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 and comparative analysis of a multidrug-resistant Acinetobacter bereziniae strain infecting an animal: a novel emerging one health pathogen?

Acinetobacter bereziniae has recently gained medical notoriety due to its emergence as a multidrug resistance and healthcare-associated pathogen. In this study, we report the whole-genome characterization of an A. bereziniae strain (A321) recovered from an infected semiaquatic turtle, as well as a comparative analysis of A. bereziniae strains circulating at the human-animal-environment interface. Strain A321 displayed a multidrug resistance profile to medically important antimicrobials, which was supported by a wide resistome. The novel Tn5393m transposon and a qnrB19-bearing ColE1-like plasmid were identified in A321 strain. Novel OXA-229-like ß-lactamases were detected and expression of OXA-931 demonstrated a 2-64-fold increase in the minimum inhibitory concentration for ß-lactam agents. Comparative genomic analysis revealed that most A. bereziniae strains did not carry any antimicrobial resistance genes (ARGs); however, some strains from China, Brazil, and India harbored six or more ARGs. Furthermore, A. bereziniae strains harbored conserved virulence genes. These results add valuable information regarding the spread of ARGs and mobile genetic elements that could be shared not only between A. bereziniae but also by other bacteria of clinical interest. This study also demonstrates that A. bereziniae can spill over from anthropogenic sources into natural environments and subsequently be transmitted to non-human hosts, making this a potential One Health bacteria that require close surveillance.

Genomic Insights into Pluralibacter gergoviae Sheds Light on Emergence of a Multidrug-Resistant Species Circulating between Clinical and Environmental Settings.

Pluralibacter gergoviae is a member of the Enterobacteriaceae family that has been reported sporadically. Although P. gergoviae strains exhibiting multidrug-resistant profiles have been identified an in-depth genomic analysis focusing on antimicrobial resistance (AMR) has been lacking, and was therefore performed in this study. Forty-eight P. gergoviae strains, isolated from humans, animals, foods, and the environment during 1970-2023, were analyzed. A large number of single-nucleotide polymorphisms were found, indicating a highly diverse population. Whilst P. gergoviae strains were found to be circulating at the One Health interface, only human and environmental strains exhibited multidrug resistance genotypes. Sixty-one different antimicrobial resistance genes (ARGs) were identified, highlighting genes encoding mobile colistin resistance, carbapenemases, and extended-spectrum ß-lactamases. Worryingly, the co-occurrence of mcr-9.1, blaKPC-2, blaCTX-M-9, and blaSHV-12, as well as mcr-10.1, blaNDM-5, and blaSHV-7, was detected. Plasmid sequences were identified as carrying clinically important ARGs, evidencing IncX3 plasmids harboring blaKPC-2, blaNDM-5, or blaSHV-12 genes. Virulence genotyping underlined P. gergoviae as being a low-virulence species. In this regard, P. gergoviae is emerging as a new multidrug-resistant species belonging to the Enterobacteriaceae family. Therefore, continuous epidemiological genomic surveillance of P. gergoviae is required.

Gram-negative bacterial diversity and evidence of international clones of multidrug-resistant strains in zoo animals.

Enterobacterales and Pseudomonas aeruginosa have been colonizing or infecting wild hosts and antimicrobial-resistant strains are present in mammals and birds. Furthermore, international high-risk clones of multidrug-resistant Escherichia coli are identified and the implications of multidrug-resistant Gram-negative bacteria in zoo animals are discussed.

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.

One Health Spread of 16S Ribosomal RNA Methyltransferase-Harboring Gram-Negative Bacterial Genomes: An Overview of the Americas.

Aminoglycoside antimicrobials remain valuable therapeutic options, but their effectiveness has been threatened by the production of bacterial 16S ribosomal RNA methyltransferases (16S-RMTases). In this study, we evaluated the genomic epidemiology of 16S-RMTase genes among Gram-negative bacteria circulating in the American continent. A total of 4877 16S-RMTase sequences were identified mainly in Enterobacterales and nonfermenting Gram-negative bacilli isolated from humans, animals, foods, and the environment during 1931-2023. Most of the sequences identified were found in the United States, Brazil, Canada, and Mexico, and the prevalence of 16S-RMTase genes have increased in the last five years (2018-2022). The three species most frequently carrying 16S-RMTase genes were Acinetobacter baummannii, Klebsiella pneumoniae, and Escherichia coli. The armA gene was the most prevalent, but other 16S-RMTase genes (e.g., rmtB, rmtE, and rmtF) could be emerging backstage. More than 90% of 16S-RMTase sequences in the Americas were found in North American countries, and although the 16S-RMTase genes were less prevalent in Central and South American countries, these findings may be underestimations due to limited genomic data. Therefore, whole-genome sequence-based studies focusing on aminoglycoside resistance using a One Health approach in low- and middle-income countries should be encouraged.

Klebsiella quasipneumoniae subsp. similipneumoniae ST1859 O5:KL35 from Soil: First Report of qnrE1 in the Environment.

A Klebsiella quasipneumoniae subsp. similipneumoniae strain, named S915, belonging to the ST1859 O5:KL35, and harboring the plasmid-mediated quinolone resistance qnrE1 gene, was isolated from a soil sample cultivated with lettuce in Brazil. The core genome multilocus sequence typing analysis revealed that S915 strain was most related to a clinical strain of Brazil. Comparative genomic analysis showed that ST1859 O5:KL35 strains have been circulating in clinical settings and are closely related to multidrug resistance and multimetal tolerance. Strain S915 presented a plasmid contig co-harboring the qnrE1 gene and tellurite tolerance operon. The region harboring the qnrE1 gene (ISEcp1-qnrE1-araJ-ahp) shared high similarity with others from infected humans, ready-to-eat dish, and food-producing animals in Brazil. This is the first report of the plasmid-mediated qnrE1 gene in the environment. Our findings evidence the initial dissemination of the qnrE1 gene in the environment by the introduction of a clinical strain, which may be spread to different sectors, representing a One Health challenge.

Predicting tigecycline susceptibility in multidrug-resistant Klebsiella species and Escherichia coli strains of environmental origin.

Tigecycline (TGC) is an important antimicrobial agent used as a last resort for difficult-to-treat infections mainly caused by carbapenem-resistant Enterobacteriaceae, but TGC-resistant strains are emerging, raising concerns. In this study, 33 whole-genome characterized multidrug-resistant (MDR) strains (Klebsiella species and Escherichia coli) positive mainly to mcr-1, bla, and/or qnr from the environment were investigated for TGC susceptibility and mutations in TGC resistance determinants, predicting a genotype-phenotype relationship. TGC minimum inhibitory concentrations (MICs) of Klebsiella species and E. coli ranged from 0.25 to 8 and 0.125 to 0.5 mg/L, respectively. In this context, KPC-2-producing Klebsiella pneumoniae ST11 and Klebsiella quasipneumoniae subsp. quasipneumoniae ST4417 strains were resistant to TGC, while some E. coli strains of ST10 clonal complex positive for mcr-1 and/or blaCTX-M exhibited reduced susceptibility to this antimicrobial. Overall, neutral and deleterious mutations were shared among TGC-susceptible and TGC-resistant strains. A new frameshift mutation (Q16stop) in RamR was found in a K. quasipneumoniae strain and was associated with TGC resistance. Deleterious mutations in OqxR were identified in Klebsiella species and appear to be associated with decreased susceptibility to TGC. All E. coli strains were determined as susceptible, but multiple point mutations were identified, highlighting deleterious mutations in ErmY, WaaQ, EptB, and RfaE in strains exhibiting decreased susceptibility to TGC. These findings demonstrate that resistance to TGC is not widespread in environmental MDR strains and provide genomic insights about resistance and decreased susceptibility to TGC. From a One Health perspective, the monitoring of TGC susceptibility should be constant, improving the genotype-phenotype relationship and genetic basis.

Electrobiochemical skills of Pseudomonas aeruginosa species that produce pyocyanin or pyoverdine for glycerol oxidation in a microbial fuel cell.

Pseudomonas aeruginosa can produce pigments, which mediate external electron transfer (EET). Depending on the mediator, this species can be explored in bioelectrosystems to harvest energy or to obtain chemicals from residual organic compounds. This study has compared the performance of microbial fuel cells (MFCs) inoculated with a Pseudomonas aeruginosa isolate, namely EW603 or EW819, which produce pyocyanin and pyoverdine, respectively. The efficiency of these MFCs in glycerol, a typical residue of biodiesel production, were also compared. The MFCs exhibited different performances. The maximum voltage was 411 and 281 mV m2, the power density was 40.1 and 21.3 mW m-2, and the coulombic efficiency was 5.16 and 1.49% for MFC-EW603 and MFC-EW819, respectively. MFC-EW603 and MFC-EW819 achieved maximum current at 560 and 2200 Ω, at 141.2 and 91.3 mA m-2, respectively. When the system was operated at the respective maximum current output, MFC-EW603 consumed the total glycerol content (11 mmol L-1), and no products could be detected after 50 h. In turn, acetic and butyric acids were detected at the end of MFC-EW819 operation (75 h). The results suggested that P. aeruginosa metabolism can be steered in the MFC to generate current or microbial products depending on the pigment-producing strain and the conditions applied to the system, such as the external resistance. In addition, gene cluster pathways related to phenazine production (phzA and phzB) and other electrogenic-related genes (mexGHI-opmB) were identified in the strain genomes, supporting the findings. These results open new possibilities for using glycerol in bioelectrochemical systems.

Multidrug-resistant Shiga toxin-producing Escherichia coli and hybrid pathogenic strains of bovine origin.

Antimicrobial-resistant Escherichia coli strains have been circulating in various sectors and can be cross-transferred between them. Among pathogenic E. coli strains, Shiga toxin-producing E. coli (STEC) and hybrid pathogenic E. coli (HyPEC) emerged as responsible for outbreaks worldwide. As bovine are reservoir of STEC strains, these pathogens primarily spread to food products, exposing humans to risk. Therefore, this study aimed to characterize antimicrobial-resistant and potentially pathogenic E. coli strains from fecal samples of dairy cattle. In this regard, most E. coli strains (phylogenetic groups A, B1, B2, and E) were resistant to ß-lactams and non-ß-lactams and were classified as multidrug-resistant (MDR). Antimicrobial resistance genes (ARGs) related to multidrug resistance profiles were detected. Furthermore, mutations in fluoroquinolone and colistin resistance determinants were also identified, highlighting the deleterious mutation His152Gln in PmrB that may have contributed to the high level (> 64 mg/L) of colistin resistance. Virulence genes of diarrheagenic and extraintestinal pathogenic E. coli (ExPEC) pathotypes were shared among strains and even within the same strain, evidencing the presence of HyPEC (i.e., ExPEC/STEC), which were assigned as unusual B2-ST126-H3 and B1-ST3695-H31. These findings provide phenotypic and molecular data of MDR, ARGs-producing, and potentially pathogenic E. coli strains in dairy cattle, contributing to the monitoring of antimicrobial resistance and pathogens in healthy animals and alerting to potential bovine-associated zoonotic infections.

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.