Phylogenomics analysis of multidrug-resistant Elizabethkingia anophelis in industrial wastewater treatment plant.

AIMS: This study investigated the phylogenetic relatedness of multidrug-resistant Elizabethkingia anophelis recovered from an industrial wastewater treatment plant (WWTPi). METHODS AND RESULTS: The wastewater samples were plated in brain heart infusion agar (4 mg/L ceftazidime, 8 mg/L meropenem, and 2 mg/L polimixin). Four isolates recovered from four stages of WWTPi (influent, aeration, decantation, and treated effluent) were identified and evaluated of susceptibility profiles in the VITEK 2 system. These strains identified as E. meningoseptica were confirmed to be E. anophelis by whole genomic sequencing (Miseq-Illumina) and showed antimicrobial resistance genes of ß-lactams, aminoglycosides, and tetracycline's classes. The ribosomal multilocus sequence typing showed that they belong to the rST 65620 together with clinical strains. The phylogenomic tree revealed the similarity of our strains to those belonging to sublineage 11 and the single nucleotide polymorphism analysis confirmed that they belong to a single clade. CONCLUSIONS: To the best of our knowledge, this is the first study reporting the persistence of multidrug-resistant E. anophelis sublineage 11 along the wastewater treatment.

Occurrence of Klebsiella pneumoniae ST244 and ST11 extensively drug-resistant producing KPC, NDM, OXA-370 in wastewater, Brazil.

AIMS: To investigate the occurrence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and their clonal relationships from hospital and municipal wastewater treatment plants (WWTPs). METHODS AND RESULTS: Eighteen Kl. pneumoniae strains recovered from three WWTPs were identified by matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF). The antimicrobial susceptibility were evaluated by disk-diffusion and the carbapenemases production by Carbapenembac®. The carbapenemases genes were investigated by real-time PCR and the clonal relationship through multilocus sequence typing (MLST). Thirty nine % (7/18) of isolates were classified as multidrug-resistant (MDR), 61.1% (11/18) extensively drug-resistant (XDR), and 83.3% (15/18) showed carbapenemase activity. Three carbapenemase-encoding genes were found, blaKPC (55%), blaNDM (27.8%) and blaOXA-370 (11.1%) as well five sequencing types ST11, ST37, ST147, ST244, and ST281. ST11 and ST244, sharing four alleles were grouped into clonal complex 11 (CC11). CONCLUSIONS: Our results show the importance of monitoring antimicrobial resistance in WWTPs effluents to minimize the risk of spreading bacterial load and ARGs in aquatic ecosystems, using advanced treatment technologies to reduce these emerging pollutants at WWTPs.

Mobile resistome of microbial communities and antimicrobial residues from drinking water supply systems in Rio de Janeiro, Brazil.

Antibiotic resistance genes (ARGs) are widespread in the environment due to the overuse of antibiotics and other pollutants, posing a threat to human and animal health. In this study, we evaluated antimicrobial residues, bacterial diversity and ARGs in two important watersheds, Guandu and São João, that supply drinking water to Rio de Janeiro city, Brazil. In addition, tap water samples were collected from three different cities in Rio de Janeiro State, including the metropolitan area of Rio de Janeiro city. Clarithromycin, sulfamethoxazole and azithromycin were found in untreated water and drinking water in all samples. A greater abundance of Proteobacteria was observed in Guandu and São João watersheds, with most of the sequences belonging to the Gammaproteobacteria class. A plasmidome-focused metagenomics approach revealed 4881 (Guandu), 3705 (São João) and 3385 (drinking water) ARGs mainly associated with efflux systems. The genes encoding metallo-ß-lactamase enzymes (blaAIM, blaGIM, blaIMP, and blaVIM) were detected in the two watersheds and in drinking water samples. Moreover, we demonstrated the presence of the colistin resistance genes mcr-3 and mcr-4 (both watersheds) and mcr-9 (drinking water and Guandu) for the first time in Brazil. Our data emphasize the importance of introducing measures to reduce the disposal of antibiotics and other pollutants capable of promoting the occurrence and spread of the microbial resistome on aquatic environments and predicting possible negative impacts on human health.

The Genome Sequence of Brucella intermedia DF13, a 2,4-Dichlorophenoxyacetic Acid-Degrading Soil Bacterium Isolated in Brazil.

Brucella intermedia/Ochrobactrum intermedium strain DF13 was isolated from Brazilian soil and is able to degrade 2,4-dichlorophenoxyacetic acid (2,4-D). Here, we report on its genome sequence, with 4,570,268 bp and a 57.8% G+C content.

Genomic Analysis of Multidrug-Resistant Enterococcus faecium Harboring vanA Gene from Wastewater Treatment Plants.

The emergence of vancomycin-resistant Enterococcus faecium (Efm) harboring vanA gene and multidrug-resistant determinants is a relevant public health concern. It is an opportunistic pathogen responsible for nosocomial infections widely distributed in the environment, including wastewater treatment plants (WWTPs). Our study addresses a genomic investigation of vanA-carrying Efm from WWTPs in Brazil. Samples from five WWTPs supplied with sewage from different sources were evaluated. Here we present whole-genome sequencing of eight vanA-Efm isolates performed on Illumina MiSeq platform. All these isolates presented multidrug-resistant profile, and five strains were from treated wastewater. Multiple antimicrobial resistance genes (ARGs) were found, such as aph(3')-IIIa, ant(6')-Ia, erm(B), and msrC, some of them being allocated in plasmids. The virulence profile was predominantly constituted by efaAfm and acm genes and all isolates, except for one, were predicted as human pathogens. Multilocus sequence typing analysis revealed a new allele and five different STs, three previously described (ST32, ST168, and ST253) and two novel ones (ST1893 and ST1894). Six strains belonged to CC17, often associated with hospital outbreaks. As far as our knowledge, no genomic studies of vanA-Efm recovered from WWTPs revealed isolates belonging to CC17 in Brazil. Therefore, our findings point to the environmental spread of Efm carrying multiple ARGs.

Genetic Relatedness of NDM-Producing Klebsiella pneumoniae Co-Occurring VIM, KPC, and OXA-48 Enzymes from Surveillance Cultures from an Intensive Care Unit.

Klebsiella pneumoniae is an opportunistic bacterial pathogen most commonly associated with nosocomial infections, especially in intensive care unit (ICU) patients. Routine surveillance cultures for carbapenemase-producing Enterobacteriaceae have become a common practice for hospital infection prevention. The objective of this study was to investigate the genetic relatedness of carbapenem-resistant K. pneumoniae carrying blaNDM gene across multilocus sequence typing (MLST) scheme. Surveillance rectal swabs from 4,463 ICU patients admitted to the Rio de Janeiro hospital (March 2016-2017) were screened on CHROMagar mSuperCARBA. Of these, 631 isolates were subjected to VITEK 2 system for phenotypic microbial identification and antibiotic susceptibility testing. Out of 631 isolates, 108 were identified as K. pneumoniae, 103 of which were confirmed by PCR of 16S-23S rDNA internal transcribed spacer (ITS). Eleven blaNDM-positive isolates were subsequently screened for blaKPC, blaBKC, blaIMP, blaVIM, blaSPM, blaOXA-48, and mcr-1-8 genes. Twenty-seven percent (3/11) revealed co-occurrence with KPC, OXA-48, and VIM, 46% (5/11) with KPC and VIM, and 18% (2/11) with VIM type. No strains harbored the blaBKC, blaSPM, blaIMP, and mcr-1 to 8 resistance genes. All 11 isolates were resistant to ß-lactams, ciprofloxacin 90%, tigecycline 82%, gentamicin 73%, and amikacin 18%, and were classified as multidrug resistant (MDR), extensively drug resistant (XDR), and pandrug resistant (PDR). Molecular epidemiology data based on MLST revealed 11 different STs, 8 of which were novel and 3 were previously described. Six out of the eight new STs were associated with MDR and PDR strains and two clonal complexes were reported, including CC258 and CC15. The coexistence of NDM-producing K. pneumoniae and other carbapenemase has been frequently described worldwide. Moreover, we report for the first time K. pneumoniae co-harboring up to four carbapenemases from active surveillance cultures.

Genomic basis of antibiotic resistance in Vibrio parahaemolyticus strain JPA1.

A multi-resistant strain of Vibrio parahaemolyticus was isolated from a tropical estuary in Rio de Janeiro, Brazil. Genome sequencing was conducted to establish the molecular basis of antibiotic resistance in this organism. The genetic content of this strain revealed it to be a non-virulent lineage that nevertheless possesses several antibiotic resistance determinants.

Virulence determinants in genetically heterogeneous populations of Aeromonads recovered from an urban lagoon.

The diversity and distribution of Aeromonas spp. associated with virulence profiles from the Rodrigo de Freitas Lagoon were investigated using phylogenetic analysis of gyrB/rpoB gene sequences for speciation. The concatenated gyrB/rpoB gene sequences clustered into five species: Aeromonas punctata/caviae (n = 37), A. hydrophila (n = 10), A. dhakensis (n = 16), A. jandaei (n = 1) and A. enteropelogenes/trota (n = 3). The virulence genes (atc/aerA/hlyA/asp/amp) resulted in 19 virulence profiles, distributed heterogeneously among the five Aeromonas species. Out of the 67 isolates, 16% presented five distinct profiles carrying four virulence genes and 7% showed all genes investigated. The hemolytic genes were detected as follows: act 54% (37/67), aerA 36% (24/67), hlyA 26% (18/67) and proteolytic genes such as asp 36% (24/57) and amp in 85% (57/67) were widely distributed in lagoon sampling stations. Meanwhile, 88% (59/67) and 92% (62/67) of the isolates showed hemolytic and proteolytic activity, respectively. Our results demonstrated that concatenated sequences of the gyrB and rpoB genes showed to be an adequate approach for the Aeromonas speciation and prevalence. The high heterogeneity of virulence genes among the species resulted in several virulence profiles, as well as high percentages of hemolytic and proteolytic activity, demonstrating the necessity of further epidemiological surveys of Aeromonas species pathogenicity in an aquatic recreational lagoon.

Genomic basis of antibiotic resistance in Vibrio parahaemolyticus strain JPA1

A multi-resistant strain of Vibrio parahaemolyticus was isolated from a tropical estuary in Rio de Janeiro, Brazil. Genome sequencing was conducted to establish the molecular basis of antibiotic resistance in this organism. The genetic content of this strain revealed it to be a non-virulent lineage that nevertheless possesses several antibiotic resistance determinants.

Perfil de resistência aos antibióticos e prevalência dos genes qacEΔ1 e sul1 em Pseudomonas aeruginosa de efluente hospitalar / Antibiotic resistance pattern and prevalence of qacEΔ1 and sul1 genes in Pseudomonas aeruginosa from hospital wastewater

Introdução: Efluentes hospitalares representam riscos à saúde pública e ambiental devido à presença de microrganismos patogênicos, drogas e produtos químicos. Pseudomonas aeruginosa é um patógeno oportunista frequentemente encontrado no ambiente hospitalar. Objetivo: Avaliar o resistoma de isolados de P. aeruginosa da estação de tratamento de esgoto hospitalar (ETEH) de um complexo hospitalar na cidade do Rio de Janeiro. Método: Vinte isolados dos cinco estágios da ETEH foram identificados como P. aeruginosa pelo sequenciamento do gene 16S rRNA. A suscetibilidade aos antibióticos foi determinada segundo o CLSI e os genes qacEΔ1 e sul1 foram detectados pela PCR. Resíduos de sulfonamidas foram pesquisados por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial. Resultados: Foi demonstrada a presença de sulfametoxazol em nível inferior a 50 ng∙L−1, resistência às sulfonamidas (80%) seguida pelas quinolonas (50%) e 13 perfis de suscetibilidade aos antimicrobianos. Os genes qacEΔ1-sul1 foram detectados em 100% dos isolados, sugerindo a presença de integrons de classe 1 em toda a ETEH. Conclusões: Os resultados sinalizaram limitações no tratamento e a propagação de genes de resistência nas etapas da ETEH. Esses dados contribuem com órgãos competentes no desenho de ações preventivas frente aos impactos negativos à saúde pública.

Introduction: Hospital effluents may pose great environmental risk due to the presence of pathogenic microorganisms, drugs and chemical components. Pseudomonas aeruginosa is an opportunistic pathogen frequently found in hospital environment. Objective: To evaluate the resistome of P. aeruginosa from the hospital wastewater treatment plant (HWTP) in a hospital complex of Rio de Janeiro city. Method: Twenty isolates from the five stages of the HWTP were identified as P. aeruginosa by 16S rRNA gene sequencing analysis. Susceptibility to antibiotics was determined according to CLSI and qacEΔ1 and sul1 genes were detected by PCR. Sulphonamide residues were investigated by high performance liquid chromatography coupled to sequential mass spectrometry. Results: The sulfamethoxazole has been demonstrated at a level below 50 ng L-1. Sulfonamide resistance (80%) has been demonstrated followed by quinolone class (50%) and 13 susceptibility patterns to antimicrobials. The qacEΔ1-sul1 genes were detected in 100% of isolates suggesting the presence of class 1 integrons in the whole HWTP. Conclusions: The results signalized limitations of HWTP and propagation of resistance genes in all stages of the HWTP. These data also contribute to the environmental sanitary surveillance in the design of prevention actions against negative impact on the public health.

Detection of human fecal contamination by nifH gene quantification of marine waters in the coastal beaches of Rio de Janeiro, Brazil.

The identification of fecal pollution in aquatic ecosystems is one of the requirements to assess the possible risks to human health. In this report, physicochemical parameters, Escherichia coli enumeration and Methanobrevibacter smithii nifH gene quantification were conducted at 13 marine waters in the coastal beaches of Rio de Janeiro, Brazil. The pH, turbidity, dissolved oxygen, temperature, and conductivity, carried out by mobile equipment, revealed varied levels due to specific conditions of the beaches. The bioindicators' enumerations were done by defined substrate method, conventional, and real-time PCR. Six marine beach sites (46 %) presenting E. coli levels in compliance with Brazilian water quality guidelines (<2500 MPN/100 mL) showed nifH gene between 5.7 × 109 to 9.5 × 1011 copies. L-1 revealing poor correlation between the two approaches. To our knowledge, this is the first inquiry in qPCR using nifH gene as a biomarker of human-specific sources of sewage pollution in marine waters in Brazil. In addition, our data suggests that alternative indicator nifH gene could be used, in combination with other markers, for source tracking studies to measure the quality of marine ecosystems thereby contributing to improved microbial risk assessment.

Fecal pollution source tracking in waters intended for human supply based on archaeal and bacterial genetic markers.

The determination of fecal pollution sources in aquatic ecosystems is essential to estimate associated health risks. In this study, we evaluate eight microbial source tracking (MST) markers including host-specific Bacteroidales and Methanobrevibacter spp. for discrimination between human, bovine, equine, and swine fecal contamination in waters intended for human supply. Overall, the novel host-specific archaeal and bacterial primers proposed in this study demonstrated high sensitivity and specificity. Markers for the Archaea domain were more prevalent in the fecal and water samples studied. We conclude that the investigations regarding the sources of fecal pollution in public water supplies can contribute to improve the quality of human health. To our knowledge, this is the first analysis using both archaeal and bacterial fecal MST markers on tropical water bodies of Rio de Janeiro city, Brazil.

Detection of fluoroquinolone resistance by mutation ingyrA gene of Campylobacterspp. isolates from broiler and laying (Gallus gallus domesticus) hens,from Rio de Janeiro State, Brazil / Detecção de resistência às fluoroquinolonas através da mutação no gene gyrA em Campylobacter spp. isolados de frangos de corte e galinhas (Gallus gallus domesticus) poedeiras, no Estado do Rio de Janeiro, Brasil

Poultry are considered to be the main reservoir of Campylobacterspp. bacteria, an important pathogen for humans. Many studies have reported a rapid selection of fluoroquinolone-resistant strains following the widespread use of these antimicrobials in poultry production and human medicine. The main mechanism of fluoroquinolone resistance in Campylobacteris a mutation in the Quinolone Resistance Determinant Region (QRDR) in the gyrA gene, which codes for the subunit of the enzyme DNA gyrase, the target for fluoroquinolone. The aim of this study was to investigate the mutation in QRDR in the gyrA gene of Campylobacterstrains previously isolated from broiler carcasses and feces of laying hens. Thirty-eight strains of C. jejuniand 19 C. colistrains (n=57), previously characterized as resistant to ciprofloxacin and enrofloxacin by the disk diffusion method and minimum inhibitory concentration (MIC), were selected. For detection of the mutation, a fragment of 454pb QRDR in the gyrA gene was used for direct sequencing. All strains presented the QRDR mutation in the gyrA gene at codon 86 (Thr-86-Ile), which confers resistance to fluoroquinolones. Other known silent mutations were observed. This genotypic characterization of fluoroquinolone resistance inCampylobacterstrains has confirmed the prior phenotypic detection of the resistance. The Thr-86-Ile mutation was observed in all samples confirming that this is the predominant mutation in enrofloxacin and ciprofloxacin resistant strains of C. jejuniand C. coli.

As aves são consideradas o principal reservatório deCampylobacterspp., um importante patógeno para humanos e muitos estudos têm relatado uma rápida seleção de cepas resistentes às fluoroquinolonas após o uso destes antimicrobianos na produção avícola e na medicina humana. O principal mecanismo de resistência às fluoroquinolonas em Campylobacterconsiste na mutação na Região Determinantes de Resistência às Quinolonas (RDRQ) do gene gyrA, que codifica para a subunidade A da enzima DNA girase, alvo das fluoroquinolonas. O objetivo deste estudo foi investigar a mutação na RDRQ do gene gyrA em cepas de Campylobacterpreviamente isolados de carcaças de frangos de corte e fezes de galinhas poedeiras. Foram selecionadas 38 cepas de C. jejunie 19 cepas de C. coli(n=57), previamente caracterizadas como resistentes à ciprofloxacina e enrofloxacina, pelo método da difusão em disco e pela determinação da concentração inibitória mínima. Para detecção da mutação, foi utilizado sequenciamento direto de um fragmento de 454pb da RDRQ do gene gyrA gerado por PCR. Todas as cepas apresentaram a mutação na RDRQ do gene gyrA no códon 86 (Tre-86-Ile), que confere resistência às fluoroquinolonas e outras mutações silenciosas foram observadas. A caracterização genotípica da resistência às fluoroquinolonas em Campylobacterconfirmou a prévia detecção fenotípica dessa resistência e a mutação Tre-86-Ile foi observada na totalidade das amostras, comprovando ser esta a mutação predominante em cepas de C. jejunie C. coliresistentes à enrofloxacina e ciprofloxacina.

Species-level identification of Bacillus strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis.

The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.