First Report of OXA-181-Producing Enterobacterales Isolates in Latin America.

We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the ß-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

Occurrence of blaNDM-7 and association with blaKPC-2, blaCTX-M15, aac, aph, mph(A), catB3 and virulence genes in a clinical isolate of Klebsiella pneumoniae with different plasmids in Brazil.

To characterize phenotypically and genotypically an isolate of multidrug-resistant (MDR) K. pneumoniae from a patient with septicemia in a hospital in Recife-PE, Brazil, resistance and virulence genes were investigated using PCR and sequencing the amplicons, and the plasmid DNA was also sequenced. The K74-A3 isolate was resistant to all ß-lactams, including carbapenems, as well as to aminoglycosides and quinolones. By conducting a PCR analysis and sequencing, the variants blaNDM-7 associated with blaKPC-2 and the cps, wabG, fim-H, mrkD and entB virulence genes were identified. The analysis of plasmid revealed the presence of blaCTX-M15, aac(3)-IVa, aph(3')-Ia, aph(4)-Ia, aac(6')ib-cr, mph(A) and catB3, and also the plasmids IncX3, IncFIB, IncQ1, ColRNAI and ColpVC. To our knowledge, this is the first report of the blaNDM-7 gene in Recife-PE and we suggest that this variant is located in IncX3. These results alert us to the risk of spreading an isolate with a vast genetic arsenal of resistance, in addition to which several plasmids are present that favor the horizontal transfer of these genes.

Novel small IncX3 plasmid carrying the blaKPC-2 gene in high-risk Klebsiella pneumoniae ST11/CG258.

This study used whole-genome sequencing (WGS) and PFGE to analysis KPC-2-producing Klebsiella pneumoniae strains from clinical specimens collected in Brazilian hospitals. The study identifies the emergence of a novel small IncX3 plasmid (pKPB11), 12,757-bp in length, in a high-risk K. pneumoniae ST11/CG258 lineage, a successful clonal group in Brazil, carrying the blaKPC-2 gene on a non-Tn4401 genetic element (NTEKPC-Ic). Comparative analysis of the pKPB11 showed that this plasmid reduced its size, losing part of its conjugation apparatus. The pKPB11 was also compared to another strain sequenced in this study (KPC89) that had the hybrid IncX3-IncU plasmid (pKP89), of approximately 45 kb in length, similarly carrying the blaKPC-2 gene on NTEKPC-Ic. To the best of our knowledge, pKPB11 is the first example of small IncX3 plasmid found in a high-risk KPC-2-producing K. pneumoniae ST11/CG258.

IncX3 plasmid harboring a non-Tn4401 genetic element (NTEKPC) in a hospital-associated clone of KPC-2-producing Klebsiella pneumoniae ST340/CG258.

IncX-type plasmids have achieved clinical significance for their contribution in the dissemination of genes confering resistance to carbapenems (most blaKPC- and blaNDM-type genes) and polymyxins (mcr-type genes), both antibiotics considered last resort for multidrug-resistant Gram-negative infections. In this study, we report the identification and complete sequence analysis of an IncX3 plasmid (designated pKP1194a) carrying a non-Tn4401 genetic element (NTEKPC) of tnpR-tnpA (partial)-blaKPC-2-ΔISKpn6/traN, originating from a hospital-associated lineage of K. pneumoniae belonging to the ST340/CG258, with epidemiological link to Brazil.

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.