Polymyxin resistance in Enterobacter cloacae complex in Brazil: phenotypic and molecular characterization.

Enterobacter cloacae complex isolates have been reported as an important nosocomial multidrug resistance pathogen. In the present study, we investigated antimicrobial susceptibility and the colistin-resistance rates, their genetic determinants and clonality among clinical E. cloacae complex isolates from different Brazilian states. For this, an initial screening was carried out on 94 clinical isolates of E. clocacae complex received between 2016 and 2018 by LAPIH-FIOCRUZ, using EMB plates containing 4 µg/mL of colistin, followed MIC determination, resulting in the selection of 26 colistin-resistant isolates from the complex. The presence of carbapenemases encoding genes (blaKPC, blaNDM and blaOXA-48), plasmidial genes for resistance to polymyxins (mcr1-9) and mutations in chromosomal genes (pmrA, pmrB, phoP and phoQ) described as associated with resistance to polymyxin were screened by PCR and DNA sequencing. Finally, the hsp60 gene was sequenced to identify species of the E. cloacae complex and genetic diversity was evaluated by PFGE and MLST. The results have shown that among 94 E. cloacae complex isolates, 19 (20.2%) were colistin-resistant. The resistant strains exhibited MIC ranging from 4 to 128 µg / mL and E. hormaechei subsp. steigerwaltii was the prevalent species in the complex (31,6%), followed by E. cloacae subsp. cloacae (26,3%). The antimicrobials with the highest susceptibility rate were gentamicin (21%) and tigecycline (26%). Carbapenemases encoding genes (blaKPC n = 5, blaNDM n = 1) were detected in 6 isolates and mcr-9 in one. Among the modifications found in PmrA, PmrB, PhoP e PhoQ (two-component regulatory system), only the S175I substitution in PmrB found in E. cloacae subsp cloacae isolates were considered deleterious (according to the prediction of PROVEAN). By PFGE, 13 profiles were found among E. cloacae complex isolates, with EcD the most frequent. Furthermore, by MLST 10 ST's, and 1 new ST, were identified in E. cloacae. In conclusion, no prevalence of clones or association among carbapenemase production and polymyxin resistance was found between the E. cloacae. Thereby, the results suggest that the increased polymyxin-resistance is related to the selective pressure exerted by the indiscriminate use in hospitals. Lastly, this study highlights the urgent need to elucidate the mechanism involved in the resistance to polymyxin in the E. cloacae complex and the development of measures to control and prevent infections caused by these multiresistant bacteria.

Comparative analysis of the antimicrobial resistance and virulence traits in ESBL-producing-Klebsiella pneumoniae ST307 strains colonizing the gastrointestinal tract and causing a fatal bloodstream infection in a leukemia patient.

Klebsiella pneumoniae is an opportunistic pathogen that can colonize the gastrointestinal tract (GIT) of humans. The mechanisms underlying the successful translocation of this pathogen to cause extra-intestinal infections remain unknown, although virulence and antimicrobial resistance traits likely play significant roles in the establishment of infections. We investigated K. pneumoniae strains isolated from GIT colonization (strains Kp_FZcol-1, Kp_FZcol-2 and Kp_FZcro-1) and from a fatal bloodstream infection (strain Kp_HM-1) in a leukemia patient. All strains belonged to ST307, carried a transferable IncF plasmid containing the blaCTX-M-15 gene (pKPN3-307 TypeA-like plasmid) and showed a multidrug-resistance phenotype. Phylogenetic analysis demonstrated that Kp_HM-1 was more closely related to Kp_FZcro-1 than to the other colonizing strains. The Kp_FZcol-2 genome showed 81 % coverage with the Kp_HM-1 246,730 bp plasmid (pKp_HM-1), lacking most of its putative virulence genes. Searching public genomes with similar coverage, we observed the occurrence of this deletion in K. pneumoniae ST307 strains recovered from human colonization and infection in different countries. Our findings suggest that strains lacking the putative virulence genes found in the pKPN3-307 TypeA plasmid are still able to colonize and infect humans, highlighting the need to further investigate the role of these genes for the adaptation of K. pneumoniae ST307 in distinct human body sites.

CABGen: A Web Application for the Bioinformatic Analysis of Bacterial Genomes.

Due to recent developments in NGS technologies, genome sequencing is generating large volumes of new data containing a wealth of biological information. Understanding sequenced genomes in a biologically meaningful way and delineating their functional and metabolic landscapes is a first-level challenge. Considering the global antimicrobial resistance (AMR) problem, investments to expand surveillance and improve existing genome analysis technologies are pressing. In addition, the speed at which new genomic data is generated surpasses our capacity to analyze it with available bioinformatics methods, thus creating a need to develop new, user-friendly and comprehensive analytical tools. To this end, we propose a new web application, CABGen, developed with open-source software. CABGen allows storing, organizing, analyzing, and interpreting bioinformatics data in a friendly, scalable, easy-to-use environment and can process data from bacterial isolates of different species and origins. CABGen has three modules: Upload Sequences, Analyze Sequences, and Verify Results. Functionalities include coverage estimation, species identification, de novo genome assembly, and assembly quality, genome annotation, MLST mapping, searches for genes related to AMR, virulence, and plasmids, and detection of point mutations in specific AMR genes. Visualization tools are also available, greatly facilitating the handling of biological data. The reports include those results that are clinically relevant. To illustrate the use of CABGen, whole-genome shotgun data from 181 bacterial isolates of different species collected in 5 Brazilian regions between 2018 and 2020 were uploaded and submitted to the platform's modules.

Epidemiology and antibiotic resistance trends in clinical isolates of Pseudomonas aeruginosa from Rio de janeiro - Brazil: Importance of mutational mechanisms over the years (1995-2015).

Pseudomonas aeruginosa is a major health concern globally and treating infections caused by MDR-isolates unarguably a humongous challenge that remains an unmet need in modern medicine. To determine patterns and mechanisms of antimicrobial resistance and its spread over the years in Rio de Janeiro, Brazil, 88 P. aeruginosa isolates were selected from 1995 to 2015. Phenotypic and genotypic characterization of antimicrobial resistance was evaluated and isolates were submitted to clonality by PFGE and MLST. PFGE analysis showed a great variability of clonal groups mainly over the past 10 years of this study. STs predominant in the early years (ST804, ST1860, ST487 and ST1602) associated to multidrug resistance (MDR) phenotype were replaced by ST277, ST244, ST1945, ST1791 with extensive drug resistance (XDR) in last years, with significant increase in resistance to carbapenems, fluoroquinolones and aminoglycosides. Colistin resistance was detected in 3.5%. The main mechanisms of antimicrobial resistance were mutational mechanisms (mutations in oprD, mexT and gyrA genes). We found the ESBL genes blaTEM (n = 2), blaSHV (n = 3) and blaCTX (n = 1).The carbapenemases genes was present in ST277 (blaSPM, n = 3), ST1560 (blaKPC, n = 3) and ST1944 (blaKPC, n = 2). The 16S RNA methylase gene (rmtD) was found in five isolates belonged to ST277. In conclusion, molecular epidemiological investigation reveals an increase of antimicrobial resistance in P. aeruginosa over 21 years in Rio de Janeiro with higher population structure and occurrence of high risk clone in the last years. The mutational mechanisms of resistance were present in all XDR isolates.

CTX-M-15-producing uropathogenic Escherichia coli isolates at Rio de Janeiro, Brazil: Molecular epidemiology and MALDI-TOF MS.

Because of the high prevalence of CTX-M-15-producing Escherichia coli isolates causing urinary tract infections in Rio de Janeiro, we have investigated bla-CTX-M-15 gene presence, as well as CTX-M-15 production, in 32 E. coli isolates recovered from the urine of outpatients assisted at a public hospital located in the west zone of Rio. Molecular epidemiology was assessed by PFGE and phylo-typing methods. The work highlights the good performance of MALDI-TOF MS as an alternative tool to detect extended-spectrum beta-lactamases among CTX-M-15-producing E. coli isolates.

Introduction of NDM-1 and OXA-370 from Brazil into Chile in strains of Klebsiella pneumoniae isolated from a single patient.

Carbapenemase-producing Enterobacteriaceae have rapidly disseminated worldwide and can colonize patients in healthcare centers. As in Chile the first isolations of NDM-1 and OXA-370 carbapenemases were related with a patient arriving from Brazil, the genetic relatedness of Klebsiella pneumoniae strains producers of these enzymes and isolated in both countries was assessed. PFGE analyses revealed that the isolates were clonally related, illustrating how travel contributes to the spread of multidrug-resistant microorganisms. In addition, the occurrence of three different carbapenemases in three different K. pneumoniae strains isolated from a single patient is described.

Draft genome sequence of KPC-2-producing Pseudomonas aeruginosa recovered from a bloodstream infection sample in Brazil.

OBJECTIVES: Klebsiella pneumoniae carbapenemase (KPC) is the most widespread carbapenemase in Enterobacteriaceae in Brazil. Although its presence is not common in Pseudomonas aeruginosa, it has been increasingly reported. Here we report a draft genome sequence of a KPC-producing P. aeruginosa strain recovered from a bloodstream infection sample in Brazil. METHODS: The antimicrobial susceptibility of KPC-producing P. aeruginosa CCBH17348 was evaluated by the disk diffusion method, Etest and broth microdilution. Carbapenemase production was confirmed by colorimetric assay (Carba NP) and PCR. Genomic DNA was sequenced by Illumina MiSeq sequencing and was assembled using the A5-Miseq pipeline, and gene annotation was performed using RAST 2.0. The database ResFinder 2.1, CRISPRFinder and MLST website were used to identify resistance genes, clustered regularly interspaced short palindromic repeats (CRISPRs) and sequence types (STs), respectively. RESULTS: Isolate CCBH17348 was considered multidrug-resistant, was susceptible to fluoroquinolones, gentamicin and polymyxin, and belonged to a newly described ST (ST2584), carrying an IncQ1 plasmid with blaKPC-2 and aph(3')-VI genes. Other genes associated with resistance and virulence found in the genome were blaOXA-50, blaPAO, fosA, catB, mutation in oprD and mexT (MexEF-OprN efflux regulator), and exotoxin-encoding genes (exoS, exoY and exoT). CONCLUSIONS: This study highlights the potential risk of new STs of P. aeruginosa carrying blaKPC-2 and the potential spread of blaKPC-2 in an IncQ1 plasmid.

Genomic characterization of an extensively drug-resistant KPC-2-producing Klebsiella pneumoniae ST855 (CC258) only susceptible to ceftazidime-avibactam isolated in Brazil.

In this study, we describe the genetic features of an XDR KPC-2-producing Klebsiella pneumoniae isolate by using whole-genome sequencing, its clinical-epidemiological context and susceptibility against antimicrobial combinations. The isolate belongs to clonal complex 258 and harbors several acquired genes and mutations associated with antimicrobial resistance.

Carbapenem-resistant Acinetobacter pittii strain harboring blaOXA-72 from Brazil.

In this study, we report the isolation of OXA-72-producing Acinetobacter pittii in Brazil. A carbapenem-resistant A. pittii strain was recovered from a hospitalized female patient from Espírito Santo, Southeastern Brazil. PCR screening and DNA sequencing allowed us to identify the presence of blaOXA-72. We observed blaOXA-72 in a ~11kb plasmid and flanked by XerC/XerD-binding sites.

Complete genome sequence of Haloferax gibbonsii strain ARA6, a potential producer of polyhydroxyalkanoates and halocins isolated from Araruama, Rio de Janeiro, Brasil.

Haloferax gibbonsii strain ARA6 is a haloarchaea isolated from saline saltern samples from Vermelha lake, located in Araruama region, Rio de Janeiro, Brazil. Its genome displays 66,2% G+C content and is composed by one circular chromosome of 2,945,391 bp and four circular plasmids comprising 993,063 bp. This genomic information shows H. gibbonsii's potential for biotechnological applications and can also contribute to assign evolutionary traits in the genus Haloferax.

Identification of immunogenic proteins of the bacterium Acinetobacter baumannii using a proteomic approach.

PURPOSE: Acinetobacter baumannii is an important opportunistic pathogen that causes pneumoniae, urinary tract infections, and/or septicemia in immunocompromised patients. This pathogen is frequently associated with nosocomial outbreaks worldwide and has become particularly problematic because of its prevalence and resistance patterns to several antibiotics. In the present study, we used an immunoproteome-based approach to identify immunogenic proteins located on the surface of A. baumannii for the development of a possible immunotherapy against this devastating bacterial infection. EXPERIMENTAL DESIGN: Sera from patients with A. baumannii infections (n = 50) and from a control group of healthy individuals (n = 3) were analyzed for reactivity against A. baumannii outer membrane proteins (OMPs) using Western blot analysis. To identify potential immunogenic proteins in A. baumannii, OMPs were separated by 2DE, and reactive sera from infected patients were randomly selected and divided into two different pools, each containing 15 sera. Finally, MALDI-TOF/TOF mass spectrometric analysis was employed to identify the corresponding proteins. RESULTS: This analysis identified six immunoreactive proteins: OmpA, Omp34kDa, OprC, OprB-like, OXA-23, and ferric siderophore receptor protein. Notably, these proteins are highly abundant on the bacterial surface and involved in virulence, antibiotic resistance, and growth. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support the notion that the proteins identified in the present immunoproteome study could serve as antigen candidates for the development of vaccines and passive immunotherapies against A. baumannii infections.