Toward a Universal Unit for Quantification of Antibiotic Resistance Genes in Environmental Samples.

Surveillance of antibiotic resistance genes (ARGs) has been increasingly conducted in environmental sectors to complement the surveys in human and animal sectors under the "One-Health" framework. However, there are substantial challenges in comparing and synthesizing the results of multiple studies that employ different test methods and approaches in bioinformatic analysis. In this article, we consider the commonly used quantification units (ARG copy per cell, ARG copy per genome, ARG density, ARG copy per 16S rRNA gene, RPKM, coverage, PPM, etc.) for profiling ARGs and suggest a universal unit (ARG copy per cell) for reporting such biological measurements of samples and improving the comparability of different surveillance efforts.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil.

BACKGROUND: Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES: This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS: Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS: We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS: This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Concentration and Variety of Carbapenemase Producers in Recreational Coastal Waters Showing Distinct Levels of Pollution.

Carbapenemase-producing bacteria cause difficult-to-treat infections related to increased mortality in health care settings. Their occurrence has been reported in raw sewage, sewage-impacted rivers, and polluted coastal waters, which may indicate their spread to the community. We assessed the variety and concentration of carbapenemase producers in coastal waters with distinct pollution levels for 1 year. We describe various bacterial species producing distinct carbapenemases not only in unsuitable waters but also in waters considered suitable for primary contact.

Heterologous Expression and Functional Characterization of the Exogenously Acquired Aminoglycoside Resistance Methyltransferases RmtD, RmtD2, and RmtG.

The exogenously acquired 16S rRNA methyltransferases RmtD, RmtD2, and RmtG were cloned and heterologously expressed in Escherichia coli, and the recombinant proteins were purified to near homogeneity. Each methyltransferase conferred an aminoglycoside resistance profile consistent with m(7)G1405 modification, and this activity was confirmed by in vitro 30S methylation assays. Analyses of protein structure and interaction with S-adenosyl-l-methionine suggest that the molecular mechanisms of substrate recognition and catalysis are conserved across the 16S rRNA (m(7)G1405) methyltransferase family.

Analysis of distinct blaKPC-encoding plasmids in an Enterobacter kobei strain recovered from recreational coastal water.

In this study we present an in-depth characterization of two blaKPC-2 encoding plasmids found in the Enterobacter kobei FL23 strain recovered from recreational coastal water. The plasmids belong to distinct incompatibility groups and carry a diverse collection of resistance genes. Furthermore, the genetic context of the blaKPC-2 gene was different in each of them. While pEkFL23-IncX3 presents a new Tn4401k, a new isoform, similar to Tn4401b but with a truncated tnpA and a deleted tnpR; pEkFL23-IncU/P6 carries a new isoform of a non-Tn4401 element (NTEKPC), named NTEKPC-IIh. Its difference from NTEKPC-IId is the truncated Tn3 resolvase upstream blaKPC-2. Capacity of conjugation, maintenance rates and fitness cost of both replicons were also assessed. Both were transferred after mating assays, whereas only pEkFL23-IncX3 was transferred under the adverse conditions of Marine broth at 25 °C as a mating platform. A remarkable stability of both plasmids was observed in the parental and transconjugant strains. Finally, both replicons did not impose a significant fitness cost to their transformant hosts, with pEkFL23-IncU/P6 conferring a statistically significant (p < 0.05) advantage in head-to-head competitions. Our findings show that E. kobei FL23 is a disquieting case of a carbapenem-resistant bacteria identified in a community setting, being a possible silent disseminator of two seemingly stable and metabolic weightless multidrug resistance plasmids.

Candida spp. isolated from recreational coastal waters of Rio de Janeiro - Brazil: Focus on antifungal resistance and virulence attributes.

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1-5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87-3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers.

Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae testing susceptible to cefepime by reference methods.

ß-Lactam susceptibility of 499 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae, determined by reference broth microdilution, demonstrated that 14.4% of isolates were categorized as cefepime susceptible according to current CLSI breakpoints. Ceftazidime- and meropenem-susceptible isolates were also observed (2.6 and 3.0%, respectively). Cefepime-susceptible KPC-producing isolates may confuse laboratory staff and clinicians in their therapeutic choices.

Irrigation Ponds as Sources of Antimicrobial-Resistant Bacteria in Agricultural Areas with Intensive Use of Poultry Litter.

Poultry litter is widely used worldwide as an organic fertilizer in agriculture. However, poultry litter may contain high concentrations of antibiotics and/or antimicrobial-resistant bacteria (ARB), which can be mobilized through soil erosion to water bodies, contributing to the spread of antimicrobial resistance genes (ARGs) in the environment. To better comprehend this kind of mobilization, the bacterial communities of four ponds used for irrigation in agricultural and poultry production areas were determined in two periods of the year: at the beginning (low volume of rainfall) and at the end of the rainy season (high volume of rainfall). 16S rRNA gene sequencing revealed not only significantly different bacterial community structures and compositions among the four ponds but also between the samplings. When the DNA obtained from the water samples was PCR amplified using primers for ARGs, those encoding integrases (intI1) and resistance to sulfonamides (sul1 and sul2) and ß-lactams (blaGES, blaTEM and blaSHV) were detected in three ponds. Moreover, bacterial strains were isolated from CHROMagar plates supplemented with sulfamethoxazole, ceftriaxone or ciprofloxacin and identified as belonging to clinically important Enterobacteriaceae. The results presented here indicate a potential risk of spreading ARB through water resources in agricultural areas with extensive fertilization with poultry litter.

Description of a new non-Tn4401 element (NTEKPC-IIe) harboured on IncQ plasmid in Citrobacter werkmanii from recreational coastal water.

OBJECTIVES: Here we describe an IncQ1-like plasmid carrying blaKPC-2 in a new non-Tn4401 element found in Citrobacter werkmanii recovered from coastal water. METHODS: In vitro and in silico approaches were used to assess antimicrobial resistance determinants, as well as blaKPC-2 vicinities. RESULTS: The LB-887 isolate showed a multidrug-resistant phenotype and was identified as C. werkmanii. Resistome analysis identified further acquired resistance determinants to ß-lactams, aminoglycosides, sulphonamides/trimethoprim, tetracyclines, chloramphenicol, macrolides, rifampicin and fluoroquinolones. Plasmidome included incompatibility groups IncA, IncC2, IncR, Col and IncQ families. The blaKPC-2 was inserted on a new variant of NTEKPC-II, called here NTEKPC-IIe, carried by an InQ1-like plasmid of 7930 kb (pKPC-LB887). NTEKPC-IIe differed from NTEKPC-IId by the complete absence of ISKpn6-tnpA. The InQ1-like backbone harbouring this element had been described in Enterobacterales recovered from clinical and environmental settings. CONCLUSION: Unravelling genetic structures related to blaKPC dissemination in different settings may provide clues on the main forces driving evolution of this important resistance determinant. Indeed, the occurrence of blaKPC in a new NTEKPC variant from an environmental source highlights the ongoing evolution of this mobile genetic element. In addition, blaKPC carriage on a small and highly mobilizable IncQ plasmid in C. freundii complex from recreational water, similar to others found in clinical isolates, may suggest its relevance for blaKPC-2 dissemination among different compartments.

qnrVC occurs in different genetic contexts in Klebsiella and Enterobacter strains isolated from Brazilian coastal waters.

OBJECTIVES: In contrast to other qnr families, qnrVC has been reported mainly in Vibrio spp. and inserted in class 1 integrons. This study aimed to identify the variants of qnrVC genes detected in Klebsiella pneumoniae carbapenemase-2-producing Enterobacter and Klebsiella strains isolated from Brazilian coastal waters and the genetic contexts associated with their occurrence. METHODS: qnrVC variants were identified by Sanger sequencing. Stains were typified by pulsed-field gel electrophoresis. Antimicrobial susceptibility testing, conjugation assays, and whole genome sequencing (WGS) were applied to identify the strains' antimicrobial resistance profile, qnrVC and blaKPC-2 co-transference, and qnrVC genetic context. RESULTS: qnrVC1 was identified in 15 Enterobacter and 3 Klebsiella, and qnrVC4 in 2 Enterobacter strains. Pulsed-field gel electrophoresis revealed 12 clonal profiles of Enterobacter and one of Klebsiella. Strains were resistant to aminoglycosides, beta-lactams, fosfomycin, quinolones, and sulfamethoxazole-trimethoprim. Co-transference of qnrVC and blaKPC-2 were obtained from five representative Enterobacter strains, which showed resistance to ampicillin and amoxicillin-clavulanate, and reduced susceptibility to extended-spectrum cephalosporins, meropenem, and ciprofloxacin. WGS analysis from representative strains revealed one K. quasipneumoniae subsp. similipneumoniae, one E. soli, four E. kobei, and seven isolates belonging to Enterobacter Taxon 3. Long-read WGS showed qnrVC and blaKPC-2 were carried by the same replicon on Klebsiella and Enterobacter strains, and the qnrVC association with not previously described genetic environments composed of insertion sequences and truncated genes. These contexts occurred in small- and high-molecular-weight plasmids belonging to IncFII, IncP6, pKPC-CAV1321, and IncU groups. CONCLUSION: Our results suggest that the dissemination of qnrVC among Enterobacterales in Brazilian coastal waters is associated with several genetic recombination events.

Multidrug-resistant Klebsiella quasipneumoniae subsp. similipneumoniae carrying blaNDM-blaCTX-M15 isolated from flies in Rio de Janeiro, Brazil.

OBJECTIVES: Flies have been implicated in the dispersal of medically important bacteria including members of the genus Klebsiella between different environmental compartments. The aim of this study was to retrieve and characterize antibiotic-resistant bacteria from flies collected near to hospitals. METHODS: Flies were collected in the vicinity of medical facilities and examined for bacteria demonstrating phenotypic resistance to ceftriaxone, followed by determination of phenotypic and genotypic resistance profiles. In addition, whole genome sequencing followed by phylogenetic analysis and resistance genotyping were performed with the multidrug-resistant (MDR) strain Lemef23, identified as Klebsiella quasipneumoniae subsp. similipneumoniae. RESULTS: The strain Lemef23, classified by multiple locus sequence typing as novel ST 3397, harboured numerous resistance genes. The blaNDM was located on a Tn3000 element, a common genetic platform for the carriage of this gene in Brazil. Inference of phylogenetic orthology of strain Lemef23 and other clinical isolates suggested an anthropogenic origin. CONCLUSIONS: The findings of this study support the role of flies as vectors of MDR bacteria of clinical importance and provide the first record of blaNDM-1 and blaCTXM-15 in a Brazilian isolate of K. quasipneumoniae subsp. similipneumoniae, demonstrating the value of surveying insects as reservoirs of antibiotic resistance.

Enrichment of potential pathogens in marine microbiomes with different degrees of anthropogenic activity.

Anthropogenic activities in coastal marine ecosystems can lead to an increase in the abundance of potentially harmful microorganisms in the marine environment. To understand anthropogenic impacts on the marine microbiome, we first used publicly available microbial phylogenetic and functional data to establish a dataset of bacterial genera potentially related to pathogens that cause diseases (BGPRD) in marine organisms. Representatives of low-, medium- and highly impacted marine coastal environments were selected, and the abundance and composition of their microbial communities were determined by quantitative PCR and 16 S rRNA gene sequencing. In total, 72 BGPRD were cataloged, and 11, 36 and 37 BGPRD were found in low-, medium- and highly human-impacted ecosystems, respectively. The absolute abundance of BGPRD and the co-occurrence of antibiotic resistance genes (AGR) increased with the degree of anthropogenic perturbation in these ecosystems. Anthropogenically impacted coastal microbiomes were compositionally and functionally distinct from those of less impacted sites, presenting features that may contribute to adverse outcomes for marine macrobiota in the Anthropocene era.

Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results.

OBJECTIVES: The aim of this study was to evaluate the presence of carbapenemases in a Klebsiella pneumoniae collection and the performance of the modified Hodge test (MHT) to correctly identify this phenotype. METHODS: Twenty-eight K. pneumoniae clinical isolates with reduced susceptibility to carbapenems were evaluated. Antimicrobial susceptibility and molecular typing were performed by agar dilution and PFGE, respectively. The MHT was performed using both standard and high inoculum of test organisms. Imipenem hydrolysis was investigated by spectrophotometric assays and carbapenemase-encoding genes were identified by PCR and amplicon sequencing. Porin loss was investigated by both PCR and SDS-PAGE. RESULTS: Susceptibility rates for imipenem, meropenem and ertapenem were 93%, 57% and 11%, respectively. The PFGE analysis showed seven unrelated genotypes. By testing standard inoculum and ertapenem or meropenem discs, 25% (n = 7) and 21% (n = 6) of the isolates were classified as carbapenemase producers, respectively. When a higher inoculum was employed, these rates increased to 54% (n = 15) and 43% (n = 12), respectively. No imipenem hydrolysis was detected. PCRs identified bla(CTX-M) in 27 (96%) isolates, of which 2 isolates also carried bla(GES-1.) SDS-PAGE and PCR assays revealed that all isolates had lost at least one outer membrane protein, except for a single isolate that was found to express both OmpK35 and OmpK36. CONCLUSIONS: False detection of carbapenemase production was observed by the MHT possibly as a result of extended-spectrum beta-lactamase (ESBL) production coupled with porin loss as reported before. Clinical laboratories must be aware of this fact, especially in geographical areas where ESBL-producing isolates are highly prevalent.

Efflux pumps expression and its association with porin down-regulation and beta-lactamase production among Pseudomonas aeruginosa causing bloodstream infections in Brazil.

BACKGROUND: Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile. RESULTS: Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC beta-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance. CONCLUSIONS: Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary beta-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates.

Predictors of carbapenemase-producing bacteria occurrence in polluted coastal waters.

The spread of carbapenemase-producing bacteria is a worldwide concern as it challenges healthcare, especially considering the insufficient development of antimicrobials. These microorganisms have been described not only in hospitals, but also in several environmental settings including recreational waters. Community exposure to antimicrobial-resistant bacteria through recreation might be relevant for human health, but risk assessment studies are lacking. Absence of effective and feasible monitoring in recreational aquatic matrices contributes to such a knowledge gap. Here, we aimed at assessing predictors of occurrence of medically relevant carbapenemase-producing bacteria in coastal waters. We quantitatively assessed recovery of carbapenemase-producing Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp. and Aeromonas spp. in superficial coastal waters showing distinct pollution history across one year, and registered data regarding tide regimen, 7-days pluviosity, salinity, pH, water temperature. We analyzed data using General Estimating Equation (GEE) to assess predictors of such occurrence. Our results suggest that the sampling site had the strongest effect over concentration of these antimicrobial-resistant microorganisms, followed by pollution indexes and tide regimen. Increased salinity, advanced sampling time, water temperature, rainfall and decrease of pH were related to decrease concentrations. We provide a list of factors that could be easily monitored and further included in models aiming at predicting occurrence of carbapenemase producers in coastal waters. Our study may encourage researchers to further improve this list and validate the model proposed, so that monitoring and future public policies can be developed to control the spread of antimicrobial resistance in the environment.

Diversity of beta-lactamases produced by ceftazidime-resistant Pseudomonas aeruginosa isolates causing bloodstream infections in Brazil.

A retrospective survey was conducted to characterize beta-lactamases in a collection of 43 ceftazidime-resistant Pseudomonas aeruginosa isolates recovered from patients with bloodstream infections hospitalized at a Brazilian teaching hospital between January and December 2005. Resistance rates for carbapenems, aminoglycosides, and quinolones were over 80%, with only colistin remaining active against all isolates. Pulsed-field gel electrophoresis analysis identified seven different genotypes. AmpC overproduction was found to be the sole beta-lactamase-mediated mechanism responsible for ceftazidime resistance in four isolates (9.3%). Nine isolates (20.9%) produced an extended-spectrum beta-lactamase (ESBL), either GES-1 (n = 7, 16.3%) or CTX-M-2 (n = 2, 4.6%). Carbapenemase activity was detected in 30 (70%) additional isolates. Among those isolates, two isolates (4.6%) produced the ESBL GES-5, possessing the ability to hydrolyze imipenem; a single isolate (2.3%) produced the metallo-beta-lactamase (MBL) IMP-1; and 27 isolates produced the MBL SPM-1 (62.8%). None of the isolates coproduced both ESBL and MBL. Insertion sequence elements ISCR4 and ISCR1 were associated with bla(SPM-1) and bla(CTX-M-2) genes, respectively, whereas the bla(GES-1) and bla(GES-5) genes were part of class 1 integron structures. This study underlines the spread of MBL- and ESBL-producing P. aeruginosa isolates as an important source of ceftazidime resistance in Brazil.

Outbreak of OXY-2-Producing Klebsiella oxytoca in a renal transplant unit.

We describe a Klebsiella oxytoca infection outbreak in a renal transplant unit that involved seven patients. All strains belonged to a single pulsed-field gel electrophoresis pattern and were resistant to amoxicillin-clavulanate, cefuroxime, piperacillin-tazobactam, and aztreonam but susceptible to ceftriaxone, ceftazidime, cefepime, and imipenem. Chromosomal beta-lactamase hyperproduction was caused by a point mutation in the bla(OXY-2) gene promoter region.

Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates.

The emergence of metallo-beta-lactamase (MBL)-producing isolates is a challenge to routine microbiology laboratories, since there are no standardized methods for detecting such isolates. The aim of this study was to evaluate the accuracy of different phenotypic methods to detect MBL production among Pseudomonas spp., Acinetobacter spp., and enterobacterial isolates, including GIM, IMP, SIM, SPM, and VIM variants. A total of 46 genetically unrelated Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter sp., and enterobacterial strains producing distinct MBLs were tested. Nineteen strains were included as negative controls. The inhibition of bacterial growth and beta-lactam hydrolysis caused by MBL inhibitors (IMBL) also were evaluated. The isolates were tested for MBL production by both a double-disk synergy test (DDST) and a combined disk assay (CD) using imipenem and ceftazidime as substrates in combination with distinct IMBL. One hundred percent sensitivity and specificity were achieved by DDST using 2-mercaptopropionic acid in combination with ceftazidime and imipenem for the detection of MBL production among P. aeruginosa and Acinetobacter species isolates, respectively. The CD test showed the same results for detecting MBL-producing enterobacteria by combining imipenem and EDTA, with a 5.0-mm-breakpoint increase in the size of the inhibition zone. Our results indicate that both phenotypic methods to detect MBL-producing isolates should be based on the genera to be tested, regardless of the enzyme produced by such isolates, as well as on the local prevalence of MBL producers.