Primer reporte de Enterobacterales dobles productores de carbapenemasas en hospitales de Paraguay. Año 2021

RESUMEN Las carbapenemasas se encuentran ampliamente distribuidas en nuestro país, tanto en bacilos gramnegativos fermentadores como no fermentadores. Durante 2021, se ha reportado incremento de cepas con estas enzimas. Con el objetivo de evaluar la doble producción de carbapenemasas en Enterobacterales y comunicar su circulación, fue puesta a punto una PCR convencional múltiple. Estudio retrospectivo en 128 aislamientos provenientes de 20 centros colaboradores de la Red Nacional de Vigilancia de la RAM (Capital, Central e interior del país), remitidos al LCSP entre febrero y setiembre de 2021, para confirmación y genotipificación de carbapenemasas. Se realizaron pruebas fenotípicas y colorimétricas con sustratos específicos, y pruebas genotípicas (PCR convencional múltiple) para la detección simultánea de varios genes de resistencia (bla NDM, bla KPC, bla OXA-48-like, bla IMP y bla VIM). De los 128 aislamientos estudiados, 107 correspondieron a Klebsiella pneumoniae, 14 a Enterobacter cloacae complex, entre otros; aislados en mayor frecuencia de muestras de orina (30%), respiratorias (30%), sangre y catéter (24%). Los genes de resistencia a los carbapenemes detectados fueron: bla NDM (77,3%), bla KPC (17,2%); siendo confirmada la doble producción de carbapenemasas en 7 aislamientos (5,5%) provenientes de 4 centros diferentes de la capital de país y uno de Central; 6 de ellas (K. pneumoniae) con bla NDM+bla KPC y 1 (E. cloacae complex) con bla NDM+bla OXA-48-like; confirmando circulación de Enterobacterales dobles productores de carbapenemasas en el país (KPC+NDM y OXA+NDM); hallazgos que obligan a proveer de capacidades de detección, de manera a que se puedan tomar medidas oportunas y eficaces de contención y control.

ABSTRACT Carbapenemases are widely distributed in our country, both in fermenting and non-fermenting gram-negative bacilli. During 2021, an increase in strains with these enzymes has been reported. In order to evaluate the double production of carbapenemases in Enterobacterales and communicate their circulation, a multiple conventional PCR was set up. Retrospective study carried out in 128 isolates from 20 collaborating centers of the National AMR Surveillance Network (Capital, Central and interior of the country), sent to the LCSP between February and September 2021, for confirmation and genotyping of carbapenemases. Phenotypic and colorimetric tests were performed with specific substrates, as well as genotypic tests (multiple conventional PCR) for the simultaneous detection of several resistance genes (blaNDM, blaKPC, blaOXA-48-like, blaIMP and blaVIM). Of the 128 isolates studied, 107 corresponded to Klebsiella pneumoniae, 14 to Enterobacter cloacae complex, among others; isolated in higher frequency from urine (30%), respiratory (30%), blood and catheter (24%) samples. The genes for resistance to carbapenems detected were: blaNDM (77.3%), blaKPC (17.2%); the double production of carbapenemases was confirmed in 7 isolates (5.5%) from 4 different centers in the capital of the country and one in Central; 6 of them (K. pneumoniae) with blaNDM + blaKPC and 1 (E. cloacae complex) with blaNDM + blaOXA-48-like; confirming circulation of double Enterobacterales producers of carbapenemases in the country (KPC + NDM and OXA + NDM); findings that require the provision of detection capabilities, so that timely and effective containment and control measures can be taken.

Contribution of acrB upregulation & OmpC/Ompk36 loss over the presence of blaNDM towards carbapenem resistance development among pathogenic Escherichia coli & Klebsiella spp.

Background & objectives: The global spread of carbapenem-resistant Enterobacteriaceae (CRE) is an emerging clinical problem. Hence, in this study, the plausible role of extended-spectrum beta-lactamases (ESBLs)/carbapenemases, OmpC/Ompk36, acrB and their combinations was explored among CRE. Methods: The minimum inhibitory concentration (MIC) of meropenem, enzyme-phenotypes (ESBLs/IR and metallo-beta-lactamase (MBL)/non-MBL carbapenemase), genotypes (blaTEM, blaSHV and blaCTX-M; blaNDM and blaVIM; blaKPC and blaOXA-48-like variants), acrB and outer membrane protein (OMP) expressions were analyzed with a total of 101 non-duplicate clinical isolates, obtained from various samples of patients visiting two tertiary care units of Eastern India during May 2013 - October 2016. This included Escherichia coli (n=36) and Klebsiella pneumoniae (n=65), categorized into two groups, namely Group I (resistant to all carbapenems; n=93; E. coli=34 and Klebsiella spp.=59) and Group II (non-resistant to all the carbapenems; n=8; E. coli=2 and Klebsiella spp.=6). Results: Though 88.17 per cent of Group I isolates exhibited ESBL property, the presence of carbapenemase activity (70.96%) and that of blaNDM gene (42/66: 63.63%) indicated their contributions towards the emergence of CRE. Further, porin loss and/or efflux pump activation among ESBL/carbapenemase-producing isolates heightened the MIC of meropenem from 64 to 256 mg/l (range exhibited by only ESBL/carbapenemase-producing isolates) to >256 mg/l. Interpretation & conclusions: These findings implied the major contribution of porin loss and/or efflux pump activation over the presence of ESBLs/carbapenemases in imparting carbapenem resistance in pathogenic bacteria.

Blandm-1 Gene Carriage In The Icu Of Tertiary Care Hospital In North India

BACKGROUND:- New Delhi metallo-β-lactamase 1 (NDM-1) enzyme leads to multidrug resistance and has been detected from bacteria in many countries. The study was done To detect bacteria carrying blaNDM-1 gene from intensive care unit (ICU) patients and correlate the change with increasing duration of ICU stay. METHODS:- Blood and urine samples were collected from 140 patients thrice- 0-2 days, 3-7 days and 7 days of ICU admission. All bacterial isolates resistant to meropenem were evaluated for Metallo-β-lactamase (MBL) production. blaNDM-1 gene was detected using Real Time PCR from the MBLproducers. RESULT:- Overall, 458 samples (229 each) of blood and urine were collected and 75 gram negative bacteria were isolated. From these, 46.7% (35/75) strains were found to be carbapenem resistant and blaNDM-1 gene was detected in 17.3% (13/75). CONCLUSION:- High prevalence of bacteria carrying blaNDM-1 gene was seen in ICU patients increasing the burden on healthcare.

Epidemiology of diabetic foot infections in a reference tertiary hospital in India

Abstract Introduction The present study attempts to examine the microbial profile and antibiotic susceptibility of diabetic foot infections in the intensive care unit of a tertiary referral centre for diabetic foot. As part of the study, we also attempted to find the prevalence of blaNDM-like gene among carbapenem-resistant gram negative infections. Methodology A prospective study of 261 patients with diabetic foot infections was performed during the period between January 2014 and June 2014. Results A total of 289 isolates were obtained from 178 tissue samples from 261 patients, 156 (59.7%) males and 105 (40.2%) females, with a mean age of 58 years (-15 years), having diabetic foot infection. No growth was seen in thirty eight (17.6%) tissue samples. Out of the total samples, 44.3% were monomicrobial and 55.7% were polymicrobial. Gram negative pathogens were predominant (58.5%). Seven of the total isolates were fungal; 0.7% showed pure fungal growth and 1.7% were mixed, grown along with some bacteria. The most frequently isolated bacteria were Staphylococcus aureus (26.9%), followed by Pseudomonas aeruginosa (20.9%). Of the 58.5% gram negative pathogens, 16.5% were Enterobacteriaceae resistant to carbapenems. Among these isolates, 4 (25%) were positive for blaNDM-like gene. Among the rest, 18.6% were carbapenem-resistant Pseudomonas, among which 4 (36.3%) were blaNDM. Among the Staphylococci, 23.7% were methicillin-resistant Staphylococcus aureus. Conclusions Our results support the recent view that gram negative organisms, depending on the geographical location, may be predominant in DFIs. There is an increase in multidrug-resistant pathogens, especially carbapenem resistance and this is creeping rapidly. We need to be more judicious while using empiric antibiotics.

Detection of blaNDM-1 in Stenotrophomonas maltophilia isolated from Brazilian soil

This study reports the presence of the blaNDM-1 gene in an isolate of Stenotrophomonas maltophilia obtained from a Brazilian soil, inside an IncA/C plasmid with ~ 45 Kb. To the best of our knowledge, this is the second report in the world and the first in Brazil of NDM-producing bacterium isolated from soil.

Drug resistance and genetic background of New Delhi metallo-β-lactamase ( NDM )-producing bacterial strains in Lishui area of Zhejiang province / 中华临床感染病杂志

Objective To analyze the drug resistance phenotype and genetic background of New Delhi metallo-β-lactamase (NDM)-producing bacterial strains in Lishui area of Zhejiang province.Methods The imipenem-resistant Enterobacteriaceae strains were isolated from January 2012 to December 2016 in Lishui Municipal Central Hospital of Zhejiang Province.Mrieux Vitek 2 Compact system was used to identified strains and PCR was used to screen for blaNDMgene.Susceptibility was detected by K-B method and MICs were obtained by Vitek 2 with GN13 cards.Plasmids typing was carried out by DNA sequencing of the replication initiator with the transconjugates as templates.The blaNDMgenetic contexts were detected by PCR and complete genome sequencing.Results A total of 102 strains of carbapenem-resistant enterobacteria (CRE), mainly Escherichia coli and Enterobacter cloacae, were isolated, of which 15 were positive for blaNDMwith a positive detection rate of 14.7%.The resistance rate to β-lactam antibiotics was 100%, and the resistance rates to aztreonam, compound sulfamethoxazole , tobramycin and gentamicin were all ＞80%;and the resistance rate to quinolones was ＞50%.Among the 15 NDM-producing strains , 12 strains were positive for Hodge test, and 2 strains of Enterobacter cloacae and 1 strain of Escherichia coli were negative. There were 11 strains of blaNDM-1and 4 strains of blaNDM-1.A variety of plasmid types such as IncX 3, IncFIIγ and IncA/C were detected, and 4 blaNDM-5genes were located in the IncX3 plasmid.Four blaNDMsurrounding gene structures were found, of which 8 blaNDM-1genes were located in ISAb125-hyp-blaNDM-1-bleMBL-TrpF-DsbC-IS26, while blaNDM-5was located in IS3000-IS5-blaNDM-5-bleMBL-TrpF-DsbC-IS26 structure, and the former was reported for the first time in China.Conclusion NDM-producing bacterial strains in Lishui area are prevalent at a low level and have high sensitivity to fosfomycin and polymyxin .The blaNDMgene may have multiple sources, but IncX3 plasmid is still the main source for gene transfer.Some new types of blaNDM-1 gene structure have been also found in this study.

Finding of a NDM-1 carbapenems-producing Leclercia adcarboxglata / 临床检验杂志

Objective To investigate the resistance mechanism of a carbapenems-resistant Leclercia adcarboxglata.Methods The species was identified by the automatic microbial analyzer,matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA sequence analysis.The conventional drug susceptibility test was detected with automatic microbial analyzer,and the minimum inhibitory concentration (MIC) for imipenem was examined by E-test.The phenotypes of carbapenemase were detected by modified carbapenem inactivation method (mCIM) and the genotypes of resistance genes were detected by polymerase chain reaction (PCR) and DNA sequencing.The characteristics of the carried plasmid were analyzed by conjugation test and S1pulsed-field gel electrophoresis (S1-PFGE).Results The clinical isolates of Leclercia adcarboxglata were resistant to imipenem,other beta-lactam antibiotics(except aztreonam) and aminoglycosides,but sensitive to quinolones and sulfonamides.The conjugation test resulted in a drug-resistance spectrum of the receptor strain E.coli J53 similar to Leclercia adcarboxglata bacteria.The phenotype of carbapenemase was positive.PCR amplification and sequencing analysis showed that blaNDM-1,blaTEM and aac (6')-Ib were detectable in the isolates simultaneously,while the conjugon only carried blaNDM-1.S1-PFGE revealed that Leclercia adcarboxglata carried 3 plasmids.Conclusion The carbapenems resistance of Leclercia adcarboxglata may contribute to carrying blaNDM-1 gene which may exist in an around 100 kb plasmid transmitted with conjugation.

Study on plasmid-mediated blaNDM-1 gene in carbapenem-resistant Citrobacter freundii / 中国感染与化疗杂志

Objective To investigate the transmission of blaNDM-1 gene in carbapenem-resistant Citrobacter freundii. Methods A total of 18 strains of NDM-1-producing C. freundii were collected from the First Affiliated Hospital of Kunming Medical University during the period from June 2012 to October 2014. The isolates were identified and subjected to antimicrobial susceptibility testing with VITEK 2 System. Conjugation experiments, pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to determine the transferability of plasmids. Results The antibiotic susceptibility results showed that all the NDM-1-producing C. freundii isolates were resistant to penicillins, cephalosporins and carbapenems. All isolates exhibited different level resistance to other antibiotics. Conjugation experiments revealed that the plasmids harboring blaNDM-1 in 13 strains were transformed into E. coli 600, and exhibited carbapenem resistance. PFGE and Southern blot hybridization found that blaNDM-1 was located on a 33.3 kb plasmid in 16 isolates and on 33.3-54.7 kb plasmid in 2 isolates. Conclusions Our findings suggest that plasmids contribute to the horizontal dissemination of blaNDM-1 gene in carbapenemresistant C. freundii.

Estudo genotípico e fenotípico de bacilos Gram-negativos produtores de carbapenemase do tipo New Delhi metalo-ß-lactamase / Genotypic and fenotypic study of Gram-negative bacilli producers carbapenemase type New Delhi metallo--ß-lactamase

Os carbapenêmicos são os antimicrobianos mais amplamente utilizados no tratamento empírico de infecções graves por bacilos Gram-negativos. A pressão seletiva gerada pelo uso desses antimicrobianos ao longo das últimas três décadas contribuiu para a disseminação de enterobactérias e Gram-negativos não fermentadores produtores de carbapenemases, particularmente as do tipo KPC e NDM. Os genes que codificam essas enzimas usualmente estão localizados em plasmídeos e/ou transpósons. A hipótese atualmente mais aceita é que o gene blaNDM-1 seja uma quimera criada em Acinetobacter baumannii. A NDM-1 foi descrita em paciente proveniente da Índia e subsequentemente evidenciou-se sua ampla disseminação nesse país. A epidemiologia que tem sido observada nos casos detectados na Europa e Estados Unidos tem sido viagem à Índia, ou seja, sem casos autóctones. No Brasil, os primeiros casos foram identificados no Rio Grande do Sul, e a seguir no Rio de Janeiro e em São Paulo. Diferentemente dos casos da Europa e América do Norte, os casos do Brasil não tem relação epidemiológica com a Índia. O sequenciamento integral dos plasmídeos e cromossomos albergando o gene blaNDM permitirá entender como ocorre a disseminação desse mecanismo de resistência no Brasil. Para isso, foi avaliado o perfil de susceptibilidade dos isolados, bem como a capacidade conjugativa e clonalidade. Das vinte e oito amostras utilizadas neste trabalho, treze delas pertencem à espécie Enterobacter hormaechei, uma à espécie Citrobacter freundii, sete à espécie Escherichia coli, quatro à Klebsiella pneumoniae e três ao gênero Acinetobacter spp. Os primeiros isolados incluídos neste estudo (Escherichia coli e Enterobacter hormaechei produzindo NDM-1) foram isolados em agosto de 2013, de uma mesma amostra de swab retal de um paciente do Rio de Janeiro que nunca viajou para o exterior. O sequenciamento completo do DNA plasmidial utilizando a plataforma Illumina e a anotação de ambos os plasmídeos albergando o gene blaNDM-1 revelou que estes pertencem a grupos de incompatibilidade diferentes, IncFIIK (E. hormaechei) e IncX3 (E. coli), e abrigam um novo transpóson composto designado Tn3000. A comparação da sequência nucleotídica do Tn3000 com aquelas disponíveis no GenBank evidencia que a mesma estrutura está presente em plasmídeos de isolados da cidade de Porto Alegre e também em diferentes continentes. As espécies de Acinetobacter (A. radioresistens, A. ursingii e A. guillouiae) isoladas em São Paulo e Porto Alegre, possuem o gene blaNDM-1 albergados em um mesmo plasmídeo não tipável de 41.087 pb. A avaliação da clonalidade dos isolados de Enterobacter hormaechei "subsp. oharae" mostrou dois perfis diferentes através da técnica de PFGE, sendo que todos os microrganismos foram isolados de um surto no mesmo hospital no Rio de Janeiro. Isolados de Klebsiella pneumoniae de uma mesma paciente internada em hospital em Salvador, de sítios distintos - swab retal, hemocultura e urina, em ordem cronológica - obtiveram o mesmo perfil clonal pela técnica de PFGE. O mesmo ocorreu com três isolados de Escherichia coli, de um mesmo paciente do Rio de Janeiro, em amostras de swab retal. Os achados deste estudo evidenciam que no Brasil, Nepal, Marrocos e Índia há uma disseminação do gene blaNDM-1 mediada por um novo elemento móvel designado Tn3000 em enterobactérias. A detecção de um mesmo plasmídeo em diferentes espécies de Acinetobacter evidencia que neste gênero bacteriano, no Brasil, a disseminação do gene blaNDM-1 ocorre por conjugação.

Carbapenems are the antimicrobials most widely used in the empirical treatment of severe infections caused by Gram-negative bacilli. The selective pressure generated by the use of these antibiotics over the last three decades has contributed to the spread of enterobacteria and Gram-negative non-fermenting producing carbapenemases, mainly KPC and NDM. Genes encoding these enzymes are usually located in plasmids and/or transposons. Currently the most accepted hypothesis is that the blaNDM-1 gene is a chimera created in Acinetobacter baumannii. The NDM-1 was described in a patient from India and subsequently was reported to be broadly disseminate in this country. The epidemiology that has been observed in cases detected in Europe and United States is traveling to India, but no autochthonous cases. In Brazil, the first cases were identified in Rio Grande do Sul, and then in Rio de Janeiro and São Paulo. Differently from the cases described in Europe and North America, the cases from Brazil have no epidemiological link with India. The complete sequencing of plasmids and chromosomes harboring blaNDM gene will understanding how the dissemination of this resistance mechanism in Brazil occurs. In this work we will be evaluate the susceptibility profile of the isolates, and their conjugal capacity and clonality. Of the twenty-eight samples used in this study, thirteen of them belong to the species Enterobacter hormaechei, one to Citrobacter freundii, seven to Escherichia coli, four to Klebsiella pneumoniae and three to the genus Acinetobacter sp. The first two isolates included in this study (Escherichia coli and Enterobacter hormaechei) were isolated in August 2013, from the same rectal swab sample from a patient from Rio de Janeiro that never traveled abroad. Complete sequencing of plasmid DNA using Illumina platform and annotation of both plasmids harboring the blaNDM-1 gene revealed that they belong to different incompatibility groups, IncFIIK (E. hormaechei) and IncX3 (E. coli), and are harbor to a new transposon designated Tn3000. The comparison of the Tn3000 nucleotide sequence with those available at GenBank shows that the same structure is present in plasmids from other Porto Alegre and also in different continents. The Acinetobacter species (A. radioresistens, A. ursingii and A. guillouiae) isolated in São Paulo and Porto Alegre, have the blaNDM-1 gene harbored in a single non-typing plasmid of 41,087 bp. The evaluation of clonal relationship of Enterobacter hormaechei "subsp. oharae" showed two different profiles by PFGE technique; of note all microorganisms were isolated from an outbreak in the same hospital in Rio de Janeiro. Isolates of Klebsiella pneumoniae from a single patient hospitalized in Salvador, from different anatomical sites - rectal swab, blood culture and urine, in chronological order - obtained the same clonal profile by the PFGE technique. The same occurred with three Escherichia coli isolates, from the same patient from Rio de Janeiro, in swab rectal strains. Our findings suggest that in Brazil, Nepal, Morocco and India there is a spread of blaNDM-1 gene mediated by Tn3000 in enterobacteria. The detection of a same plasmid in different species of Acinetobacter shows that in this bacterial genus, in Brazil, the dissemination of the blaNDM-1 gene occurs by conjugation.

Construction of blaNDM-1gene deletion mutant of Enterobacter cloacae and analysis of its biological characteristics / 中华微生物学和免疫学杂志

Objective To construct a blaNDM-1gene deletion mutant of Enterobacter cloacae and to analyze its biological characteristics. Methods The blaNDM-1gene deletion mutant was constructed by using Red homologous recombination technology and verified by PCR and RT-qPCR. Antimicrobial susceptibility profiles,growth curves and in vitro competition abilities of the original strain and the blaNDM-1gene deletion mutant were analyzed. Results PCR,DNA sequencing and RT-qPCR showed that the blaNDM-1gene dele-tion mutant was successfully constructed. Antimicrobial susceptibility test showed that the original strain was resistant to imipenem,meropenem and ertapenem, while the blaNDM-1gene deletion mutant was sensitive to all. The original strain and the blaNDM-1gene deletion mutant had similar growth curves in Luria-Bertani liq-uid medium. In vitro competition experiment revealed that the competitive index of them was 0.69. Conclu-sion Red homologous recombination technology can be used to knockout the blaNDM-1gene of Enterobacter cloacae,which is associated with antimicrobial resistance and competitiveness.

Detección de los genes de carbapenemasas blaKPC y blaNDM en aislamientos de Klebsiella pneumoniae del Hospital General San Juan de Dios de la ciudad de Guatemala

La resistencia a los antibióticos constituye uno de los problemas más relevantes de salud pública en todo el mundo. Las enterobacterias productoras de carbapenemasas representan la mayor amenaza. Las carbapenemasas son potentes enzimas que inactivan los antibióticos carbapenémicos y en general, a todos los antibióticos betalactámicos. Las consecuencias para el tratamiento de las infecciones causadas por estas bacterias son relevantes, ya que los carbapenemes son de las últimas opciones disponibles para bacterias multirresistentes. Esta investigación tuvo como objetivos determinar por medio de la reacción en cadena de la polimerasa (PCR) punto final, la presencia de los genes de carbapenemasas blaKPC (Klebsiella pneumoniae carbapenemasa) y blaNDM (New Delhi Metalobetalactamasa) en aislamientos de K. pneumoniae del Hospital General San Juan de Dios de la ciudad de Guatemala, caracterizar el tipo de muestra y definir el servicio del hospital donde se aislaron este tipo de bacterias. Se analizaron 54 aislamientos de K. pneumoniae resistentes a carbapenemes (imipenem y/o meropenem), 49 (91%) fueron portadoras del gen blaNDM. Estas bacterias se aislaron con más frecuencia en muestras de sangre (37%) y orina (14%). En esta investigación, el 53% de aislamientos se obtuvieron de pacientes de servicios de intensivos. Los resultados de este estudio indican que K. pneumoniae portadora del gen blaNDM se ha diseminado dentro del Hospital General San Juan de Dios, desde el primer caso reportado hace cinco años, poniendo en riesgo de muerte a los pacientes, especialmente a los hospitalizados en los servicios de intensivos.

Resistance to antibiotics is one of the most important public health problems worldwide. Enterobacteriaceae producing carbapenemases pose the greatest threat. The carbapenemases are powerful enzymes that inactivate carbapenem antibiotics and generally all beta-lactam antibiotics. The consequences for the treatment of infections caused by these bacteria are important because carbapenems are the latest options available for multidrug-resistant bacteria. This research aimed to determine through endpoint polymerase chain reaction (PCR), the presence of the carbapenemases encoding genes blaKPC (Klebsiella pneumoniae carbapenemase) and blaNDM (New Delhi metallolactamase) in K. pneumoniae isolates from San Juan de Dios General Hospital located in Guatemala City; and to characterize the sample type and define the service of the hospital where such bacteria were isolated 54 K. pneumoniae isolates resistant to carbapenems (imipenem and / or meropenem), were analyzed. From these, 49 (91 %) were detected as blaNDM gene carriers. These bacteria were isolated more frequently in blood samples (37%) and urine (14%). In this research, 53% of isolates were obtained from patients hospitalized in intensive care units. This study demonstrates that K. pneumoniae carrying the blaNDM gene has spread within San Juan de Dios General Hospital, since the first reported case five years ago, risking the life of patients, especially of those hospitalized in intensive care services.

High-throughput DNA sequencing of Klebsiella pneumoniae plasmid pNDM-LJ / 实用医学杂志

Objective To investigate the relationship between the resistance of the Klebsiella pneumoni-ae and the Klebsiella pneumoniae plasmid pNDM-LJ carrying blaNDM-1 by high-throughput DNA sequencing. Methods High-throughput DNA sequencing was carried out by the Illumina Miseq platform , and sequencing data were assembled by Edena software. Contigs were annotated by the RAST server and analyzed by the BLAST server. Results The plasmid pNDM-LJ was 54-kb in size with a GC content of 49%. The plasmid encoded 52 putative functional genes and belonged to the IncX3 group in incompatible classifications. Analysis of the plasmid sequence revealed high similarity with other IncX3 plasmids. The blaNDM-1 gene was located in a complicated gene environment possibly constructed by several transposition events. The 5′ and 3′ ends of the blaNDM-1 gene were adjacent to the ISAba125 and IS 26 respectively , forming a 10.8-kb transposon-like structure. Conclusion The plasmid pNDM-LJ carried the blaNDM-1 gene being resistant to carbapenems and played a possibly impor-tant role in transmission of blaNDM-1 in China.

occurrence of blaNDM-1 & absence of blaKPC genes encoding carbapenem resistance in uropathogens from a tertiary care centre from north India.

Background & objectives: Carbapenem resistance mediated by carbapenemases is increasingly being reported worldwide. This study was conducted to know the occurrence of important carbapenem resistance encoding genes in gram-negative bacilli (GNB) causing complicated urinary tract infection (CUTI), and to look at the genetic diversity of these isolates. Methods: The study was carried out on 166 consecutive carbapenem resistant uropathogens (CRU) isolated from cases with CUTI during 2008 and 2012. Carbapenemase production was characterized phenotypically and polymerase chain reaction was used to detect blaVIM, blaIMP, blaKPC, and blaNDM-1. BOX- PCR was done on 80 randomly selected isolates for molecular typing. Results: The blaVIM gene was present in 34 (43.6%), blaIMP in five (6.4%) and none of the isolates from 2008 had blaNDM-1 or blaKPC genes. Among the isolates from 2012, blaNDM-1 gene was present in 47 (53.4%), blaVIM in 19 (24.4%), blaIMP in one (1.1%) and none had blaKPC. There were nine isolates during the two years which had multiple genes encoding carbapenemases; while 66 did not have any of the genes tested. Of the 80 isolates subjected to BOX-PCR, 58 could be used for analysis and showed, presence of multiple clusters of carbapenem resistant isolates and absence of a single dominant clone. Interpretation & conclusions: The blaNDM-1 gene was absent in our isolates obtained during 2008 but was present amongst Enterobacteriaceae isolated in 2012. The blaKPC gene was also not found. Nine isolates obtained during the two years had multiple genes encoding carbapenemases confirming the previous reports of emergence of GNB containing genes encoding multiple carbapenemases. Typing using BOX-PCR indicated that this emergence was not because of clonal expansion of a single strain, and multiple strains were circulating at a single point of time.

Complete sequencing of an IncX3 plasmid carrying blaNDM-5 allele reveals an early stage in the dissemination of the blaNDM gene.

Purpose: The aim of the present study was to perform molecular characterisation of the blaNDM plasmids and to understand the mechanism of its spread among pathogenic bacteria. Materials and Methods: Seventy-six non-repetitive carbapenem-resistant isolates which were collected during Nov 2011 to April 2013 from four hospitals in Chennai were analyzed for the presence of the blaNDM gene by PCR. Further, the genetic context of the blaNDM gene was analyzed by PCR specifi c to ISAba125 and bleMBL gene. One of the blaNDM plasmid was completely sequenced in the Illumina HiSeq platform. Results: Twenty-three isolates consisting of 8 Escherichia coli, 8 Klebsiella pneumoniae, 3 Klebsiella oxytoca, 3 Acinetobacter baumanii and 1 Pseudomonas aeruginosa were found to carry the blaNDM gene. In 18 isolates the blaNDM gene was associated with a bleMBL gene and the ISAba125 element. The complete sequencing of pNDM-MGR194 revealed an IncX3 replication type plasmid, with a length of 46,253 bp, an average GC content of 47% and 59 putative ORFs. The iteron region contained the blaNDM5 gene and the bleMBL, trpF and dsbC genes downstream and an IS5 inserted within the ISAba125 element upstream. Conclusion: This is the fi rst report where the blaNDM gene insertion in a plasmid is not accompanied by other resistance gene determinants. These observations suggest that the IncX3 plasmid pNDM-MGR194 is an early stage in the dissemination of the blaNDM.

High prevalence of multidrug-resistance in Acinetobacter baumannii and dissemination of carbapenemase-encoding genes blaOXA-23-like, blaOXA-24-like and blaNDM-1 in Algiers hospitals

Objective:To assess and characterize antibiotic resistance in Acinetobacter baumannii strains recovered from 5 health-care facilities in Algiers. Methods:Antibiotic susceptibility testing was performed by agar diffusion and agar dilution methods, resistance genes were identified by PCR and sequencing, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Results:Among 125 tested isolates, 117 (93.6%) were multidrug-resistant, of which 94 (75.2%) were imipenem resistant. The blaADC and blaOXA-51-like genes were detected in all isolates, in association with ISAba1 sequence in 84%and 8%(imipenem resistant) of isolates, respectively. The blaOXA-23-like and blaOXA-24-like carbapenemase genes were detected in 67.02%and 20.21%of imipenem-resistant isolates, respectively. The blaOXA-23-like gene is linked to ISAba1 or ISAba4 elements. The metallo-β-lactamase NDM-1 gene was found in 10 (10.6%) imipenem-resistant strains from three hospitals, it is linked to ISAba125 element in nine strains. Extended spectrumβ-lactamases production was not detected. Imipenem and cefotaxime resistance phenotypes could not be transferred to Escherichia coli by conjugation. Outer membrane protein CarO gene was not detected in four imipenem-resistant isolates. The aac(6’)-Ib, sul1, sul2, tetA and tetB genes were present in 5.31%, 36.17%, 77.65%, 1.06%and 65.92%of strains, respectively. Class 1 integrons were detected in 23.4%strains. ERIC-PCR typing showed a genetic diversity among blaOXA-23-like and blaOXA-24-like positive strains, while clonality was observed among blaNDM-1 positives. Conclusions:This study highlighted the high prevalence of imipenem resistance in Acinetobacter baumannii in Algiers hospitals mediated mainly by blaOXA-23-like, blaOXA-24-like, and blaNDM-1 genes.

Multidrug resistant NDM-1 metallo-beta-lactamase producing Klebsiella pneumoniae sepsis outbreak in a neonatal intensive care unit in a tertiary care center at central India.

Objective: The objective of the following study is to detect genes encoding carbapenem resistance in Klebsiella pneumoniae sepsis outbreak in a neonatal intensive care unit (NICU). Materials and Methods: Antibiotic sensitivity test was performed by standard Kirby Bauer disc diffusion technique and minimum inhibitory concentrations of antibiotics was determined by VITEK-2. Polymerase chain reaction (PCR) assays and sequencing was used to determine the presence of beta-lactamase encoding genes. Conjugation experiments were performed to determine the transferability of beta-lactamase. Isolate relatedness were determined by repetitive-element PCR (REP), enterobacterial repetitive intergenic consensus (ERIC) PCR and random amplifi ed polymorphic deoxyribonucleic acid (RAPD). Results: All the isolates were completely resistant to the second and third generation cephalosporins tested as well as carbapenems. Susceptibility profi ling of the isolates indicated that 100% retained susceptibility to tigecycline and colistin. Conjugation experiments indicated that blaNDM-1 was transferable and likely through a plasmid-mediated event. All the isolates showed the presence of blaNDM-1 with co association of blaCTX-M-15. REP-PCR, ERIC-PCR and RAPD revealed a single clonal type circulating in NICU environment. Conclusion: Co-production of NDM-1 with CTX-M-15 in K. pneumoniae isolates was detected for the fi rst time in our NICU. Transmission of plasmid carrying these resistant genes to other members of Enterobacteriaceae will increase the incidence of multidrug resistance. Early detection of these genes will help in prevention and adequate infection control by limiting the spread of these organisms.