Unveiling novel threats: Urban river isolation of Aeromonas veronii with unusual VEB-28 extended-spectrum ß-lactamase and distinct mcr variants.

Aeromonas spp. are frequently encountered in aquatic environments, with Aeromonas veronii emerging as an opportunistic pathogen causing a range of diseases in both humans and animals. Recent reports have raised public health concerns due to the emergence of multidrug-resistant Aeromonas spp. This is particularly noteworthy as these species have demonstrated the ability to acquire and transmit antimicrobial resistance genes (ARGs). In this study, we report the genomic and phenotypic characteristics of the A. veronii TR112 strain, which harbors a novel variant of the Vietnamese Extended-spectrum ß-lactamase-encoding gene, blaVEB-28, and two mcr variants recovered from an urban river located in the Metropolitan Region of São Paulo, Brazil. A. veronii TR112 strain exhibited high minimum inhibitory concentrations (MICs) for ceftazidime (64 µg/mL), polymyxin (8 µg/mL), and ciprofloxacin (64 µg/mL). Furthermore, the TR112 strain demonstrated adherence to HeLa and Caco-2 cells within 3 h, cytotoxicity to HeLa cells after 24 h of interaction, and high mortality rates to the Galleria mellonella model. Genomic analysis showed that the TR112 strain belongs to ST257 and presented a range of ARGs conferring resistance to ß-lactams (blaVEB-28, blaCphA3, blaOXA-912) and polymyxins (mcr-3 and mcr-3.6). Additionally, we identified a diversity of virulence factor-encoding genes, including those encoding mannose-sensitive hemagglutinin (Msh) pilus, polar flagella, type IV pili, type II secretion system (T2SS), aerolysin (AerA), cytotoxic enterotoxin (Act), hemolysin (HlyA), hemolysin III (HlyIII), thermostable hemolysin (TH), and capsular polysaccharide (CPS). In conclusion, our findings suggest that A. veronii may serve as an environmental reservoir for ARGs and virulence factors, highlighting its importance as a potential pathogen in public health.

Biodiversity of carbapenem-resistant bacteria in clinical samples from the Southwest Amazon region (Rondônia/Brazil).

Brazil is recognized for its biodiversity and the genetic variability of its organisms. This genetic variability becomes even more valuable when it is properly documented and accessible. Understanding bacterial diversity through molecular characterization is necessary as it can improve patient treatment, reduce the length of hospital stays and the selection of resistant bacteria, and generate data for health and epidemiological surveillance. In this sense, in this study, we aimed to understand the biodiversity and molecular epidemiology of carbapenem-resistant bacteria in clinical samples recovered in the state of Rondônia, located in the Southwest Amazon region. Retrospective data from the Central Public Health Laboratories (LACEN/RO) between 2018 and 2021 were analysed using the Laboratory Environment Manager Platform (GAL). Seventy-two species with carbapenem resistance profiles were identified, of which 25 species carried at least one gene encoding carbapenemases of classes A (blaKPC-like), B (blaNDM-like, blaSPM-like or blaVIM-like) and D (blaOXA-23-like, blaOXA-24-like, blaOXA-48-like, blaOXA-58-like or blaOXA-143-like), among which we will highlight Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Serratia marcescens, and Providencia spp. With these results, we hope to contribute to the field by providing epidemiological molecular data for state surveillance on bacterial resistance and assisting in public policy decision-making.

Clonal dissemination of highly virulent Serratia marcescens strains producing KPC-2 in food-producing animals.

Serratia marcescens is a Gram-negative bacterium presenting intrinsic resistance to polymyxins that has emerged as an important human pathogen. Although previous studies reported the occurrence of multidrug-resistance (MDR) S. marcescens isolates in the nosocomial settings, herein, we described isolates of this extensively drug-resistant (XDR) species recovered from stool samples of food-producing animals in the Brazilian Amazon region. Three carbapenem-resistant S. marcescens strains were recovered from stool samples of poultry and cattle. Genetic similarity analysis showed that these strains belonged to the same clone. Whole-genome sequencing of a representative strain (SMA412) revealed a resistome composed of genes encoding resistance to ß-lactams [blaKPC-2, blaSRT-2], aminoglycosides [aac(6')-Ib3, aac(6')-Ic, aph(3')-VIa], quinolones [aac(6')-Ib-cr], sulfonamides [sul2], and tetracyclines [tet(41)]. In addition, the analysis of the virulome demonstrated the presence of important genes involved in the pathogenicity of this species (lipBCD, pigP, flhC, flhD, phlA, shlA, and shlB). Our data demonstrate that food-animal production can act as reservoirs for MDR and virulent strains of S. marcescens.

Genetic and biochemical characterization of BIM-1, a novel acquired subgroup B1 MBL found in a Pseudomonas sp. strain from the Brazilian Amazon region.

OBJECTIVES: To characterize a novel acquired MBL, BIM-1, in a Pseudomonas #2 (subgroup P. guariconensis) strain isolated from the Aurá river located in the Brazilian Amazon hydrographic basin. METHODS: WGS using an Illumina® MiSeq System was used to characterize the genome of Pseudomonas sp. IEC33019 strain. Southern blotting/hybridization assays were performed to confirm the location of the MBL-encoding gene, blaBIM-1 (Belém Imipenemase). Antimicrobial susceptibility testing, cloning, and biochemical and phenotypic characterization were performed to determine BIM-1 kinetics. RESULTS: The IEC33019 strain showed high resistance rates to ß-lactams, ciprofloxacin and aminoglycosides, being susceptible only to polymyxins and susceptible, increased exposure to aztreonam. WGS analysis revealed a novel acquired MBL-encoding gene, blaBIM-1, found as a gene cassette inserted into a class 1 integron (In1326) that also carried qnrVC1 and aadA11e. In1326 was located in a complex transposon, Tn7122, carried by a 52.7 kb conjugative plasmid (pIEC33019) with a toxin/antitoxin system (vapB/vapC). BIM-1 belongs to the molecular subgroup B1 and shares 70.2% and 64.9% similarity with SIM-1 and IMP-1, respectively. Kinetics analysis of BIM-1 showed hydrolytic activity against all ß-lactams tested. CONCLUSIONS: BIM-1 is a novel acquired MBL encoded by a gene carried by mobile genetic elements, which can be transferred to other Gram-negative bacilli (GNB). Because the IEC33019 strain was recovered from a river impacted by a populous metropolitan region with poor basic sanitation and served by limited potable freshwater, it would be important to establish the role of the BIM-1-producing GNB as nosocomial pathogens and/or as colonizers of the riverside population in this geographical region.

Candida-associated denture stomatitis: clinical, epidemiological, and microbiological features.

OBJECTIVE: The identification of Candida spp. in denture stomatitis, the clinical manifestations, and the antifungal susceptibility profile lead to a correct and individualized therapeutic management of the patients. This study is aimed at investigating the clinical manifestations and epidemiological and microbiological characteristics of Candida-associated denture stomatitis. DESIGN: The samples were obtained by swabbing the oral mucosa of the subjects and then seeded onto Sabouraud Dextrose Agar and onto CHROMagar® Candida plates. The identification at the species level was confirmed by Matrix Assisted Laser Desorption Time of Flight Mass Spectrometry. Clinical classification was performed according to the criteria proposed by Newton (1962): (i) pinpoint hyperemia, (ii) diffuse hyperemia, and (iii) granular hyperemia. For carrying out the antifungal susceptibility testing, we adopted the CLSI M27-S4 protocol. RESULTS: C. albicans was the most prevalent species in our study. Regarding non-albicans Candida species, C. glabrata was the most common species isolated from the oral mucosa (n = 4, 14.8%), while in the prosthesis, it was C. tropicalis (n = 4, 14.8%). The most prevalent clinical manifestation was pinpoint hyperemia and diffuse hyperemia. Candida albicans, C. glabrata, and C. parapsilosis were susceptible to all the tested antifungals. Concerning fluconazole and micafungin, only two strains showed dose-dependent sensitivity (minimum inhibitory concentration (MIC), 1 µg/mL) and intermediate sensitivity (MIC, 0.25 µg/mL). One C. tropicalis strain was resistant to voriconazole (MIC, 8 µg/mL). CONCLUSIONS: C. albicans was the most common species found in oral mucosa and prosthesis. The tested antifungal drugs showed great activity against most isolates. The most prevalent clinical manifestations were Newton's type I and type II.

Genomic Analysis of Klebsiella pneumoniae ST258 Strain Coproducing KPC-2 and CTX-M-14 Isolated from Poultry in the Brazilian Amazon Region.

This study aimed to characterize a Klebsiella pneumoniae strain (KP411) recovered from the stool samples of poultry (Gallus gallus) in the Brazilian Amazon Region. The whole-genome sequencing of KP411 revealed the presence of an important arsenal of antimicrobial resistance genes to ß-lactams (blaCTX-M-14, blaTEM-1B, blaKPC-2, blaSVH-11), aminoglycosides [aph(3″)- Ib, aph(6)-Id, aph(3')-Ia], sulfonamides (sul1, sul2), quinolones (oqxAB), fosfomycin (fosAKP), and macrolides [mph(A)]. Furthermore, our analyses revealed that the KP411 strain belongs to the ST258 clonal lineage, which is one of the main epidemic clones responsible for the dissemination of KPC-2 worldwide. Our data suggest that food-producing animals may act as reservoirs of multidrug-resistant K. pneumoniae belonging to the ST258 clone, and, consequently, contribute to their dissemination to humans and the environment.

Characterization of a Carbapenem-Resistant BKC-1-Producing Clinical Isolate Belonging to the Pseudomonas putida Group from Brazil.

Since its first report, the class A Brazilian Klebsiella carbapenemase (BKC) has been detected only among Enterobacterales isolates from Brazilian hospitals. In this study, we characterized a multidrug-resistant Pseudomonas juntendi clinical isolate and identified a 43.3-kb plasmid carrying blaBKC-1 and a class 1 integron (In1996) containing the arr-2, qnrVC1, dfrA21, and aac(6')-Ib' gene cassettes. Our results confirm the ability of Pseudomonas putida group isolates to acquire antimicrobial resistance determinants and further act as resistance reservoirs.

Genome sequencing of an XDR Klebsiella pneumoniae ST101 strain isolated from a Brazilian Amazon river.

OBJECTIVE: Herein, this study aimed to perform the genomic characterization of a blaKPC-2 positive Klebsiella pneumoniae (KP1.1JP) strain isolated from the surface water of river located the Brazilian Amazon region. METHODS: Antimicrobial susceptibility testing was performed following BrCAST/EUCAST recommendations. Genomic DNA was extracted and sequenced using the Illumina® NextSeq platform and the assembly of the generated reads was performed using the SPAdes software. Research on the sequence type, resistance and virulence encoding genes, and plasmid replicon typing was carried out. RESULTS: The KP1.1JP strain was resistant to all ß-lactams, aminoglycosides, and fluoroquinolones tested. The genome size was 5 626 346 bp, distributed in 203 contigs and a guanine and cytosine content of 57.02%. The values of N50 and N75 were 285 583 bp and 173 927 bp, respectively. We verified that KP1.1JP belongs to ST101 and carries genes encoding resistance to ß-lactams (blaCTX-M-15, blaTEM-1B, blaOXA-1, blaSVH-182, and blaKPC-2), aminoglycosides [aac(3')-IIa, aph(3')-Vla], fluoroquinolones [aac(6')-Ib-cr], phenicol (catA1, catA2, catB3), tetracycline [tet(D)], trimethoprim (dfrA14), and fosfomycin (fosA). Additionally, the following virulence encoding genes were also detected: mrkABCDFHIJ (Fimbria type 3); fimABCDRFGHIK (Fimbria type 1); entABCDEFS and fepABCDG (siderophores); iroN, irp1, and irp2 (salmochelins); fyuA and ybtAEPQSTUX (yersiniabactin); and iutA (aerobactin). CONCLUSIONS: We report the occurrence of a K. pneumoniae ST101 strain carrying blaKPC-2 gene in an Amazon river in Brazil. The genomic characteristics of this strain will contribute to a better understanding of the spread of pathogens of clinical importance in the environment based on a One Health perspective.

Exploring the Bacteriome and Resistome of Humans and Food-Producing Animals in Brazil.

The epidemiology of antimicrobial resistance (AMR) is complex, with multiple interfaces (human-animal-environment). In this context, One Health surveillance is essential for understanding the distribution of microorganisms and antimicrobial resistance genes (ARGs). This report describes a multicentric study undertaken to evaluate the bacterial communities and resistomes of food-producing animals (cattle, poultry, and swine) and healthy humans sampled simultaneously from five Brazilian regions. Metagenomic analysis showed that a total of 21,029 unique species were identified in 107 rectal swabs collected from distinct hosts, the highest numbers of which belonged to the domain Bacteria, mainly Ruminiclostridium spp. and Bacteroides spp., and the order Enterobacterales. We detected 405 ARGs for 12 distinct antimicrobial classes. Genes encoding antibiotic-modifying enzymes were the most frequent, followed by genes related to target alteration and efflux systems. Interestingly, carbapenemase-encoding genes such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1 were identified in distinct hosts. Our results revealed that, in general, the bacterial communities from humans were present in isolated clusters, except for the Northeastern region, where an overlap of the bacterial species from humans and food-producing animals was observed. Additionally, a large resistome was observed among all analyzed hosts, with emphasis on the presence of carbapenemase-encoding genes not previously reported in Latin America. IMPORTANCE Humans and food production animals have been reported to be important reservoirs of antimicrobial resistance (AMR) genes (ARGs). The frequency of these multidrug-resistant (MDR) bacteria tends to be higher in low- and middle-income countries (LMICs), due mainly to a lack of public health policies. Although studies on AMR in humans or animals have been carried out in Brazil, this is the first multicenter study that simultaneously collected rectal swabs from humans and food-producing animals for metagenomics. Our results indicate high microbial diversity among all analyzed hosts, and several ARGs for different antimicrobial classes were also found. As far as we know, we have detected for the first time ARGs encoding carbapenemases, such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1, in Latin America. Thus, our results support the importance of metagenomics as a tool to track the colonization of food-producing animals and humans by antimicrobial-resistant bacteria. In addition, a network surveillance system called GUARANI, created for this study, is ready to be expanded and to collect additional data.

Kinetics Analysis of ß-Lactams Hydrolysis by OXA-50 Variants of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunist pathogen usually associated with life threatening infections and exhibits a set of intrinsic and acquired antimicrobial mechanisms. Although resistance to penicillins-like compounds is commonly associated with the chromosomal Pseudomonas-derived cephalosporinases ß-lactamase, the real contribution of OXA-50, a second chromosomally encoded ß-lactamase, remains unclear. In this study, we characterized the biochemical properties of OXA-50, OXA-488, and OXA-494. Both oxacilinases differ from OXA-50 in two amino acids each. The blaOXA-50, blaOXA-488, and blaOXA-494 were cloned into pET26b+ that was transformed into Escherichia coli DH5α strain, expressed in E. coli BL21 strain, and then purified for obtaining the hydrolytic parameters. Benzylpenicillin was the preferential substrate instead of oxacillin. Besides, OXA-488 showed a threefold increase in catalytic efficiency for benzylpenicillin, and it was twofold more efficient in hydrolyzing imipenem, compared with OXA-50, although such carbapenemase activity was considered weak. In addition, OXA-488 and OXA-494 showed an increased affinity for penicillins, which contributed to the increased catalytic efficiency against ampicillin, especially OXA-488. Chromosomally encoded resistance mechanisms are usually overshadowed by acquired mechanisms. However, understanding their real contribution is essential to comprehend the versatile profiles verified in P. aeruginosa isolates. Such information can help to choose the best therapy in a scenario of limited options.

Large Scale Genome-Centric Metagenomic Data from the Gut Microbiome of Food-Producing Animals and Humans.

The One Health concept is a global strategy to study the relationship between human and animal health and the transfer of pathogenic and non-pathogenic species between these systems. However, to the best of our knowledge, no data based on One Health genome-centric metagenomics are available in public repositories. Here, we present a dataset based on a pilot-study of 2,915 metagenome-assembled genomes (MAGs) of 107 samples from the human (N = 34), cattle (N = 28), swine (N = 15) and poultry (N = 30) gut microbiomes. Samples were collected from the five Brazilian geographical regions. Of the draft genomes, 1,273 were high-quality drafts (≥90% of completeness and ≤5% of contamination), and 1,642 were medium-quality drafts (≥50% of completeness and ≤10% of contamination). Taxonomic predictions were based on the alignment and concatenation of single-marker genes, and the most representative phyla were Bacteroidota, Firmicutes, and Proteobacteria. Many of these species represent potential pathogens that have already been described or potential new families, genera, and species with potential biotechnological applications. Analyses of this dataset will highlight discoveries about the ecology and functional role of pathogens and uncultivated Archaea and Bacteria from food-producing animals and humans. Furthermore, it also represents an opportunity to describe new species from underrepresented taxonomic groups.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

Trichosporon asahii causing subcutaneous mycoses in an immunocompetent patient: case report and a minireview.

Trichosporon spp. are a constituent of the normal flora of humans that can cause both superficial and invasive infections, mainly in immunocompromised and immunocompetent hosts, respectively. Herein, we a report of Trichosporon asahii causing subcutaneous fungal infection (SFI) in an immunocompetent patient after carpal tunnel surgery. Although susceptible to fluconazole, the treatment of SFI failed even using high doses of this azole. The skin lesion improved following the administration of voriconazole. We conducted a literature minireview searching reports on SFI in immunocompetent patients to check for epidemiological, diagnostic, therapeutic, and outcome characteristics. A total of 32 cases were reported. Despite being uncommon, the clinical suspicion and early diagnosis of SFI in immunocompetent patients undergoing previous surgery are important. Our study indicated that the azoles are the most active antifungal agents against Trichosporon spp., except for fluconazole, and voriconazole can be considered the first therapeutic option.

Role of ISKpn23 in blaBKC-1 Expression and Mobilization.

This study aimed to verify the role of ISKpn23 in the expression and mobilization of blaBKC-1 and aph(3')-VIi. Five constructs related to the natural blaBKC-1 genetic background in plasmid p60136 were made and submitted for antimicrobial susceptibility testing and quantitative reverse transcription-PCR. Transposition of ISKpn23-blaBKC-1 was investigated using transposition assays involving a 9.7-kb nonconjugative plasmid carrying blaBKC-1 (p60136) and a transfer-proficient plasmid (pOX38-Gen). The presence of ISKpn23 had a crucial role in blaBKC-1 expression, resulting in increased ß-lactam MICs. While we detected mobilization of p60136 by the pOX38-Gen plasmid, transposition of ISKpn23-blaBKC-1 was not observed.

Decreased susceptibility to imipenem and ceftazidime in early virulent Raoultella spp. strains retrieved from human intestinal infections.

The genus Raoultella spp. is comprised of four species, namely, R. electrica, R. ornithinolytica, R. planticola, and R. terrigena, which are rarely reported to cause infections in humans. This study aimed to characterize six strains of Raoultella spp. isolated from stool samples from patients with diarrhea. The strains included in the study were previously identified by biochemical methods as K. pneumoniae, during a surveillance study conducted in 1987. In the present study, the strains were re-identified by MALDI TOF and 16S rRNA sequencing and subsequently subjected to virulence gene screening by PCR, hemolytic activity, biofilm formation, hypermucoviscosity phenotype, capacity to interact with Caco-2 cells, and antimicrobial susceptibility test. Our results revealed that, among the six strains, three were identified as R. ornithinolytica and three as R. planticola. The genes related to iron uptake systems (aero1, aero2, iutA, entB, and ybtS) and adhesin (mrkD) were found in all strains. Furthermore, all strains demonstrated the ability to interact in vitro with Caco-2 cells and form biofilms. In general, the strains studied were sensitive to the antimicrobials tested; however, it was possible to observe high MICs for imipenem compared to ertapenem and meropenem and high minimal inhibitory concentrations (MICs) for ceftazidime, except for one strain. Our results show the occurrence of virulent strains of Raoultella spp. with high MICs for imipenem and ceftazidime causing diarrhea. We hope that our findings can contribute to the understanding of the evolution of this species since, as far as we know, these are the oldest isolates reported so far.

Genomic analyses of ciprofloxacin-resistant Neisseria gonorrhoeae isolates recovered from the largest South American metropolitan area.

We sequenced 13 Neisseria gonorrhoeae isolates exhibiting distinct susceptibility profiles and which were recovered over 12 years in the metropolitan region of São Paulo, Brazil. Whole Genome Sequencing (WGS) was performed on an Illumina MiSeq™ 2 × 300 bp paired-end reads. Bioinformatics analyses were carried out using CGE, PATRIC, and BLAST databases for manual curation of obtained genomes. Multilocus sequence typing (MLST) analysis identified seven STs, namely ST1580, ST1590, ST1901, ST1902, ST8161, ST9363, and ST15640. Moreover, a diversity of mutations was observed in MtrR/G45D-A39T, PIB/G120K-A121S, and PBP1/L421P. Mutations associated with sulfonamides (DHPS/R228S) and rifampicin (RNAP/H552N) were also detected, as well as tetracycline resistance determinants, namely rpsJ/V57M and tet(M). The results presented herein can contribute to the knowledge of N. gonorrhoeae strains circulating in Sao Paulo, Brazil.

Spread of multidrug-resistant Acinetobacter baumannii isolates belonging to IC1 and IC5 major clones in Rondônia state.

In Brazil, carbapenem-resistant A. baumannii (CRAB) is a critical pathogen showing high carbapenem resistance rates. Currently, there is little epidemiological data on A. baumannii isolated in the Northern Brazilian region. Herein, this study aimed to characterize the resistance mechanisms of CRAB isolates recovered from hospitalized patients in the state of Rondônia in 2019. Most of CRAB were considered as extensively drug-resistant, and some of them showed high MICs for minocycline. Only polymyxins showed a satisfactory activity. All isolates carried blaOXA-23 and were included in 14 distinct clusters, with the predominance of clonal group A (29%). The IC1 was the most frequent clonal group, followed by IC5 and IC4. Here, we firstly reported the epidemiological scenario of CRAB in the state of Rondônia, located in the Brazilian Amazon region. The high frequency of CRAB presenting XDR phenotype is of great concern, due to limited therapeutical options, especially in the actual pandemic scenario, in which we observed an overcrowding of ICU beds. Such results are essential to better characterize the epidemiology of CRAB in the entire Brazilian territory.

pmrCAB Recombination Events among Colistin-Susceptible and -Resistant Acinetobacter baumannii Clinical Isolates Belonging to International Clone 7.

Acinetobacter baumannii is a successful nosocomial pathogen due to its genomic plasticity. Homologous recombination allows genetic exchange and allelic variation among different clonal lineages and is one of the mechanisms associated with horizontal gene transfer (HGT) of resistance determinants. The main mechanism of colistin resistance in A. baumannii is mediated through mutations in the pmrCAB operon. Here, we describe two A. baumannii clinical isolates belonging to International Clone 7 (IC7) that have undergone recombination in the pmrCAB operon and evaluate the contribution of mobile genetic elements (MGE) to this phenomenon. Isolates 67569 and 72554 were colistin susceptible and resistant, respectively, and were submitted for short- and long-read genome sequencing using Illumina MiSeq and MinION platforms. Hybrid assemblies were built with Unicycler, and the assembled genomes were compared to reference genomes using NUCmer, Cortex, and SplitsTree. Genomes were annotated using Prokka, and MGEs were identified with ISfinder and repeat match. Both isolates presented a 21.5-kb recombining region encompassing pmrCAB. In isolate 67659, this region originated from IC5, while in isolate 72554 multiple recombination events might have happened, with the 5-kb recombining region encompassing pmrCAB associated with an isolate representing IC4. We could not identify MGEs involved in the mobilization of pmrCAB in these isolates. In summary, A. baumannii belonging to IC7 can present additional sequence divergence due to homologous recombination across clonal lineages. Such variation does not seem to be driven by antibiotic pressure but could contribute to HGT mediating colistin resistance. IMPORTANCE Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales, the presence of mcr-type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB. We have recently demonstrated that distinct pmrCAB sequences are associated with different A. baumannii International Clones (IC). In this study, we identified the presence of homologous recombination as an additional cause of genetic variation in this operon, which, to the best of our knowledge, was not mediated by mobile genetic elements. Even though this phenomenon was observed in both colistin-susceptible and -resistant isolates, it has the potential to contribute to the spread of resistance-conferring alleles, leading to reduced susceptibility to this last-resort antimicrobial agent.

A new mutation in mgrb mediating polymyxin resistance in Klebsiella variicola.

Polymyxin resistance is a public health concern - present in humans, animals and the environment - caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola (K. variicola). This study performed microbiological and genomic characterisation of three polymyxin-resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing using Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial-resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.