalence of ESBL producing K. pneumonia was 21% [30/138]. Their pattern of antimicrobial susceptibility showed that 90% was resistant to [Sulphamethoxazole / Trimethoprim], 70% was resistant to [Amoxicillin / Clavulanate], 63.3% was resistant to Cefotaxime and Ceftazidime, 60% was resistant to Amikacin, 46.7% was resistant to Doxycycline, Cefoxitin, Ceftriaxone and Levofloxacin, 40% was resistant to Cefepime, 20% was resistant to Ertapenem and [Sulbactam / Cefoperazone], 13.3% was resistant to [Piperacillin / Tazobactam], 10% was resistant to [Imipenem/Cilastatin] and Gentamycin and 6.7% was resistant to Meropenem and Ciprofloxacin. Among the ESBL producing K. pneumoniae, three out of 30 [10%] and 16 out of 30 [53.3%] were positive for bla SHV and bla CTX-M genes respectively. It could be concluded that ESBL producing isolates of K. pneumonia have been increasingly recognized in the hospital settings in Egypt and are associated with multiple drug resistance. Thus, molecular identification of the genes encoding beta lactamases would be essential for a reliable epidemiological investigation of their transmission in hospitals and antimicrobial resistance

Detection of extended-spectrum beta -lactamases in Klebsiella pneumoniae: comparison of phenotypic characterization methods

Extended-spectrum beta -lactamase producing K. pneumoniae is a serious threat to the patients. This manuscript shows the comparison of phenotypic characterization methods used for ESBL K. pneumoniae and frequency distribution of these isolates in various clinical samples. Eleven different types of pathological samples collected on various time intervals were analyzed. K. pneumoniae were identified with API 20E system [bioMerieux] and initial screening of ESBL K. pneumoniae was performed using the ceftazidime antimicrobial disc. Double-disc synergy test [DDST] and CLSI confirmatory test were compared for the phenotypic detection of ESBL K. pneumoniae. A total number of 214 ESBL producing K. pneumoniae were isolated from various clinical samples. Frequency distribution of ESBL producing K. pneumoniae was found to be highest among blood 117 [54.7%] and urine 46 [21.5%] samples. Data regarding the use of various interventions among these patients showed most common presence of intravenous line 209 [97.7%] and urinary catheters 46 [21.5%]. Comparison of DDST and CLSI confirmatory test showed that the DDST detected 145 [67.8%] isolates while 213 [99.5%] ESBL K. pneumoniae were characterized by CLSI confirmatory test. The use of CLSI confirmatory test is very efficient in the early detection of ESBL K. pneumoniae especially when the facilities for molecular characterization are not available

AmpC β-lactamases in nosocomial isolates of Klebsiella pneumoniae from India.

Background & objectives: AmpC β-lactamases are clinically significant since these confer resistance to cephalosporins in the oxyimino group, 7-α methoxycephalosporins and are not affected by available β-lactamase inhibitors. In this study we looked for both extended spectrum β-lactamases (ESBL) and AmpC β-lactamases in Klebsiella pneumoniae clinical isolates. Methods: One hundred consecutive, non-duplicate clinical isolates of K. pneumoniae collected over a period of one year (June 2008 - June 2009) were included in the study. An antibiotic susceptibility method was used with 10 antibiotics for Gram-negative infections which helped in screening for ESBL and AmpC β-lactamases and also in confirmation of ESBL production. The detection of AmpC β-lactamases was done based on screening and confirmatory tests. For screening, disc diffusion zones of cefoxitin <18 mm was taken as cefoxitin resistant. All cefoxitin resistant isolates were tested further by AmpC disk test and modified three dimensional test. Multiplex-PCR was performed for screening the presence of plasmid-mediated AmpC genes. Results: Of the 100 isolates of K. pneumoniae studied, 48 were resistant to cefoxitin on screening. AmpC disk test was positive in 32 (32%) isolates. This was also confirmed with modified three dimensional test. Indentation indicating strong AmpC producer was observed in 25 isolates whereas little distortion (weak AmpC) was observed in 7 isolates. ESBL detection was confirmed by a modification of double disk synergy test in 56 isolates. Cefepime was the best cephalosporin in synergy with tazobactam for detecting ESBL production in isolates co-producing AmpC β-lactamases. The subsets of isolates phenotypically AmpC β-lactamase positive were subjected to amplification of six different families of AmpC gene using multiplex PCR. The sequence analysis revealed 12 CMY-2 and eight DHA-1 types. Interpretation & conclusions: Tazobactam was the best β-lactamase inhibitor for detecting ESBL in presence of AmpC β-lactamase as this is a very poor inducer of AmpC gene. Amongst cephalosporins, cefepime was the best cephalosporin in detecting ESBL in presence of AmpC β-lactamase as it is least hydrolyzed by AmpC enzymes. Cefepime-tazobactam combination disk test would be a simple and best method in detection of ESBLs in Enterobacteriaceae co-producing AmpC β-lactamase in the routine diagnostic microbiology laboratories.

Increase resistant rates and ESBL production between E. coli isolates causing urinary tract infection in young patients from Iran

Emerging antimicrobial resistance rates and Extended-spectrum beta-lactamase producing Escherichia coli recovered from urinary tract infections (UTI) is an increasing problem in specific regions, limiting therapeutic options. One hundred E. coli isolates causing UTI in patients with age from 2 months to 12 years admitted at CMC in the period of April 2009 to March 2010 were tested for antibiotic susceptibility using the disk diffusion method. Surprisingly high resistance rates were recorded for E. coli against TMP/SMX (84%), cefalotin (66%), cefuroxime (50%), cefixime (50%) and ceftriaxone (45%). Antimicrobial susceptibility of E. coli isolates was followed by meropenem (98%), amikacin (95%), nitrofurantoin (91%) and gentamicin (68%). Extended spectrum beta-lactamase production, was observed in 32% of community and 42% of nosocomial isolates. The results of this study and numerous observations regarding the increasing resistance to these antibiotics, in several countries, emphasize the need for local population-specific surveillance for guiding empirical therapy for UTI in children.

Incidence of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in patients with urinary tract infection / Incidência de Klebsiella pneumoniae produtoras de beta-lactamases de espectro estendido em pacientes com infecção do trato urinário

CONTEXT AND OBJECTIVES: Resistant bacteria are emerging worldwide as a threat to favorable outcomes from treating common infections in community and hospital settings. The present investigation was carried out to study the incidence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients with urinary tract infection in different seasons of the year, in order to determine the prevalence of the genes blaTEM, blaSHV and blaCTX-M, which are responsible for ESBL production among ESBL-producing K. pneumoniae, in three cities in Iran, and to investigate the antimicrobial susceptibility pattern of K. pneumoniae in different seasons. DESIGN AND SETTING: Retrospective study carried out among patients with urinary tract infections in five hospitals in Iran. METHOD: Two hundred and eighty-eight clinical isolates of K. pneumoniae were collected between March 2007 and April 2008 from five hospitals in three cities in Iran. ESBLs were identified by phenotypic and genotypic methods. ESBL-producing Klebsiella pneumoniae were evaluated against non-beta-lactam antibiotics. Genes coding for ESBLs (blaSHV, TEM and CTX-M) were screened. RESULTS: Among the 288 clinical isolates of K. pneumoniae, 37.7 percent, 46.7 percent and 15.6 percent were obtained from hospitals in Ilam, Tehran and Tabriz, respectively, of which 39.4 percent, 50.7 percent and 45.8 percent were ESBL-producing K. pneumoniae in Ilam, Milad and Emam Reza hospitals, respectively. CONCLUSION: According to the results from this study, resistance to third-generation cephalosporins is higher during the cold months than during the warm months.

CONTEXTO E OBJETIVOS: As bactérias resistentes estão surgindo em todo o mundo como uma ameaça ao resultado favorável no tratamento de infecções comuns em ambientes comunitários e hospitalares. O presente estudo foi realizado com o objetivo de avaliar a incidência de Klebsiella pneumoniae produtoras de beta-lactamases de espectro estendido (BLEEs) em pacientes com infecção do trato urinário em diferentes estações do ano, para verificar a prevalência dos genes blaTEM, blaSHV e blaCTX-M responsáveis para a produção de BLEEs em K. pneumoniae productoras de BLEEs, em três cidades do Irã, e investigar o perfil de sensibilidade antimicrobiana de K. pneumoniae em diferentes estações. DESENHO E LOCAL: Estudo retrospectivo realizado em pacientes com infecções do trato urinário em cinco hospitais no Irã. MÉTODO: Duzentos e oitenta e oito isolados clínicos de K. penumoniae foram coletados entre março de 2007 e abril de 2008 em cinco hospitais de três cidades no Irã. BLEEs foram identificados por métodos fenotípicos e genotípicos. K. pneumoniae produtoras de BLEEs foram avaliadas contra antibióticos não beta-lactâmicos. Os genes codificadores de BLEEs (blaSHV, TEM e CTX-M) foram investigados. RESULTADOS: Entre os 288 isolados clínicos de K. pneumoniae, 37,7 por cento, 46,7 por cento e 15,6 por cento eram provenientes dos hospitais em Ilam, Tehran e Tabriz, respectivamente, dos quais 39,4 por cento, 50,7 por cento e 45,8 por cento eram K. pneumoniae produtoras de BLEEs nos hospitais em Ilam, Milad e Eman Reza, respectivamente. CONCLUSÃO: De acordo com os resultados deste estudo, a resistência às cefalosporinas de terceira geração é maior nos meses frios do que nos meses quentes.