Multiclonal Expansion of Klebsiella pneumoniae Isolates Producing NDM-1 in Rio de Janeiro, Brazil.

We characterized NDM-1-producing Klebsiella isolates from Rio de Janeiro, Brazil. PCR was applied for resistance and virulence determinants. The genetic context of blaNDM was determined by S1 nuclease pulsed-field gel electrophoresis (PFGE) and hybridization. Genotyping was performed by PFGE and multilocus sequence typing (MLST). Most isolates carried multiple resistance genes and remained susceptible to amikacin, fosfomycin-trometamol, polymyxin B, and tigecycline. The spread of NDM-1-producing Klebsiella pneumoniae was not associated with clonal expansion and appears to be associated with Tn3000.

mgrB Mutations Mediating Polymyxin B Resistance in Klebsiella pneumoniae Isolates from Rectal Surveillance Swabs in Brazil.

We aimed to investigate polymyxin B (PMB) resistance and its molecular mechanisms in 126 Klebsiella pneumoniae isolates from rectal swabs in Brazil. Ten isolates exhibited PMB resistance with interruption of mgrB gene by insertion sequences or missense mutations. Most of the PMB-resistant isolates harbored blaKPC-2 (n = 8) and belonged to clonal complex 258 (CC258) (n = 7). These results highlight the importance of monitoring the spread of polymyxin-resistant bacteria in hospitals, since few options remain to treat multidrug-resistant isolates.

Detection of Carbapenemase Genes in Aquatic Environments in Rio de Janeiro, Brazil.

This study reveals the presence of different carbapenemase genes (blaKPC, blaNDM, blaGES, and blaOXA48-like genes) detected directly from water samples and clonal dispersion (by pulsed-field gel electrophoresis [PFGE] and multilocus sequence typing [MLST]) of KPC-2-producing Enterobacteriaceae in two important urban aquatic matrixes from Rio de Janeiro, Brazil, highlighting the role of aquatic environments as gene pools and the possibility of community spreading.

Clonal Dissemination of OXA-370-Producing Klebsiella pneumoniae in Rio de Janeiro, Brazil.

Enzymes of the OXA-48 family have become some of the most important beta-lactamases in the world. A new OXA-48 variant (OXA-370) was first described for an Enterobacter hormaechei strain isolated in Rio Grande do Sul (southern region of Brazil) in 2013. Here we report detection of the blaOXA-370 gene in 24 isolates belonging to three Enterobacteriaceae species (22 Klebsiella pneumoniae isolates, 1 Enterobacter cloacae isolate, and 1 Enterobacter aerogenes isolate) collected from five hospitals in Rio de Janeiro, Brazil, in 2013 and 2014. The isolates showed a multidrug resistance profile, and 12.5% were resistant to polymyxin B. Besides blaOXA-370, no other carbapenemase genes were observed by PCR, whereas blaOXA-1 was found in all isolates and 22 isolates (91.6%) possessed blaCTX-M-15. Molecular typing of the K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two clonal groups, i.e., KpA (21 isolates) and KpB (1 isolate). KpA was characterized as sequence type 16 (ST16) and KpB as ST1041 by multilocus sequence typing (MLST). ST16 has been observed for KPC-producing K. pneumoniae in Rio de Janeiro. Plasmid analysis performed with six representative OXA-370-producing isolates showed plasmids harboring the blaOXA-370 gene in all strains, ranging from 25 kb to 150 kb. This study suggests that there is an urgent need to investigate the presence of OXA-370 and dissemination of the K. pneumoniae ST16 clone carrying this gene in Brazil.

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil.

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri (CCBH11880), Enterobacter hormaechei subsp. oharae (CCBH10892) and Klebsiella pneumoniae (CCBH13327), isolated in Brazil. Besides blaNDM-1, resistance genes to aminoglycosides [aadA1, aadA2, aac(6')-Ib-cr] and quinolones (qnrA1, qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to the blaNDM-1 gene in all strains.

Draft genome sequence of a multidrug-resistant Acinetobacter baumannii ST15 (CC15) isolated from Brazil.

Acinetobacter baumannii is an important pathogen frequently associated with nosocomial outbreaks around the world. In Brazil, A. baumannii has become particularly problematic because of its prevalence and the carbapenems resistance. Here, we report the draft genome sequence of a multidrug-resistant A. baumannii(ST15/CC15) isolated in 2009 from the state of Espírito Santo (Southeast Brazil). We observed important resistance determinant genes in an estimated genome size of 4,102,788 bp with 3,862 predicted coding regions. A detailed report of the genomic data analysis might help to understand the specific features of highly successful strains belonged to a relevant complex clonal in different Brazilian geographical regions.

First report of NDM-1-producing Acinetobacter baumannii sequence type 25 in Brazil.

New Delhi metallo-ß-lactamase 1 (NDM-1) was first identified in Brazil in Enterobacter hormaechei and Providencia rettgeri in 2013. Here, we describe the first case of NDM-1-producing Acinetobacter baumannii sequence type 25 isolated from the urinary tract of a 71-year-old man who died of multiple complications, including A. baumannii infection. The NDM-1 gene was detected by quantitative PCR, and its sequence confirmed its presence in an ∼ 100-kb plasmid.

Update of the molecular epidemiology of KPC-2-producing Klebsiella pneumoniae in Brazil: spread of clonal complex 11 (ST11, ST437 and ST340).

OBJECTIVES: To perform molecular epidemiology for 113 KPC-producing Klebsiella pneumoniae isolated in 2010 from 12 Brazilian states. METHODS: The resistance profile was determined by disc diffusion and Etest. Genetic polymorphism was analysed by PFGE and multilocus sequence typing. The genetic environment of the bla(KPC) gene was determined by PCR and identification of the carrier plasmid was determined by hybridization. RESULTS: Most of the isolates were multidrug resistant, with 15% and 49% being resistant to polymyxin and tigecycline, respectively. Twenty-two sequence types (STs) were observed, with ST11, ST437 and ST340 [clonal complex 11 (CC11)] being the most prevalent (75% of isolates) observed in 10 states. bla(KPC-2) was associated with transposon Tn4401 'b' and in 36% this gene was found in IncN plasmids of 40 kb. CONCLUSIONS: In Brazil, the spread of bla(KPC-2) is occurring due to dispersion of Tn4401 'b', carried by IncN plasmids of 40 kb, and mainly the dissemination of CC11, with ST437 and ST11 playing an important role.

OXA-23-producing Acinetobacter baumannii: a new hotspot of diversity in Rio de Janeiro?

OBJECTIVES: this study focused on the population structure of OXA-23-producing Acinetobacter baumannii clinical isolates from Rio de Janeiro, Brazil. METHODS: the analysis included several genomic typing methods, including PFGE, two multilocus sequence typing (MLST) schemes, sequence group (SG) determination and bla(OXA-51-like) sequencing. The genomic context of the bla(OXA-23) gene was also evaluated using I-CeuI hybridizations and PCR assays. RESULTS: congruent clustering was obtained revealing four lineages. In accordance, four new sequence types (STs) (ST131, ST132, ST133 and ST134) were obtained with the MLST-OD scheme (associated with the Oxford Database) and four (ST79, ST15 and two new allelic profiles) with the MLST-IP scheme (developed by the Institute Pasteur). Four SGs (SG1, SG4 and two new profiles) were identified, allowing the association of 70% of the isolates with European clone II. bla(OXA-51-like) sequencing revealed the presence of bla(OXA-66), bla(OXA-69), bla(OXA-95) and bla(OXA-132). CONCLUSIONS: identification of new STs together with new SG profiles are findings suggestive of a local diversity hotspot that is worth exploring.

Occurrence of blaOXA-23 gene in imipenem-susceptible Acinetobacter baumannii.

The aim of the current study was to describe the occurrence of the blaOXA-23 gene and the ISAba1 element in imipenem-susceptible Acinetobacter baumannii strains. By performing the polymerase chain reaction mapping using combinations of ISAba1 forward primers and the blaOXA-23-like gene reverse primers, we demonstrated that the ISAba1 element did not occur upstream of the blaOXA-23 gene in five of 31 isolates, which explained the lack of resistance to imipenem despite the presence of the blaOXA-23 gene. All of the blaOXA-23-positive isolates were susceptible to imipenem and meropenem with minimal inhibitory concentration ≤ 4 µg/mL. Pulsed-field gel electrophoresis analysis revealed four genotypes among the five blaOXA-23-positive isolates. The current report of the blaOXA-23 gene in imipenem-susceptible isolates provided evidence that this gene may be silently spread in a hospital environment and highlighted the threat of undetected reservoirs of carbapenemase genes.

Population Structure of KPC-2-Producing Klebsiella pneumoniae Isolated from Surveillance Rectal Swabs in Brazil.

KPC-producing Klebsiella pneumoniae (KPC-Kp) has become an important public health issue. The previous intestinal colonization by KPC-Kp has been an important risk factor associated with the progression to infections. The objective of this study was to assess the genetic characterization of KPC-Kp isolates recovered from human rectal swabs in Brazil. We selected 102 KPC-Kp isolates collected during 2009-2013 in 11 states. Antimicrobial susceptibility was determined by disk diffusion, E-test, and broth microdilution. The resistance and virulence genes were investigated by PCR. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates were mostly resistant to ß-lactams, sulfonamides, chloramphenicol, quinolones, and aminoglycosides but susceptible to fosfomycin/trometamol, polymyxin B, and tigecycline. The blaKPC-2 was mostly associated with Tn4401b. Besides that, the isolates carried blaCTX-M, blaSHV, blaTEM, and aac(6')-Ib in high frequency and aac(3')IIa and qnr genes in moderate frequency. The PFGE revealed 26 pulsotypes and MLST performed in representative strains revealed 23 sequence types, 45% belonging to clonal complex 258 (CC258). Isolates of CC258 were found in all states. Seventy percent of the 102 KPC-Kp isolates belonged to CC258-associated pulsotypes. We describe the dissemination of KPC-2-Kp associated with Tn4401b belonging to CC258 colonizing patients in Brazil, which is also prevalent in infected patients, suggesting a clear colonization-infection correlation.

Genetic information on OXA-23-producing Acinetobacter baumannii strain CCBH15815, belonging to ST730/ST783, isolated from Brazil.

In Brazil, A. baumannii has been described as nosocomial pathogens causing hospital-acquired infections. Current WGS technologies have been useful in identifying of genetic features between Acinetobacter isolates. Here, we report the draft genome sequence of OXA-23 producing A. baumannii CCBH15815 clinical isolate, belonging to ST730/ST783, recovered from a 21-year-old hospitalised patient. We observed important resistance determinant genes, especially beta-lactamases-encoding genes, in an estimated genome size of 4,058,633 bp with 3839 predicted coding regions.

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.

Distribution of Clinical NDM-1-Producing Gram-Negative Bacteria in Brazil.

New Delhi metallo-ß-lactamase (NDM)-producing bacteria have been identified at a worrying rate in Brazil since 2013. Owing to the need to understand the extent of their spread, this study reports the dissemination of blaNDM in different species of Gram-negative bacilli in different regions and states of Brazil. A total of 81 isolates from nine states were studied, including 11 species. All isolates carried blaNDM-1 variant and were considered multidrug resistant. Colistin and amikacin were the agents with higher activity compared with the other drugs tested. The findings indicate that the NDM-1 enzyme is already widespread in the country.

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.

Rapid detection of Yersinia enterocolitica serotype O:3 using a duplex PCR assay.

Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and identification of Y. enterocolitica from various sources on selective media have been seldom successful due to several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we developed a single duplex PCR assay for the detection of Y. enterocolitica ssp. palearctica bioserotype 4/O:3 using DNA extracted from a source. We combined the primer for tufA (elongation factor Tu) with the primer for rfbC (the biosynthesis of the O side chain) in one single reaction, which showed good results when we analyzed 88 Yersinia strains and when it was tested in the DNA from stool samples of two groups of pregnant women, one comprising HIV-positive women and the other comprising of HIV-negative women. Furthermore, the duplex PCR assay was found to be 16 times better in detecting Yersinia spp. in stool samples than the culture-based method. In addition, it was found to be a rapid screening method for the detection of Y. enterocolitica serotype O:3, and it could still detect other Y. enterocolitica serotypes and Yersinia species as well. We anticipate that the duplex PCR assay could be a useful tool for hospital and veterinary surveillance studies on Yersinia worldwide.

Next-generation sequencing virulome analysis of a Yersinia enterocolitica subsp. palearctica bioserotype 4/O:3 ST18 isolated from human blood in Brazil.

Yersinia enterocolitica is a widespread Gram-negative bacterium that causes gastrointestinal disease and other clinical manifestations in humans. Potentially pathogenic Y. enterocolitica has been isolated in Brazil, from human, environmental, food, and animal sources. Herein we report a genome sequence of Y. enterocolitica subsp. palearctica strain YE 19, serotype O:3, biotype 4, sequence type 18, with virulence determinants isolated from human blood in Rio de Janeiro in 2005. The results corroborate other findings that this strain harbors a set of virulence determinants that could play a role in host pathoadaptation and may also justify the successful dissemination of bioserotype 4/O:3 in Brazil. The presence of strains harboring all of these virulence genes in Brazil is a potential threat to young children and immunocompromised individuals, for whom yersiniosis are a significant source of morbidity and mortality. The results of a genomic data analysis will help understand the virulence of Brazilian strains and provide data for Y. enterocolitica studies worldwide.

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.