Colistin-resistant Escherichia coli belonging to different sequence types: genetic characterization of isolates responsible for colonization, community- and healthcareacquired infections

The plasmid-mediated colistin-resistance gene named mcr-1 has been recently described in different countries and it became a public health challenge. Of note, few studies have addressed the spread of Escherichia coli harboring the mcr-1 gene in both, community and hospital settings. A total of seven colistin-resistant E. coli carrying mcr-1, collected from 2016 to 2018, from community (n=4), healthcare-acquired infections (n=2) and colonization (n=1) were identified in three high complexity hospitals in Sao Paulo, Brazil. These colistin-resistant isolates were screened for mcr genes by PCR and all strains were submitted to Whole Genome Sequencing and the conjugation experiment. The seven strains belonged to seven distinct sequence types (ST744, ST131, ST69, ST48, ST354, ST57, ST10), and they differ regarding the resistance profiles. Transference of mcr-1 by conjugation to E. coli strain C600 was possible in five of the seven isolates. The mcr-1 gene was found in plasmid types IncX4 or IncI2. Three of the isolates have ESBL-encoding genes (blaCTX-M-2, n=2; blaCTX-M-8, n=1). We hereby report genetically distinct E. coli isolates, belonging to seven STs, harboring the mcr-1 gene, associated to community and healthcare-acquired infections, and colonization in patients from three hospitals in Sao Paulo. These findings point out for the potential spread of plasmid-mediated colistin-resistance mechanism in E. coli strains in Brazil

Epidemiological and microbiological characterization of dissemination of plasmid-mediated resistance gene to colistin, mcr-1, at Hospital das Clínicas - FMUSP

Introdução: O gene de resistência plasmidial à colistina, mcr-1 (do inglês mobile colistin resistance) foi identificado pela primeira vez no final de 2015 e desde então foi descrito em isolados de animais, humanos e ambiente. Entretanto, estudos conduzidos em hospitais brasileiros são escassos. Objetivos: Descrever a disseminação de mcr-1 em unidades que apresentaram pacientes (caso) com infecção e/ou colonização por Enterobactérias resistentes a colistina e sensíveis a carbapenêmicos, e em pacientes hospitalizados na mesma unidade e período que os casos (contactantes), entre agosto de 2016 a janeiro de 2017. Avaliar outros mecanismos de resistência à colistina desses isolados. Material e Métodos: Foi realizada coleta de amostras com swab retal, processamento em ágar MacConkey e triagem em meio com colistina. Todos os isolados obtidos foram testados para mcr-1 por PCR e confirmados por sequenciamento Sanger. Foi criado um banco de dados no programa Epi InfoTM (CDC) com variáveis clínicas e demográficas dos pacientes. Análise univariada dos fatores de risco associados com aquisição do gene mcr-1 foi realizada e o valor de p <= 0.05 foi considerado significativo. Variáveis significativas foram colocadas em um modelo de regressão logística. Foram realizadas concentração inibitória mínima para colistina, meropenem e imipenem; extração de DNA plasmidial; PCR para plasmídeo tipo IncX4; sequenciamento total do genoma; conjugação e teste para bomba de efluxo com CCCP dos isolados positivos para o gene mcr-1. Resultados: Todos os contactantes dos 3 casos, totalizando 20 pacientes identificados entre setembro de 2016 a janeiro de 2017 foram avaliados. O tempo de hospitalização médio prévio a coleta de amostras foi de 21 dias para os 23 pacientes, 54 dias para os 3 pacientes caso e 16 dias para os 20 contactantes. O uso de meropenem, tempo de uso de meropenem e de colistina durante internação, ser submetido a cirurgia e tempo de internação prévio a coleta foram considerados fatores de risco para aquisição de mcr-1 na análise univariada. Entretanto, apenas tempo de uso meropenem foi fator de risco foi fator de risco independente. Foram obtidos 7 isolados positivos para mcr-1 de K. pneumoniae, K. quasipneumoniae, K. aerogenes, P. mirabilis, P. stuartii e M. morganii, sendo as últimas 3 espécies intrinsecamente resistentes à colistina. Porém, não foi possível transferir o gene mcr-1 por conjugação. O gene mcr-1 não foi encontrado por sequenciamento total do genoma, mas o PCR foi confirmado por sequenciamento Sanger em 5 isolados (2 K. pneumoniae, 1 K. aerogenes, 1 M. morganii e 1 P. mirabilis). Nenhum isolado foi positivo para plasmídeo tipo IncX4 por PCR nem por sequenciamento total do genoma. Dois isolados de K. pneumoniae, resistentes à colistina, ao meropenem e ao imipenem eram coprodutores de KPC-2, além de variadas ESBLs. Um isolado de K. quasipneumoniae (anteriormente K. pneumoniae pertencente ao ST-1308) foi descrito pela primeira vez no Brasil como carreador de mcr-1. Os isolados de Klebsiella spp. também apresentaram inativação de mgrB, relacionado a resistência a colistina. Todos os isolados sequenciados apresentaram diminuição de CIM para colistina na presença de inibidor de bomba de efluxo (CCCP). Os genes codificadores de AcrAB-TolC de bomba de efluxo apresentaram mutações no isolado de P. stuartii que também teve variação menor de CIM. Conclusão: O gene mcr-1 foi identificado em diferentes gêneros e espécies de Enterobactérias, incluindo as intrinsecamente resistentes à colistina. Outros mecanismos de resistência à colistina como mutação cromossômica e bomba de efluxo, foram observados em isolados carreadores de mcr-1, evidenciando a complexidade da resistência a colistina nas Enterobactérias. O tempo de uso de meropenem foi o único fator de risco independente para aquisição do gene mcr-1Introduction: The plasmid-mediated resistance gene to colistin, mcr-1 (mobile colistin resistance) was first identified by the end of 2015 and ever since was described in isolates from animals, humans and environment. However, studies conducted in Brazilian hospitals are scarce. Aims: Describe the dissemination of the mcr-1 gene in units where patients (case) with infection and/or colonized by Enterobacteria resistant to colistin and susceptible to carbapenems, and patients hospitalized at the same unit and period as the cases (contact patients), between August 2016 and January 2017. Evaluate other mechanisms of resistance to colistin in these isolates. Material and Methods: Samples were collected with rectal swabs, processed in MacConkey agar and screened on medium with colistin. All isolates obtained were tested for mcr-1 by PCR and confirmed by Sanger sequencing. A database was created in the Epi InfoTM program (CDC) with clinical and demographic variables from patients. Univariate analysis to risk factors associated to mcr-1 gene acquisition was performed and p-value <= 5 was considered significant. Significant variables went through logistic regression model. Isolates positive to mcr-1 gene by PCR were submitted to minimum inhibitory concentration determination to colistin, meropenem and imipenem; plasmid DNA extraction; whole genome sequencing; conjugation and IncX4 plasmid type by PCR. Results: All contact patients from 3 cases, totalizing 20 patients identified between September 2016 and January 2017 were evaluated. Average hospitalization time before sample collection was 21 days for the 23 patients, 54 days the 3 case patients and 16 days for the 20 contact patients. Use of meropenem, time of use of meropenem and colistin in hospital, surgical procedure and hospitalization time before sampling were considered risk factors for acquisition of mcr-1 in unadjusted analysis. Nevertheless, only time of meropenem use was an independent risk factor. Seven isolates positive for mcr-1 were obtained of K. pneumoniae, K. quasipneumoniae, K. aerogenes, P. mirabilis, P. stuartii and M. morganii, the last three are species intrinsically resistant to colistin. However, it was not possible to transfer mcr-1 gene by conjugation. The mcr-1 gene was not found through whole genome sequencing, but PCR products were confirmed by Sanger sequencing in 5 isolates (2 K. pneumoniae, 1 K. aerogenes, 1 M. morganii and 1 P. mirabilis). None of the isolates were positive for IncX4 plasmid type by PCR neither whole genome sequencing. Two K. pneumoniae isolates, resistant to colistin, meropenem and imipenem, were coproducers of KPC-2, as well as others ESBLs. One isolate of K. quasipneumoniae (former K. pneumoniae of ST-1308) here first described in Brazil and as mcr-1 carrier. The Klebsiella spp. isolates also had inactivation of mgrB, related to colistin resistance. All sequenced isolates presented lower MIC to colistin in presence of efflux pump inhibitor (CCCP). Codifying genes of AcrAB-TolC efflux pump presented mutations in P. stuartii which MIC also varied less than others. Conclusion: The mcr-1 gene was identified in different genera and species of Enterobacteria, including in intrinsically resistant ones. Other mechanisms of resistance to colistin as chromosomal mutations and efflux pump were observed in isolates carrying mcr-1, highlighting colistin resistance complexity in Enterobacteria. Time of use of meropenem was the only independent risk factor for acquisition of mcr-1 gen

Conjugative transfer of plasmid p_8N_qac(MN687830.1) carrying qacA gene from Staphylococcus aureus to Escherichia coli C600: potential mechanism for spreading chlorhexidine resistance

The methicillin resistant Staphylococcus aureus (MRSA) is recognized by its ability to acquire and transferring resistance genes through interspecies conjugative plasmids. However, transference of plasmids from Gram-positive cocci to Gram-negative bacilli is not well characterized. In this report, we describe the transfer of a conjugative plasmid carrying qacA from MRSA to Escherichia coli C600. We performed a conjugation experiment using a chlorhexidine resistant MRSA isolate (ST-105/SCCmec type III) carrying the gene qacA and qacC as the donor and a chlorhexidine susceptible E. coli C600 isolate as the receptor. Transconjugants were selected using MacConkey agar plates containing chlorhexidine in concentrations ranging from 0.25 to 16 g.L-1. To genotypically confirm the transfer of the resistance gene, the transconjugants were screened by Polymerase Chain Reaction (PCR) and submitted to Sanger’s sequencing. MRSA isolates successfully transferred the chlorhexidine resistance gene (qacA) to the recipient E. coli strain C600. The E. coli transconjugant exhibited an important reduction of chlorhexidine susceptibility, with MICs increasing from ≤ 0.25 to ≥ 16 g.L-1 after conjugation. The qacA gene was detected by PCR as well as in the Sanger’s sequencing analysis of DNA from transconjugant plasmids. To the best of our knowledge, this is the first report of the plasmid p_8N_qac(MN687830.1) carrying qacA and its transfer by conjugation from a MRSA to an E. coli. These findings increase concerns on the emergence of resistance dissemination across the genus and emphasizes the importance of continuous antiseptic stewardship

Susceptibility to chlorhexidine and mupirocin among methicillin-resistant Staphylococcus aureus clinical isolates from a teaching hospital

Despite the widespread use of chlorhexidine (CHX) to prevent infection, data regarding the in vitro action of CHX against methicillin-resistant Staphylococcus aureus (MRSA) are limited. Clinical isolates from Hospital das Clinicas, Sao Paulo, Brazil, identified during 2002/2003 and 2012/2013 were studied to describe the susceptibility to CHX and mupirocin, molecular characteristics, and virulence profile of MRSA. Susceptibility test to Mupirocin was performed by the disk diffusion method and to CHX by the agar dilution technique. PCR for virulence genes, mecA gene and Staphylococcal Cassette Chromosome mec (SCCmec) types were investigated as well. Mupirocin- and CHX-resistant isolates were sequenced using the IlluminaTM plataform. Two hundred and sixteen MRSA clinical isolates were evaluated: 154 from infected and 62 from colonized patients. Resistance to mupirocin was observed in four isolates assigned as SCCmec type III and STs (ST05; ST239 and ST105) carrying mupA and blaZ, two of them co-harboring the ileS gene. Only one isolate assigned as SCCmec type III was resistant to CHX (MIC of 8.0 μg.mL-1) and harbored the qacA gene. Resistance to chlorhexidine and mupirocin were found in isolates carrying qacA and mupA in our hospital. Since these genes are plasmid-mediated, this finding draws attention to the potential spread of resistance to mupirocin in our hospital