Catastrophic floods and antimicrobial resistance: Interconnected threats with wide-ranging impacts.

•Climate change and AMR combined worsen vulnerabilities, accelerating AMR spread.•Floods can spread AMR-related pathogens, impacting health, agriculture, and ecosystems.•Integrated strategies are needed to address climate change and AMR, enhancing sanitation.

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) RdJ clone (CC5-ST105-SCCmecII-t002) in Santa Catarina, Brazil.

The emergence of highly successful genetic lineages of methicillin-resistant Staphylococcus aureus (MRSA) poses a challenge in human healthcare due to increased morbidity and mortality rates. The RdJ clone (CC5-ST105-SCCmecII-t002 lineage), previously identified in Rio de Janeiro, Brazil, was linked to bloodstream infections and features a mutation in the aur gene (encoding aureolysin). Additionally, clinical isolates derived from this clone were more effective at evading monocytic immune responses. This study aimed to detect the RdJ clone among clinical MRSA isolated in Santa Catarina (SC) and examine its antimicrobial resistance and phagocytosis evasion capabilities. Our findings revealed the RdJ clone in 20 % of MRSA isolates, all exhibiting multiresistance. RdJ clone isolates from SC did not demonstrate a decreased rate of phagocytosis compared to CC5 non-RdJ isolates. Structural analysis suggests that the aur mutation is unlikely to significantly impact aureolysin activity. Genomic analysis of one isolate unveiled a genetic variant of the RdJ clone, sharing lineage and gene distribution but lacking the aur mutation. This study enhances the understanding of the clinical and epidemiologic risks associated with the RdJ clone and the biological mechanisms underlying its spreading in SC.

Carbapenemase-producing Klebsiella quasipneumoniae ST688 (NDM-1) and Klebsiella michiganensis ST40 (KPC-2) in food destined for hospitalized patients.

OBJECTIVES: Klebsiella spp. are leading causes of nosocomial infections. Their ability to harbour antimicrobial resistance genes makes them an important public health threat. This study aimed to report the genomic background of carbapenemase-producing Klebsiella quasipneumoniae (HV55B) and Klebsiella michiganensis (HV55D) strains isolated from fresh vegetables destined for hospitalized inpatients. METHODS: Microbiological and molecular methods were used to isolate and identify the strains, which were submitted to the antimicrobial susceptibility test and pH tolerance assays. Whole genome sequencing was performed on MiSeq and NextSeq platforms, and online available tools were applied to bioinformatic analysis of clinically relevant information. RESULTS: Both isolates were considered multidrug-resistant and tolerated pH ≥ 4 for 24 h. HV55B belonged to sequence type (ST) ST668, and presented a broad resistome and plasmids from four incompatibility groups. HV55D belonged to ST40. Both strains HV55B and HV55D were genetically close to isolates responsible for human infections around the world, which stands for the plausibility of such bacteria to cause disease in patients of the studied institution. CONCLUSIONS: Our results confirm the presence of carbapenemase-producing Klebsiella spp. in fresh foodstuffs intended for hospitalized inpatients' consumption. The genomes characterized here also provide clinically and genomically relevant information to forthcoming epidemiological surveillance studies.

Reversion of KPC-114 to KPC-2 in ceftazidime-avibactam- resistant/meropenem-susceptible Klebsiella pneumoniae ST11 is related to low mutation rates.

Klebsiella pneumoniae strains that produce Klebsiella pneumoniae Carbapenemase (KPC) variants displaying resistance to ceftazidime-avibactam (CZA) often remain susceptible to meropenem (MEM), suggesting a potential therapeutic use of this carbapenem antibiotic. However, in vitro studies indicate that these sorts of strains can mutate becoming MEM-resistant, raising concerns about the effectiveness of carbapenems as treatment option. We have studied mutation rates occurring from the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae belonging to ST11. Two-step fluctuation assays (FAs) were conducted. In brief, initial cultures of KPC-114-producing K. pneumoniae showing 1 µg/mL MEM MIC were spread on Mueller-Hinton agar plates containing 2-8 µg/mL MEM. A second step of FA, at 4-16 µg/mL MEM was performed from a mutant colony obtained at 2 µg/mL MEM. Mutation rates were calculated using maximum likelihood estimation. Parental and mutant strains were sequenced by Illumina NextSeq, and mutations were predicted by variant-calling analysis. At 8 µg/mL MEM, mutants derived from parental CZA-resistant (MIC ≥ 64 µg/mL)/MEM-susceptible (MIC = 1 µg/mL) KPC-114-positive K. pneumoniae exhibited an accumulative mutation rate of 3.05 × 10-19 mutations/cell/generation, whereas at 16 µg/mL MEM an accumulative mutation rate of 1.33 × 10-19 mutations/cell/generation resulted in the reversion of KPC-114 (S181_P182 deletion) to KPC-2. These findings highlight that the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae ST11 is related to low mutation rates suggesting a low risk of therapeutic failure. In vivo investigations are necessary to confirm the clinical potential of MEM against CZA-resistant KPC variants.IMPORTANCEThe emergence of ceftazidime-avibactam (CZA) resistance among carbapenem-resistant Klebsiella pneumoniae is a major concern due to the limited therapeutic options. Strikingly, KPC mutations mediating CZA resistance are generally associated with meropenem susceptibility, suggesting a potential therapeutic use of this carbapenem antibiotic. However, the reversion of meropenem-susceptible to meropenem-resistant could be expected. Therefore, knowing the mutation rate related to this genetic event is essential to estimate the potential use of meropenem against CZA-resistant KPC-producing K. pneumoniae. In this study, we demonstrate, in vitro, that under high concentrations of meropenem, reversion of KPC-114 to KPC-2 in CZA-resistant/meropenem-susceptible K. pneumoniae belonging to the global high-risk ST11 is related to low mutation rates.

Gut colonization by extended-spectrum ß-lactamase-producing Escherichia coli in dairy herd in Brazil: successful dissemination of a One Health clone.

The overuse of antimicrobials in livestock has contributed to the emergence and selection of clinically relevant multidrug-resistant bacteria. In Brazil, there is no conclusive information on the occurrence of Escherichia coli producing extended-spectrum ß-lactamase (ESßL) in cattle breeding, which is an important sector of agribusiness in this country. Herein, we investigated the presence of ESßL-positive E. coli strains in dairy cattle from a commercial farm with routine practice of therapeutic cephalosporins. Ninety-five rectal swab samples were collected from healthy dairy calves and cows under treatment with ceftiofur. Samples were screened for the presence of ESßL producers, and positive isolates were identified by MALDI-TOF, with subsequent screening for genes encoding ESßL variants by PCR and sequencing. The presence of ESßL (CTX-M-15)-producing E. coli was confirmed in calves, and lactating and dry cows. Most ESßL strains with genetic homologies ≥ 90% were grouped into two major PFGE clusters, confirming the suscessful expansion of clonally related lineages in animals from different lactating cycles, on the same property. Four representatives CTX-M-15-positive E. coli strains had their genomes sequenced, belonging to the clonal complex (CC) 23 and sequence type (ST) 90. A phylogeographical landscape of ST90 was performed revealing a global One Health linkage. Our results highlight the intestinal microbiota of dairy cattle as a hotspot for the spread of critical priority ESßL-producing E. coli and demonstrate that ST90 is an international clone genomically adapted to human and animal hosts, which deserve additional investigation to determine its zoonotic potential and impact in food chain.

Genome-based diagnostic of MDR Escherichia coli ONT:H19 ST10955 causing human gastrointestinal infection.

This study focuses on the genomic characterization of a multidrug-resistant Escherichia coli strain responsible for a severe gastrointestinal infection in a 33-year-old male. The patient initially received sulfamethoxazole/trimethoprim treatment, which proved ineffective. Fecal culture confirmed the presence of E. coli displaying a MDR profile to ampicillin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, and tetracycline. Serotyping identified the strain as ONT:H19. Virulence analysis indicated a highly virulent profile with numerous virulence markers. Plasmid analysis uncovered various plasmids carrying both antimicrobial resistance and virulence genes. MLST assigned the strain to ST10955. Phylogenomic analysis revealed similarity to an older Brazilian isolate, suggesting the persistence of a common lineage with evolving antimicrobial resistance. This report highlights the first identification of a multidrug-resistant ST10955 E. coli strain with a wide resistome and virulence potential, emphasizing the importance of ongoing surveillance of E. coli strains due to their potential for severe infections, resistance development, and virulence.

ESBL-Producing Enterobacterales at the Human-Domestic Animal-Wildlife Interface: A One Health Approach to Antimicrobial Resistance in Piauí, Northeastern Brazil.

The use, misuse, and overuse of antimicrobials is one of the main public health threats of the 21st century. We investigated the risk factor of the presence of extended-spectrum, cephalosporin-resistant Enterobacterales in feces of non-domestic and domestic birds and other domestic animals in Piauí State, northeast Brazil. We collected a total of 387 cloacal and rectal swab samples of free-living birds, domestic birds, and domestic mammals in five municipalities: Amarante, Água Branca, Lagoa Alegre, Parnaíba, and Teresina. A total of 59/387 (15.2%) of these samples harbored extended spectrum beta-lactamase (ESBL)-producing Enterobacterales. Using the MALDI-TOF technique, we identified fifty-seven samples as Escherichia coli and two samples as Klebsiella pneumoniae. Teresina and Parnaíba had the highest prevalence of animals with resistant bacteria (32.1% and 27.1%, respectively) and highest exposure risk factor (OR of 16.06 and 8.58, respectively, and p < 0.001 for all). Multidrug-resistant, ESBL-producing Enterobacterales were observed in 72.8% of the samples (43/59). For the free-living birds, the positive samples belonged to a great kiskadee (Pitangus sulphuratus) and a semipalmated sandpiper (Calidris pusilla) in migratory and resident species, respectively. For domestic animals, the swine samples showed the highest prevalence of antimicrobial resistance. The lack of access to veterinary care and information regarding antimicrobial therapy, along with the easy access to antimicrobials without medical prescription, favors the inadequate use of antimicrobials in Piauí.

Rapid diagnostic of multidrug-resistant sepsis pathogens directly from blood culture bottles using MALDI-TOF and the EUCAST RAST.

In this study, rapid diagnostic of multidrug-resistant (MDR) sepsis pathogens, directly from positive blood culture (BC) bottles, was evaluated by combining MALDI-TOF and the EUCAST Rapid Antimicrobial Susceptibility Testing (RAST). Carbapenemase production in Escherichia coli and Klebsiella pneumoniae isolates was also evaluated by RAST. From 171 positive BC bottles analyzed, 79 (46 %) MDR species, including E. coli (4/34, 12 %), K. pneumoniae (33/48, 69 %), Pseudomonas aeruginosa (12/12, 100 %), Acinetobacter baumannii (15/15, 100 %), and Staphylococcus aureus (14/37, 38 %) displaying resistance to beta-lactams, fluoroquinolones, aminoglycosides, and/or trimethoprim/sulphamethoxazole, were identified. In this regard, turnaround time of direct MALDI-TOF identification and RAST was < 7 h, which was significantly (p< 0.05) lower than our routine method. Carbapenemase detection by RAST displayed 100% sensitivity and 88.7 % specificity at 8 h. This protocol could offer advantages for the treatment and clinical outcomes of septic patients, improving the rapid diagnostic of sepsis by MDR pathogens.

Spread and persistence of antimicrobial resistance genes in wastewater from human and animal sources in São Paulo, Brazil.

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (blaCTX-M, blaKPC, qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes blaCTX-M, qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems.

Hybrid genome assembly of colistin-resistant mcr-1.5-producing Escherichia coli ST354 reveals phylogenomic pattern associated with urinary tract infections in Brazil.

BACKGROUND: The rapid and global spread of Escherichia coli carrying mcr-type genes at the human-animal-environmental interface has become a serious global public health problem. OBJECTIVE: To perform a genomic investigation of a colistin-resistant E. coli strain (14005RM) causing urinary tract infection, using a hybrid de novo assembly of Illumina/Nanopore sequence data, presenting phylogenomic insights into the relationship with mcr-1-positive strains circulating at the human-animal-environmental interface, in Brazil. METHODS: Genomic DNA was sequenced using both the Illumina NexSeq and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools. RESULTS: The genome assembly size was 5 333 039 bp. The mcr-1.5-positive E. coli strain 14005RM belongs to the sequence type ST354 and presented a broad resistome (antibiotics, heavy metals, disinfectants, and glyphosate) and virulome. The mcr-1.5 gene was carried by an IncI2 plasmid (p14005RM, sizing 65,458 kb). Full genome SNP-based phylogenetic analysis reveals that mcr-1.5-producing E. coli strain 14005RM is highly related (> 98% identity) to colistin-resistant mcr-1.1-positive ST354 lineages associated with urinary tract infections in Brazil since 2015. CONCLUSION: Mobile colistin resistance within the Brazilian One Health microbiosphere is mediated by mcr gene variants propagated by IncX4, IncHI2, and IncI2 plasmids, circulating among global clones of E. coli.

Genomic Features of an MDR Escherichia coli ST5506 Harboring an IncHI2/In229/blaCTX-M-2 Array Isolated from a Migratory Black Skimmer.

Migratory birds have contributed to the dissemination of multidrug-resistant (MDR) bacteria across the continents. A CTX-M-2-producing Escherichia coli was isolated from a black skimmer (Rynchops niger) in Southeast Brazil. The whole genome was sequenced using the Illumina NextSeq platform and de novo assembled by CLC. Bioinformatic analyses were carried out using tools from the Center for Genomic Epidemiology. The genome size was estimated at 4.9 Mb, with 4790 coding sequences. A wide resistome was detected, with genes encoding resistance to several clinically significant antimicrobials, heavy metals, and biocides. The blaCTX-M-2 gene was inserted in an In229 class 1 integron inside a ∆TnAs3 transposon located in an IncHI2/ST2 plasmid. The strain was assigned to ST5506, CH type fumC19/fimH32, serotype O8:K87, and phylogroup B1. Virulence genes associated with survival in acid conditions, increased serum survival, and adherence were also identified. These data highlight the role of migratory seabirds as reservoirs and carriers of antimicrobial resistance determinants and can help to elucidate the antimicrobial resistance dynamics under a One Health perspective.

Genomic scan of a healthcare-associated NDM-1-producing Citrobacter freundii ST18 isolated from a green sea turtle impacted by plastic pollution.

BACKGROUND: Carbapenemase-producing Citrobacter freundii has been reported as a leading cause of healthcare-associated infections. Particularly, C. freundii belonging to the sequence type (ST) 18 is considered to be an emerging nosocomial clone. OBJECTIVES: To report the genomic background and phylogenomic analysis of a multidrug-resistant NDM-1-producing C. freundii ST18 (strain CF135931) isolated from an endangered green sea turtle affected by plastic pollution in Brazil. METHODS: Genomic DNA was extracted and sequenced using the Illumina NextSeq platform. De novo assembly was performed by CLC Workbench, and in silico analysis accomplished by bioinformatics tools. For phylogenomic analysis, publicly available C. freundii (txid:546) genome assemblies were retrieved from the NCBI database. RESULTS: The genome size was calculated at 5 290 351 bp, comprising 5263 total genes, 4 rRNAs, 77 tRNAs, 11ncRNAs, and 176 pseudogenes. The strain belonged to C. freundii ST18, whereas resistome analysis predicted genes encoding resistance to ß-lactams (blaNDM-1, blaOXA-1, blaCMY-117, and blaTEM-1C), aminoglycosides (aph(3'')-Ib, aadA16, aph(3')-VI, aac(6')-Ib-cr, and aph(6)-Id), quinolones (aac(6')-Ib-cr), macrolides (mph(A) and erm(B)), sulphonamides (sul1 and sul2), tetracyclines (tetA and tetD), and trimethoprim (dfrA27). The phylogenomic analysis revealed that CF135931 strain is closely related to international human-associated ST18 clones producing NDM-1. CONCLUSION: Genomic surveillance efforts are necessary for robust monitoring of the emergence of drug-resistant strains and WHO critical priority pathogens within a One Health framework. In this regard, this draft genome and associated data can improve understanding of dissemination dynamics of nosocomial clones of carbapenemase-producing C. freundii beyond hospital walls. In fact, the emergence of NDM-1-producing C. freundii of global ST18 in wildlife deserves considerable attention.