Description and comparative genomic analysis of a mcr-1-carrying Escherichia coli ST683/CC155 recovered from touristic coastal water in Northeastern Brazil.

Polymyxin resistance is an emerging health issue aggravated by mcr dissemination among Enterobacterales recovered from various sources. Commensal Escherichia coli plays a key role in the spread of antimicrobial resistance in community settings and is likely to spread silently. It may transfer resistance genes to pathogenic bacteria in the gastrointestinal tract and the environment, and may cause difficult-to-treat infections, especially in immunocompromised patients. Unraveling actors disseminating resistance to last-resort antimicrobials might support the future development of control measures. Here we report the occurrence of a commensal ST683/CC155 colistin-resistant mcr-1.1-harboring E. coli (JP24) obtained from touristic coastal water. JP24's genome was sequenced and comparatively analyzed with other genomes from ST683/CC155 isolated worldwide and with mcr-carrying isolates recovered from various sources in Brazil. Besides mcr-1, JP24 carried blaCTX-M-8, tet(A), tet(34), dfrA12, sul2, sul3, aph(3')-Ia, aph(3')-IIa, aadA1, aadA2, cmlA1, Inu(G), mef(B) and mdf(a). mcr-1 and blaCTX-M-8 were transferable by IncX4 and IncI1/Iγ plasmids, respectively. Tree-based phylogeny of the ST683/CC155 isolates core genome revealed two larger clades. E. coli JP24 was grouped into a subclade together with an isolate from Thailand (ERR4221036), both carrying mcr-1. The core genome-based tree of the isolates carrying mcr-1 from Brazil revealed proximity with E. coli ECEST9 recovered from a mangrove also located in Northeastern Brazil. Accessory genome-based tree clustered most environmental isolates apart from the clinical ones and remained JP24 closer to ECEST9. High sequence conservation was observed between mcr-1-harboring plasmids detected in different species and reservoirs in Brazil and other countries. In addition to recreational coastal waters being potential sources for community exposure to antimicrobial-resistant bacteria, our findings reinforce a more prominent role of horizontal gene transfer, other than clonal expansion, in mcr dissemination in the community.

Performance of distinct phenotypic methods for carbapenemase detection: The influence of culture media.

We evaluated the performance of five phenotypic tests [Modified Hodge Test (MHT); combined-disk test (CDT) using phenylboronic acid, EDTA, and cloxacillin; CarbaNP and CarbAcinetoNP; Blue-Carba, Carbapenembac™ and Carbapenembac Metallo™] for carbapenemase detection in Gram-negative bacilli (GNB). A total of 73 carbapenemase producers and 27 non-carbapenemase producers were tested. All GNB were subcultured onto Müeller-Hinton agar (MHA), MacConkey agar (MAC), and sheep blood agar (SBA). High sensitivity (100%) and specificity (100%) was observed for MHA using CarbaNP, Blue-Carba, and Carbapenembac™. The sensitivity and specificity of CarbaNP (98.6%/100%), Blue-Carba (97.1%/91.0%), and Carbapenembac™ (100%/96.5%) were slightly lower for SBA. In contrast, unacceptable sensitivity rates of CarbaNP (71.1%) and Blue-Carba (66.6%), but not Carbapenembac™ (97.3%), were observed for MAC. The colorimetric methods showed high sensitivity and specificity to detect carbapenemase production from isolates grown on MHA or SBA. However, colonies obtained from MAC must not be tested for carbapenemase detection by colorimetric methods.

High Frequency of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Nosocomial Strains Isolated from a Teaching Hospital in Brazil.

The aim of this study was to investigate the frequency, antimicrobial sensitivity profile, and genetic characteristics of nosocomial strains of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae isolated from inpatients at a teaching hospital in Brazil. The bacterial identification, phenotypic detection of ESBL, and antimicrobial susceptibility profile were performed by the VITEK 2 automated system. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) mass spectrometry was used to confirm the identity of the species and genotyping of ESBL-producing K. pneumoniae was performed by pulsed-field gel electrophoresis (PFGE). Thirty-six ESBL-producing K. pneumoniae nosocomial strains isolated from November 2013 to August 2014 were analyzed. High resistance rates were observed for ceftriaxone, ceftazidime, cefepime, gentamicin, and ciprofloxacin. However, all isolates were susceptible to amikacin and meropenem. All strains harbored blaCTX-M-like and blaSHV-like genes. Molecular typing by PFGE showed a diversity of genotypes distributed among 25 clusters, but two isolates collected in different wards had the same genotypic profile and carried the same bla genes, so they were considered clones. The data showed that there was a high frequency of ESBL-producing K. pneumoniae multidrug-resistant among patients in the studied hospital. Furthermore, the detection of blaCTX-M-like genes in all isolates suggests that these enzymes are the major ESBL responsible for the beta-lactam resistance phenotypes among the analyzed strains.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil.

The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. ß-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Influence of Culture Media on Detection of Carbapenem Hydrolysis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

In this study, we evaluated the influence of distinct bacterial growth media on detection of carbapenemase hydrolysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. False-negative results were observed for OXA-25-, OXA-26-, and OXA-72-producing Acinetobacter baumannii isolates grown on MacConkey agar medium. The other culture media showed 100% sensitivity and 100% specificity for detecting carbapenemase.