Genetic diversity of KPC-2-producing Klebsiella pneumoniae complex from aquatic ecosystems.

During the COVID-19 pandemic, the occurrence of carbapenem-resistant Klebsiella pneumoniae increased in human clinical settings worldwide. Impacted by this increase, international high-risk clones harboring carbapenemase-encoding genes have been circulating in different sources, including the environment. The blaKPC gene is the most commonly disseminated carbapenemase-encoding gene worldwide, whose transmission is carried out by different mobile genetic elements. In this study, blaKPC-2-positive Klebsiella pneumoniae complex strains were isolated from different anthropogenically affected aquatic ecosystems and characterized using phenotypic, molecular, and genomic methods. K. pneumoniae complex strains exhibited multidrug-resistant and extensively drug-resistant profiles, spotlighting the resistance to carbapenems, ceftazidime-avibactam, colistin, and tigecycline, which are recognized as last-line antimicrobial treatment options. Molecular analysis showed the presence of several antimicrobial resistance, virulence, and metal tolerance genes. In-depth analysis showed that the blaKPC-2 gene was associated with three different Tn4401 isoforms (i.e., Tn4401a, Tn4401b, and Tn4401i) and NTEKPC elements. Different plasmid replicons were detected and a conjugative IncN-pST15 plasmid harboring the blaKPC-2 gene associated with Tn4401i was highlighted. K. pneumoniae complex strains belonging to international high-risk (e.g., ST11 and ST340) and unusual clones (e.g., ST323, ST526, and ST4216) previously linked to clinical settings. In this context, some clones were reported for the first time in the environmental sector. Therefore, these findings evidence the occurrence of carbapenemase-producing K. pneumoniae complex strains in aquatic ecosystems and contribute to the monitoring of carbapenem resistance worldwide.

Emergence of carbapenem-resistant Klebsiella pneumoniae species complex from agrifood system: detection of ST6326 co-producing KPC-2 and NDM-1.

BACKGROUND: Klebsiella pneumoniae species complex (KpSC) is an important disseminator of carbapenemase-encoding genes, mainly blaKPC-2 and blaNDM-1, from hospitals to the environment. Consequently, carbapenem-resistant strains can be spread through the agrifood system, raising concerns about food safety. Therefore, this study aimed to isolate carbapenem-resistant KpSC strains from agricultural and environmental sectors and characterize them by phenotypic, molecular, and genomic analyses. RESULTS: K. pneumoniae and Klebsiella quasipneumoniae strains isolated from soils with lemon, guava, and fig cultivation, and surface waters displayed an extensive drug resistance profile and carried blaKPC-2, blaNDM-1, or both. In addition to carbapenemase-encoding genes, KpSC strains harbor a broad resistome (antimicrobial resistance and metal tolerance) and present putative hypervirulence. Soil-derived K. pneumoniae strains were assigned as high-risk clones (ST11 and ST307) and harbored the blaKPC-2 gene associated with Tn4401b and Tn3-like elements on IncN-pST15 and IncX5 plasmids. In surface waters, the coexistence of blaKPC-2 and blaNDM-1 genes was identified in the K. pneumoniae ST6326, a new carbapenem-resistant regional Brazilian clone. In this case, blaKPC-2 with Tn4401a isoform and blaNDM-1 associated with a Tn125-like transposon were located on different plasmids. In addition, K. quasipneumoniae ST526 presented the blaNDM-1 gene associated with a Tn3000 transposon on an IncX3 plasmid. CONCLUSION: These findings alert for the transmission of carbapenemase-positive KpSC across the agricultural and environmental sectors, raising critical food safety and environmental issues. This article is protected by copyright. All rights reserved.

The relationship between water quality and the microbial virulome and resistome in urban streams in Brazil.

Urban streams that receive untreated domestic and hospital waste can transmit infectious diseases and spread drug residues, including antimicrobials, which can then increase the selection of antimicrobial-resistant bacteria. Here, water samples were collected from three different urban streams in the state of São Paulo, Brazil, to relate their range of Water Quality Indices (WQIs) to the diversity and composition of aquatic microbial taxa, virulence genes (virulome), and antimicrobial resistance determinants (resistome), all assessed using untargeted metagenome sequencing. There was a predominance of phyla Proteobacteria, Actinobacteria, and Bacteroidetes in all samples, and Pseudomonas was the most abundant detected genus. Virulence genes associated with motility, adherence, and secretion systems were highly abundant and mainly associated with Pseudomonas aeruginosa. Furthermore, some opportunistic pathogenic genera had negative correlations with WQI. Many clinically relevant antimicrobial resistance genes (ARGs) and efflux pump-encoding genes that confer resistance to critically important antimicrobials were detected. The highest relative abundances of ARGs were ß-lactams and macrolide-lincosamide-streptogramin. No statistically supported relationship was detected between the abundance of virulome/resistome and collection type/WQI. On the other hand, total solids were a weak predictor of gene abundance patterns. These results provide insights into various microbial outcomes given urban stream quality and point to its ecological complexity. In addition, this study suggests potential consequences for human health as mediated by aquatic microbial communities responding to typical urban outputs.

Genomic features of an extensively drug-resistant and NDM-1-positive Klebsiella pneumoniae ST340 isolated from river water.

The environmental contamination plays a significant role in the emergence of antimicrobial resistance. In this study, we report a genomic analysis of an extensively drug-resistant and blaNDM-1-producing Klebsiella pneumoniae (EW807) strain recovered from a surface water sample. Strain EW807 belonged to sequence type (ST) 340 and serotype O4:KL15, a high-risk clone of the clonal group 258. This strain carried a broad resistome, including blaNDM-1 and blaCTX-M-15. The core genome multilocus sequence typing phylogenetic analysis revealed that the EW807 strain was most related to strains from Brazil and the USA. An IncX3 plasmid was identified harboring the blaNDM-1 gene, while an IncFIB(K) plasmid was detected carrying the blaCTX-M-15 in addition to multidrug resistance and multimetal tolerance regions. IncX3 and IncFIB(K) plasmids shared high similarity with plasmids from a human in China and a dog in Brazil, respectively. The regions harboring the blaNDM-1 and blaCTX-M-15 genes contained sequences from the Tn3 family. These findings suggest that IncX3 plasmid could play a role in the spread of NDM-1 in a post-pandemic scenario. To the best of our knowledge, this is the first report of blaNDM-1-producing K. pneumoniae ST340 O4:KL15 strain in the environment. Therefore, the presence of high-risk clones of K. pneumoniae carrying carbapenemases in the environment requires strict surveillance.

Patterns of antimicrobial resistance and metal tolerance in environmental Pseudomonas aeruginosa isolates and the genomic characterization of the rare O6/ST900 clone.

Pseudomonas aeruginosa can harbor several virulence and antimicrobial resistance genes (ARGs). In this regard, virulent and multidrug-resistant (MDR) P. aeruginosa strains are closely related to severe infections. In addition, this species can also carry metal tolerance genes, selecting mainly antimicrobial-resistant strains. The action of several pollutants on the environment may favor the occurrence of antimicrobial-resistant and metal-tolerant strains. Therefore, the aim of this study was to characterize potentially pathogenic, antimicrobial-resistant, and/or metal-tolerant P. aeruginosa isolates from different environmental samples (waters, soils, sediments, or sands) and to perform a whole-genome sequence-based analysis of a rare clone from residual water. Environmental isolates carried virulence genes related to adherence, invasion, and toxin production, and 79% of the isolates harbored at least five virulence genes. In addition, the isolates were resistant to different antimicrobials, including important antipseudomonal agents, and 51% of them were classified as MDR, but only ARGs associated with aminoglycoside resistance were found. Furthermore, some isolates were tolerant mainly to copper, cadmium, and zinc, and presented metal tolerance genes related to these compounds. Whole-genome characterization of an isolate with unique phenotype with simultaneous resistance to antimicrobials and metals showed nonsynonymous mutations in different antimicrobial resistance determinants and revealed a classification of O6/ST900 clone as rare, potentially pathogenic, and predisposed to acquire multidrug resistance genes. Therefore, these results draw attention to the dissemination of potentially pathogenic, antimicrobial-resistant, and metal-tolerant P. aeruginosa isolates in environmental niches, alerting to a potential risk mainly to human health.

Genetic plurality of blaKPC-2-harboring plasmids in high-risk clones of Klebsiella pneumoniae of environmental origin.

International high-risk clones of Klebsiella pneumoniae are important human pathogens that are spreading to the environment. In the COVID-19 pandemic scenario, the frequency of carbapenemase-producing strains increased, which can contribute to the contamination of the environment, impacting the surrounding and associated ecosystems. In this regard, KPC-producing strains were recovered from aquatic ecosystems located in commercial, industrial, or agricultural areas and were submitted to whole-genome characterization. K. pneumoniae and Klebsiella quasipneumoniae subsp. quasipneumoniae strains were assigned to high-risk clones (ST11, ST340, ST307) and the new ST6325. Virulome analysis showed genes related to putative hypervirulence. Strains were resistant to almost all antimicrobials tested, being classified as extensively drug-resistant or multidrug-resistant. In this context, a broad resistome (clinically important antimicrobials and hazardous metal) was detected. Single replicon (IncX5, IncN-pST15, IncU) and multireplicon [IncFII(K1)/IncFIB(pQil), IncFIA(HI1)/IncR] plasmids were identified carrying the blaKPC-2 gene with Tn4401 and non-Tn4401 elements. An unusual association of blaKPC-2 and qnrVC1 and the coexistence of blaKPC-2 and mer operon (mercury tolerance) was found. Comparative analysis revealed that blaKPC-2-bearing plasmids were most similar to plasmids from Enterobacterales of Brazil, China, and the United States, evidencing the long persistence of plasmids at the human-animal-environmental interface. Furthermore, the presence of uncommon plasmids, displaying the interspecies, intraspecies, and clonal transmission, was highlighted. These findings alert for the spread of high-risk clones producing blaKPC-2 in the environmental sector and call attention to rapid dispersion in a post-pandemic world.

Occurrence and genetic characteristics of multidrug-resistant Escherichia coli isolates co-harboring antimicrobial resistance genes and metal tolerance genes in aquatic ecosystems.

Multidrug-resistant (MDR) Escherichia coli isolates (n = 50) were recovered from aquatic ecosystems, which presented high counts of E. coli and metal values within the recommended range. These isolates showed different multidrug resistance profiles, highlighting the resistance to extended-spectrum cephalosporins, polymyxins, and fluoroquinolones. Several antimicrobial resistance genes (ARGs) were found, spotlighting the presence of at least one ß-lactamase-encoding gene in each E. coli isolate. Substitutions in the quinolone resistance-determining regions and the two-component systems involving PhoP/PhoQ and PmrA/PmrB were also found. The metal tolerance gene rcnA (nickel and cobalt efflux pump) was the most prevalent. In this regard, 94% of E. coli isolates presented the co-occurrence of at least one ARG and metal tolerance gene. Furthermore, virulence genes and genetic diversity were found among MDR E. coli isolates. The emergence of potentially pathogenic isolates exhibiting multidrug resistance and metal tolerance emerged as a global health problem at the human-animal-environment interface.

Phenothiazinium dyes for photodynamic treatment present lower environmental risk compared to a formulation of trifloxystrobin and tebuconazole.

The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents.

Colistin-resistant mcr-1-positive Escherichia coli ST1775-H137 co-harboring blaCTX-M-2 and blaCMY-2 recovered from an urban stream.

The rapid dissemination of colistin resistance mcr-type genes and extended-spectrum ß-lactamase-encoding genes at the human-animal-environment interface has raised concerns worldwide. In this study, we performed a genomic investigation of a multidrug (MDR)- and colistin-resistant Escherichia coli strain recovered from an urban stream strongly affected by pollution and used for recreational purposes in Brazil. E. coli strain EW827 was resistant to clinically significant antimicrobials, including polymyxins, extended-spectrum cephalosporins, and fluoroquinolones. Whole-genome sequencing analysis revealed that EW827 strain belonged to ST1775 and carried the fimH137 allele, clinically relevant antimicrobial resistance genes (e.g., mcr-1.1, blaCTX-M-2, and blaCMY-2), tolerance genes to metals, and biocide resistance genes. Moreover, IncX4 and IncI1-ST12 replicon types were identified carrying mcr-1.1 and blaCMY-2, respectively. A novel genetic environment of the mcr-1.1 gene, in which a 258-bp ∆IS5-like was inserted in the opposite orientation upstream of the mcr-1.1-pap2 element, was also detected. Additionally, the blaCTX-M-2 gene was harbored by a Tn21-like element on the chromosome. The occurrence of MDR E. coli co-harboring mcr-1.1, blaCTX-M-2, and blaCMY-2 in urban water represents a potential risk to humans, animals, and environmental safety. Therefore, epidemiological studies are required to monitoring multidrug-resistant bacteria and their antimicrobial resistance genes in aquatic ecosystems to determine possible routes and fates of these genes.

Widespread high-risk clones of multidrug-resistant extended-spectrum ß-lactamase-producing Escherichia coli B2-ST131 and F-ST648 in public aquatic environments.

Aquatic environments are considered a reservoir for the dissemination of multidrug-resistant (MDR) bacteria, principally Escherichia coli, with the consequent spread of acquired antimicrobial resistance genes (ARGs). Widespread high-risk clones of MDR E. coli are responsible for human infections worldwide. This study aimed to characterise, through whole-genome sequencing (WGS), isolates of MDR E. coli harbouring ARGs obtained from public aquatic environments in Brazil. MDR E. coli isolates were obtained from rivers, streams and lakes that presented different Water Quality Index records and were submitted to WGS. The resistome, mobilome and virulome showed a great diversity of ARGs, plasmids and virulence genes, respectively. In addition, mutations in the quinolone resistance-determining regions of GyrA, ParC and ParE as well as several metal resistance genes (MRGs) and antibacterial biocide resistance genes (ABGs) were detected. Typing and subtyping of MDR E. coli revealed different lineages, with two belonging to widespread high-risk clones (i.e. B2-ST131-fimH30 and F-ST648-fimH27), which are grouped by core genome multilocus sequence typing (cgMLST) in clusters with E. coli lineages obtained from different sources distributed worldwide. MDR bacteria carrying MRGs and ABGs have emerged as a global human and environmental health problem. Detection of widespread high-risk clones calls for attention to the dissemination of fluoroquinolone-resistant QnrS1- and CTX-M-producing E. coli lineages associated with human infections in public aquatic environments.

Genomic insights into multidrug-resistant and hypervirulent Klebsiella pneumoniae co-harboring metal resistance genes in aquatic environments.

Klebsiella pneumoniae is one of the most important pathogens related to hospital-acquired infections. The incidence of infections by hypervirulent K. pneumoniae (hvKp), especially community-acquired infections, has been increasing in recent decades. The occurrence of multidrug-resistant (MDR) hvKp has been increasingly reported worldwide decreasing the treatment options, which is a concern. Aquatic environments have been considered a reservoir of MDR pathogens, which contribute to the spread of MDR pathogens. Therefore, this study aimed to characterize MDR-hvKp strains obtained from public aquatic environments using whole genome sequencing in Brazil. Resistome analysis showed ARGs to ß-lactams, quinolones, tetracyclines, sulfonamides, and fosfomycin as well as several metal resistance genes. Virulome analysis showed several virulence genes. Besides, genomic islands, CRISPR and prophage-related sequences were also detected. MLST analysis revealed the presence of two novel sequences types (STs) belonging to different clonal complexes (CCs) [ST4415 (CC515) and ST4416 (CC2654)], and one already described [ST661 (CC661)]. The presence of MDR-hvKp lineages in water sources belonging to STs and CCs associated with humans and animals shows the ability of these pathogens to spread to different aquatic environments. This study reports for the first time two novel STs of MDR-hvKp as well as the presence of a rare ST661 closely related to outbreaks in aquatic environments, and contributes to surveillance studies and MDR-hvKp monitoring worldwide.

High Level of Resistance to Antimicrobials and Heavy Metals in Multidrug-Resistant Pseudomonas sp. Isolated from Water Sources.

Antimicrobial-resistant bacteria (ARB) carrying antimicrobial resistance genes (ARGs) have been increasingly detected in water sources. Pseudomonas sp. are opportunistic pathogens commonly reported in water samples and different antimicrobial resistance mechanisms have been described in Pseudomonas sp., producing multidrug-resistant (MDR) phenotype. Besides, the presence of heavy metal resistance genes (HMRGs) may select ARB, which is worrying. Therefore, this study aimed to characterize the genotypes of Pseudomonas sp. isolated from different water sources. Water samples (i.e., rivers, streams, lakes and sewage treatment plants) were collected from different cities in Brazil. The bacterial identification was performed by sequencing the 16S rDNA and the resistance profile to antimicrobials and heavy metals were determined by minimum inhibitory concentration (MIC). Several ARGs, HMRGs, and plasmids were researched by PCR and the amplicons were sequenced for confirmation. A total of 23 Pseudomonas sp. isolates were obtained and identified as Pseudomonas saponiphila, Pseudomonas hunanensis, Pseudomonas aeruginosa, and Pseudomonas asiatica. These isolates presented high MICs to antimicrobials and heavy metals, being the great majority (n = 21; 91%) classified as MDR. Different clinically important ARGs were detected, such as blaGES, qnrS, qepA, tetB, aac(3')-IIa, and ant(2″)-Ia. The czcA gene was the only HMRG detected and no plasmids were found. To the best of our knowledge, this is the first report of the world of P. saponiphila carrying ARGs (i.e., blaGES, qnrS, aac(3')-IIa, tetB) and QepA-producing P. hunanensis and the first time of P. saponiphila, P. asiatica, and P. hunanensis in Brazil.

Detection of different ß-lactamases encoding genes, including blaNDM, and plasmid-mediated quinolone resistance genes in different water sources from Brazil.

Bacterial resistance occurs by spontaneous mutations or horizontal gene transfer mediated by mobile genetic elements, which represents a great concern. Resistance to ß-lactam antibiotics is mainly due to the production of ß-lactamases, and an important mechanism of fluoroquinolone resistance is the acquisition plasmid determinants. The aim of this study was to verify the presence of ß-lactamase-encoding genes and plasmid-mediated quinolone resistance genes in different water samples obtained from São Paulo state, Brazil. A high level of these resistance genes was detected, being the blaSHV, blaGES, and qnr the most prevalent. Besides that, the blaNDM gene, which codify an important and hazardous metallo-ß-lactamase, was detected.