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.

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.

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.

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.

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.