Integrative genome-centric metagenomics for surface water surveillance: Elucidating microbiomes, antimicrobial resistance, and their associations.

Surface water ecosystems are intimately intertwined with anthropogenic activities and have significant public health implications as primary sources of irrigation water in agricultural production. Our extensive metagenomic analysis examined 404 surface water samples from four different geological regions in Chile and Brazil, spanning irrigation canals (n = 135), rivers (n = 121), creeks (n = 74), reservoirs (n = 66), and ponds (n = 8). Overall, 50.25 % of the surface water samples contained at least one of the pathogenic or contaminant bacterial genera (Salmonella: 29.21 %; Listeria: 6.19 %; Escherichia: 35.64 %). Furthermore, a total of 1,582 antimicrobial resistance (AMR) gene clusters encoding resistance to 25 antimicrobial classes were identified, with samples from Brazil exhibiting an elevated AMR burden. Samples from stagnant water sources were characterized by dominant Cyanobacteriota populations, resulting in significantly reduced biodiversity and more uniform community compositions. A significant association between taxonomic composition and the resistome was supported by a Procrustes analysis (p < 0.001). Notably, regional signatures were observed regarding the taxonomic and resistome profiles, as samples from the same region clustered together on both ordinates. Additionally, network analysis illuminated the intricate links between taxonomy and AMR at the contig level. Our deep sequencing efforts not only mapped the microbial landscape but also expanded the genomic catalog with newly characterized metagenome-assembled genomes (MAGs), boosting the classification of reads by 12.85 %. In conclusion, this study underscores the value of metagenomic approaches in surveillance of surface waters, enhancing our understanding of microbial and AMR dynamics with far-reaching public health and ecological ramifications.

Unveiling the genomic landscape of Salmonella enterica serotypes Typhimurium, Newport, and Infantis in Latin American surface waters: a comparative analysis.

Surface waters are considered ecological habitats where Salmonella enterica can persist and disseminate to fresh produce production systems. This study aimed to explore the genomic profiles of S. enterica serotypes Typhimurium, Newport, and Infantis from surface waters in Chile, Mexico, and Brazil collected between 2019 and 2022. We analyzed the whole genomes of 106 S. Typhimurium, 161 S. Newport, and 113 S. Infantis isolates. Our phylogenetic analysis exhibited distinct groupings of isolates by their respective countries except for a notable case involving a Chilean S. Newport isolate closely related to two Mexican isolates, showing 4 and 13 single nucleotide polymorphisms of difference, respectively. The patterns of the most frequently detected antimicrobial resistance genes varied across countries and serotypes. A strong correlation existed between integron carriage and genotypic multidrug resistance (MDR) across serotypes in Chile and Mexico (R > 0.90, P < 0.01), while integron(s) were not detected in any of the Brazilian isolates. By contrast, we did not identify any strong correlation between plasmid carriage and genotypic MDR across diverse countries and serotypes.IMPORTANCEUnveiling the genomic landscape of S. enterica in Latin American surface waters is pivotal for ensuring public health. This investigation sheds light on the intricate genomic diversity of S. enterica in surface waters across Chile, Mexico, and Brazil. Our research also addresses critical knowledge gaps, pioneering a comprehensive understanding of surface waters as a reservoir for multidrug-resistant S. enterica. By integrating our understanding of integron carriage as biomarkers into broader MDR control strategies, we can also work toward targeted interventions that mitigate the emergence and dissemination of MDR in S. enterica in surface waters. Given its potential implications for food safety, this study emphasizes the critical need for informed policies and collaborative initiatives to address the risks associated with S. enterica in surface waters.

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.

WHO Critical Priority Escherichia coli as One Health Challenge for a Post-Pandemic Scenario: Genomic Surveillance and Analysis of Current Trends in Brazil.

The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens is a critical issue that is no longer restricted to hospital settings. The rapid spread of critical priority pathogens in Brazil is notably worrying, considering its continental dimension, the diversity of international trade, livestock production, and human travel. We conducted a nationwide genomic investigation under a One Health perspective that included Escherichia coli strains isolated from humans and nonhuman sources, over 45 years (1974-2019). One hundred sixty-seven genomes were analyzed extracting clinically relevant information (i.e., resistome, virulome, mobilome, sequence types [STs], and phylogenomic). The endemic status of extended-spectrum ß-lactamase (ESBL)-positive strains carrying a wide diversity of blaCTX-M variants, and the growing number of colistin-resistant isolates carrying mcr-type genes was associated with the successful expansion of international ST10, ST38, ST115, ST131, ST354, ST410, ST648, ST517, and ST711 clones; phylogenetically related and shared between human and nonhuman hosts, and polluted aquatic environments. Otherwise, carbapenem-resistant ST48, ST90, ST155, ST167, ST224, ST349, ST457, ST648, ST707, ST744, ST774, and ST2509 clones from human host harbored blaKPC-2 and blaNDM-1 genes. A broad resistome to other clinically relevant antibiotics, hazardous heavy metals, disinfectants, and pesticides was further predicted. Wide virulome associated with invasion/adherence, exotoxin and siderophore production was related to phylogroup B2. The convergence of wide resistome and virulome has contributed to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. IMPORTANCE A One Health approach for antimicrobial resistance must integrate whole-genome sequencing surveillance data of critical priority pathogens from human, animal and environmental sources to track hot spots and routes of transmission and developing effective prevention and control strategies. As part of the Grand Challenges Explorations: New Approaches to Characterize the Global Burden of Antimicrobial Resistance Program, we present genomic data of WHO critical priority carbapenemase-resistant, ESBL-producing, and/or colistin-resistant Escherichia coli strains isolated from humans and nonhuman sources in Brazil, a country with continental proportions and high levels of antimicrobial resistance. The present study provided evidence of epidemiological and clinical interest, highlighting that the convergence of wide virulome and resistome has contributed to the persistence and rapid spread of international high-risk clones of E. coli at the human-animal-environmental interface, which must be considered a One Health threat that requires coordinated actions to reduce its incidence in humans and nonhuman hosts.

Antimicrobial Resistance in Farm Animals in Brazil: An Update Overview.

In animal husbandry, antimicrobial agents have been administered as supplements to increase production over the last 60 years. Large-scale animal production has increased the importance of antibiotic management because it may favor the evolution of antimicrobial resistance and select resistant strains. Brazil is a significant producer and exporter of animal-derived food. Although Brazil is still preparing a national surveillance plan, several changes in legislation and timely programs have been implemented. Thus, Brazilian data on antimicrobial resistance in bacteria associated with animals come from official programs and the scientific community. This review aims to update and discuss the available Brazilian data on this topic, emphasizing legal aspects, incidence, and genetics of the resistance reported by studies published since 2009, focusing on farm animals and derived foods with the most global public health impact. Studies are related to poultry, cattle, and pigs, and mainly concentrate on non-typhoid Salmonella, Escherichia coli, and Staphylococcus aureus. We also describe legal aspects of antimicrobial use in this context; and the current occurrence of genetic elements associated with resistance to beta-lactams, colistin, and fluoroquinolones, among other antimicrobial agents. Data here presented may be useful to provide a better understanding of the Brazilian status on antimicrobial resistance related to farm animals and animal-derived food products.

Colistin resistance emerges in pandrug-resistant Klebsiella pneumoniae epidemic clones in Rio de Janeiro, Brazil.

Klebsiella pneumoniae is an important human pathogen, able to accumulate and disseminate a variety of antimicrobial resistance genes. Resistance to colistin, one of the last therapeutic options for multi-drug-resistant bacteria, has been reported increasingly. Colistin-resistant K. pneumoniae (ColRKp) emerged in two hospitals in Rio de Janeiro state, Brazil in 2016. The aim of this study was to investigate if these ColRKp isolates were clonally related when compared between hospitals, to identify the molecular mechanisms of colistin resistance, and to describe other antimicrobial resistance genes carried by isolates. Twenty-three isolates were successively recovered, and the whole-genome sequence was analysed for 10, each of a different pulsed-field gel electrophoresis (PFGE) type. Although some PFGE clusters were found, none of them included isolates from both hospitals. Half of the isolates were assigned to CC258, three to ST152 and two to ST15. One isolate was pandrug resistant, one was extensively drug resistant, and the others were multi-drug resistant. Colistin resistance was related to mutations in mgrB, pmrB, phoQ and crrB. Eleven new mutations were found in these genes, including two nucleotide deletions in mgrB. All isolates were carbapenem resistant, and seven were associated with carbapenemase carriage (blaKPC-2 in six isolates and blaOXA-370 in one isolate). All isolates had a blaCTX-M, and two had a 16S ribosomal RNA methyltransferase encoding gene (armA and rmtB). ColRKp were composed of epidemic clones, but cross-dissemination between hospitals was not detected. Colistin resistance emerged with several novel mutations amid highly resistant strains, further restricting the number of drugs available and leading to pandrug resistance.

Evidence for Clonally Associated Increasing Rates of Azithromycin Resistant Neisseria gonorrhoeae in Rio de Janeiro, Brazil.

Azithromycin is one of the drugs used in the combined therapy for syndromic treatment of gonorrhoea in many countries, including Brazil. Our research group, which receives isolates from clinical laboratories since 2006, has detected, after 2016, a tendency of rising rates of azithromycin resistance, with isolates showing higher minimal inhibitory concentrations (MICs) than those previously reported in this country. In this study, we report the susceptibility to azithromycin of 93 N. gonorrhoeae isolates obtained between 2014 and 2017. Strains with MIC ≥2 µg/mL were characterized according to azithromycin resistance mechanisms and strain typing. Results indicate that azithromycin resistance has emerged in all these years in unrelated MLST-STs, but after 2016 a clonal complex connected with ST1901 has been more frequently detected, grouping isolates with MIC varying from 2 to 64 µg/mL, with DelA mutations at the mtrR promoter region associated or not with mutations at rrl alleles. High rates of azithromycin resistance may compromise the use of this drug in the combined therapy with ceftriaxone. Inclusion of Rio de Janeiro in the Brazilian gonococcal surveillance program is important to evaluate if this data indicates an epidemiological phenomenon in the country.

Draft Genome Sequence of a Tetracycline-Resistant Plesiomonas shigelloides Strain Isolated from Aquaculture-Reared Tilapia.

We hereby present the 3.7-Mb draft genome sequence of Plesiomonas shigelloides strain FM82, isolated from a tilapia (Oreochromis niloticus) reared in a fish farm in Rio de Janeiro, Brazil. P. shigelloides strain FM82 carries antimicrobial resistance, biofilm, and CRISPR-related genes.

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.

Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples

Abstract The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false–positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

Modified Carba NP test for the detection of carbapenemase production in gram-negative rods: optimized handling of multiple samples.

The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false-positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II.

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.