Genomic characterization of New Delhi metallo-beta-lactamase-producing species of Morganellaceae, Yersiniaceae, and Enterobacteriaceae (other than Klebsiella) from Brazil over 2013-2022.

Over the last decade, New Delhi metallo-beta-lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed-field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM-1 allele was predominant (n = 29), but blaNDM-5 was identified in an E. coli specimen with a novel ST, and the blaNDM-7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr-1-producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended-spectrum ß-lactamases were common: cefotaximase from Munich (CTX-M)-15 (n = 10), CTX-M-2 (n = 4), and CTX-M-8 (n = 3) were detected, and the mcr-1-producing E. coli was found to co-produce both CTX-M-8 and CTX-M-55 ß-lactamases. The mcr-9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM-producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options.

Whole genome sequencing of Bacillus anthracis isolated from animal in the 1960s, Brazil, belonging to the South America subclade.

Bacillus anthracis causes anthrax disease and can affect humans and other animals. This zoonotic disease has an impact on the economic and health aspects. B. anthracis population is divided into three major clades: A (with worldwide distribution), B, and C (restricted to specific regions). Anthrax is most common in agricultural regions of central and southwestern Asia, sub-Saharan Africa, Southern and Eastern Europe, the Caribbean, and Central and South America. Here, we sequenced by short and long reads technologies to generate a hybrid assembly of a lineage of B. anthracis recovered from animal source in the 1960s in Brazil. Isolate identification was confirmed by phenotypic/biochemical tests and MALDI-TOF MS. Antimicrobial susceptibility was performed by in-house broth microdilution. B. anthracis IAL52 was susceptible to penicillin, amoxicillin, doxycycline, levofloxacin, and tetracycline but non-susceptible to ciprofloxacin. IAL52 was classified as sequence type ST2, clade A.Br.069 (V770 group). Sequencing lineages of B. anthracis, especially from underrepresented regions, can help determine the evolution of this critical zoonotic and virulent pathogen.

Genomic Diversity of NDM-Producing Klebsiella Species from Brazil, 2013-2022.

Background: Since its first report in the country in 2013, NDM-producing Enterobacterales have been identified in all the Brazilian administrative regions. In this study, we characterized by antimicrobial susceptibility testing and by molecular typing a large collection of NDM-producing Klebsiella isolates from different hospitals in Brazil, mainly from the state of Sao Paulo, over the last decade. Methods: Bacterial isolates positive for blaNDM-genes were identified by MALDI-TOF MS and submitted to antimicrobial susceptibility testing by disk diffusion or broth microdilution (for polymyxin B). All isolates were submitted to pulsed-field gel electrophoresis, and isolates belonging to different clusters were submitted to whole genome sequencing by Illumina technology and downstream analysis. Mating out assays were performed by conjugation, plasmid sizes were determined by S1-PFGE, and plasmid content was investigated by hybrid assembly after MinIon long reads sequencing. Results: A total of 135 NDM-producing Klebsiella were identified, distributed into 107 different pulsotypes; polymyxin B was the only antimicrobial with high activity against 88.9% of the isolates. Fifty-four isolates presenting diversified pulsotypes were distributed in the species K. pneumoniae (70%), K. quasipneumoniae (20%), K. variicola (6%), K. michiganensis (a K. oxytoca Complex species, 2%), and K. aerogenes (2%); blaNDM-1 was the most frequent allele (43/54, 80%). There was a predominance of Clonal Group 258 (ST11 and ST340) encompassing 35% of K. pneumoniae isolates, but another thirty-one different sequence types (ST) were identified, including three described in this study (ST6244 and ST6245 for K. pneumoniae, and ST418 for K. michiganensis). The blaNDM-1 and blaNDM-7 were found to be located into IncF and IncX3 type transferable plasmids, respectively. Conclusions: Both clonal (mainly driven by CG258) and non-clonal expansion of NDM-producing Klebsiella have been occurring in Brazil in different species and clones, associated with different plasmids, since 2013.

Genomic diversity of NDM-producing Klebsiella species from Brazil, 2013-2022

Since its first report in the country in 2013, NDM-producing Enterobacterales have been identified in all the Brazilian administrative regions. In this study, we characterized by antimicrobial susceptibility testing and by molecular typing a large collection of NDM-producing Klebsiella isolates from different hospitals in Brazil, mainly from the state of Sao Paulo, over the last decade. Methods: Bacterial isolates positive for blaNDM-genes were identified by MALDI-TOF MS and submitted to antimicrobial susceptibility testing by disk diffusion or broth microdilution (for polymyxin B). All isolates were submitted to pulsed-field gel electrophoresis, and isolates belonging to different clusters were submitted to whole genome sequencing by Illumina technology and downstream analysis. Mating out assays were performed by conjugation, plasmid sizes were determined by S1-PFGE, and plasmid content was investigated by hybrid assembly after MinIon long reads sequencing. Results: A total of 135 NDM-producing Klebsiella were identified, distributed into 107 different pulsotypes; polymyxin B was the only antimicrobial with high activity against 88.9% of the isolates. Fifty-four isolates presenting diversified pulsotypes were distributed in the species K. pneumoniae (70%), K. quasipneumoniae (20%), K. variicola (6%), K. michiganensis (a K. oxytoca Complex species, 2%), and K. aerogenes (2%); blaNDM-1 was the most frequent allele (43/54, 80%). There was a predominance of Clonal Group 258 (ST11 and ST340) encompassing 35% of K. pneumoniae isolates, but another thirty-one different sequence types (ST) were identified, including three described in this study (ST6244 and ST6245 for K. pneumoniae, and ST418 for K. michiganensis). The blaNDM-1 and blaNDM-7 were found to be located into IncF and IncX3 type transferable plasmids, respectively. Conclusions: Both clonal (mainly driven by CG258) and non-clonal expansion of NDM-producing Klebsiella have been occurring in Brazil in different species and clones, associated with different plasmids, since 2013.

Silent mutations in ribosomal protein genes are associated with high-risk clones of carbapenem-resistant Acinetobacter baumannii prevalent in Brazil.

OBJECTIVES: To analyze the relationship of ribosomal protein mutations and clonality of high-risk clones Acinetobacter baumannii. METHODS: Seventy-nine carbapenem-resistant A. baumannii were subjected to whole-genome sequencing (Illumina NextSeq), and codifying sequences of ribosomal proteins were extracted and screened for mutations. MALDI-TOF MS analysis (Bruker Biotyper) and Spectra data from MALDI-TOF was employed to generate a dendrogram based on principal component analysis (PCA) data. Clones were identified by Multilocus sequencing typing (MLST) based on WGS. RESULTS: Ribosomal RNA protein sequences extracted from the genomes identified mutations that were associated with clonal complexes, but most of them were silent. PCA did not cluster the isolates according to their clonality identified by MLST. CONCLUSIONS: By comparing the nucleotide and amino acid sequences of diversified A. baumannii, and Bruker Biotyper profiles, we showed that silent mutations in ribosomal RNA nucleotides are associated with clonal complexes, but since most of the mutations were silent, MALDI-TOF MS raw data was not a useful tool for typing the high-risk clones of this species.

Silent mutations in ribosomal protein genes are associated with high-risk clones of carbapenem-resistant Acinetobacter baumannii prevalent in Brazil

Objectives: To analyze the relationship of ribosomal protein mutations and clonality of high-risk clones Acinetobacter baumannii. Methods: Seventy-nine carbapenem-resistant A. baumannii were subjected to whole-genome sequencing (Illumina NextSeq), and codifying sequences of ribosomal proteins were extracted and screened for mutations. MALDI-TOF MS analysis (Bruker Biotyper) and Spectra data from MALDI-TOF was employed to generate a dendrogram based on principal component analysis (PCA) data. Clones were identified by Multilocus sequencing typing (MLST) based on WGS. Results: Ribosomal RNA protein sequences extracted from the genomes identified mutations that were associated with clonal complexes, but most of them were silent. PCA did not cluster the isolates according to their clonality identified by MLST. Conclusions: By comparing the nucleotide and amino acid sequences of diversified A. baumannii, and Bruker Biotyper profiles, we showed that silent mutations in ribosomal RNA nucleotides are associated with clonal complexes, but since most of the mutations were silent, MALDI-TOF MS raw data was not a useful tool for typing the high-risk clones of this species.

Genomic and phenotypic characterisation of antimicrobial resistance in carbapenem-resistant Acinetobacter baumannii hyperendemic clones CC1, CC15, CC79 and CC25.

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly driven by the spread of clonal lineages. High frequencies of CRAB are reported in South America, and clonal complexes CC1, CC15, CC79 and CC25 are predominant. A total of 79 non-redundant CRAB recovered from 26 Brazilian hospitals were selected for antimicrobial susceptibility testing by microdilution and whole-genome sequencing (WGS). Multilocus sequence typing (MLST), acquired antimicrobial resistance genes and phylogeny based on high-quality SNPs were extracted from WGS data. XDR (86.1%), MDR (12.7%) and one PDR isolate from CC15 (1.3%) were identified. Colistin resistance was more frequent in CC25 isolates (P < 0.01). Prevalence of CC79 (n = 22; 27.8%) CC1 (n = 21; 26.6%), CC15 (n = 21; 26.6%) and CC25 (n = 12; 15.2%) was observed. Regarding carbapenem-hydrolysing class D ß-lactamases (CHDLs), blaOXA-23 was frequently detected in CC1, CC15 and CC25 isolates, whereas blaOXA-72 was the most frequent CHDL in CC79 isolates [n = 12/22 (54.5%); P < 0.01]. High-quality SNP analysis correlated well with sequence type and revealed that CRAB clones are highly conversed and present some clone-specific resistance determinants. This study provides essential information to understand the antimicrobial resistance patterns of CRAB in Brazilian hospitals, where hyperendemic XDR-CRAB clones are disseminated. Phenotypic and genomic analysis of CRAB recovered from Brazilian hospitals revealed the predominance of XDR phenotype in the majority of international clonal complex CC79, CC1, CC15 and CC25. Dissemination of specific CRAB lineages in Brazil is suggested to be driven by their resistance determinants under antimicrobial selective pressure.

Virulence Profiling and Molecular Typing of Shiga Toxin-Producing E. coli (STEC) from Human Sources in Brazil.

Since no recent data characterizing Shiga toxin-producing E. coli (STEC) from human infections in Brazil are available, the present study aimed to investigate serotypes, stx genotypes, and accessory virulence genes, and also to perform pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) of 43 STEC strains recovered from 2007 to 2017. Twenty-one distinct serotypes were found, with serotype O111:H8 being the most common. However, serotypes less frequently reported in human diseases were also found and included a hybrid STEC/ETEC O100:H25 clone. The majority of the strains carried stx1a as the sole stx genotype and were positive for the eae gene. Regarding the occurrence of 28 additional virulence genes associated with plasmids and pathogenicity islands, a diversity of profiles was found especially among the eae-harboring strains, which had combinations of markers composed of up to 12 distinct genes. Although PFGE analysis demonstrated genetic diversity between serotypes such as O157:H7, O111:H8, O26:H11, O118:H16, and O123:H2, high genetic relatedness was found for strains of serotypes O24:H4 and O145:H34. MLST allowed the identification of 17 distinct sequence types (STs) with ST 16 and 21 being the most common ones. Thirty-five percent of the strains studied were not typeable by the currently used MLST approach, suggesting new STs. Although STEC O111:H8 remains the leading serotype in Brazil, a diversity of other serotypes, some carrying virulence genes and belonging to STs incriminated as causing severe disease, were found in this study. Further studies are needed to determine whether they have any epidemiological relevance.

Chromoblastomycosis in the Amazon region, Brazil, caused by Fonsecaea pedrosoi, Fonsecaea nubica, and Rhinocladiella similis: Clinicopathology, susceptibility, and molecular identification.

Chromoblastomycosis is a chronic subcutaneous disease caused by human contact with melanized fungi occurring mainly in tropical and subtropical zones worldwide. This study assessed 12 patients with chromoblastomycosis from Rondônia, Brazil, Amazon region. In sum, 83.3% were men, 41.6% were from Monte Negro city, median age was 52.9 years, and median time to disease progression was 12.2 years. Lesions were located on the lower limbs (75%), and verruciform was prevalent form (66.6%). After 3 years of treatment with itraconazole, two patients were considered cured. The etiological agents were identified by the molecular sequence of the ribosomal internal transcribed spacer ITS1, 5.8S, and ITS2 region and ß-tubulin genes. Eight strains were identified as Fonsecaea pedrosoi, two were F. nubica, and two were Rhinocladiella similis. The antifungal activity of five drugs was evaluated, and the most active drug was terbinafine (range minimal inhibitory concentration [MIC] 0.015-0.12 µg/ml), itraconazole (range MIC 0.03-0.5 µg/ml) and voriconazole (range MIC 0.06-0.5 µg/ml). The highest MIC was 5-fluorocytosine (range MIC 2-32 µg/ml), and amphotericin B (range MIC 0.25-2 µg/ml). In conclusion, the present study expanded the epidemiological disease database and described for the first time F. nubica and R. similis as chromoblastomycosis agents in the Brazilian Amazon region. Our results confirmed the importance of using molecular methods to identify the melanized fungi and stimulate the recognition of the disease in other places where no cases have been reported.

Genomic and phenotypic characterisation of antimicrobial resistance in carbapenem-resistant Acinetobacter baumannii hyperendemic clones CC1, CC15, CC79 and CC25

Dissemination of carbapenem-resistant Acinetobacter baumannii (CRAB) is mainly driven by the spread of clonal lineages. High frequencies of CRAB are reported in South America, and clonal complexes CC1, CC15, CC79 and CC25 are predominant. A total of 79 non-redundant CRAB recovered from 26 Brazilian hospitals were selected to perform antimicrobial susceptibility test (AST) by microdilution and whole genome sequencing (WGS). MLST, acquired resistance genes and phylogeny based on high-quality SNPs were extracted from WGS. XDR (86.1%), MDR (12.7%) and one PDR isolate from CC15 (1.3%) were identified. Colistin resistance was more frequently on CC25 isolates (p<0.01). Prevalence of CC79 (n=22; 27.8%) CC1 (n=21; 26.6%), CC15 (n=21; 26.6%), and CC25 (n=12; 15.2%) was observed. Regarding the carbapenem-hydrolyzing class D β-lactamases (CHDL), blaOXA-23 gene was frequently detected in CC1, CC15, and CC25 isolates, but blaOXA-72 gene was the most frequent CHDL in CC79 isolates (n=12/22, 54.5%; p<0.01). High-quality SNPs analysis correlated well with the ST, and revealed that CRAB clones are highly conversed and present some clone-specific resistance determinants. This study provides essential information to understand the antimicrobial resistance patterns of CRAB in Brazilian hospitals, where hyperendemic XDR CRAB clones are disseminated. Phenotypic and genomic analysis of CRAB recovered from 26 Brazilian hospitals revealed the prevalence of XDR phenotype in the majority of international clonal complex CC79, CC1, CC15 and CC25. Dissemination of specific CRAB lineages in Brazil is suggested to be driven by their resistance determinants under antimicrobial selective pressure.

Detection of pandrug-resistant ST15 acinetobacter baumannii causing bloodstream infection in an HSCT patient in Brazil

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Chromoblastomycosis in the amazon region, BRazil, caused by fonsecaea pedrosoi, fonsecaea nubica, and Rhinocladiella similis: clinicopathology, susceptibility, and molecular identification

Chromoblastomycosis is a chronic subcutaneous disease caused by human contact with melanized fungioccurring mainly in tropical and subtropical zones worldwide. This study assessed 12 patients with chro-moblastomycosis from Rondˆonia, Brazil, Amazon region. In sum, 83.3% were men, 41.6% were from MonteNegro city, median age was 52.9 years, and median time to disease progression was 12.2 years. Lesions werelocated on the lower limbs (75%), and verruciform was prevalent form (66.6%). After 3 years of treatmentwith itraconazole, two patients were considered cured. The etiological agents were identified by the molec-ular sequence of the ribosomal internal transcribed spacer ITS1, 5.8S, and ITS2 region andß-tubulin genes.Eight strains were identified asFonsecaea pedrosoi, two wereF. nubica,and two wereRhinocladiella similis.The antifungal activity of five drugs was evaluated, and the most active drug was terbinafine (range minimalinhibitory concentration [MIC] 0.015­0.12µg/ml), itraconazole (range MIC 0.03­0.5µg/ml) and voriconazole(range MIC 0.06­0.5µg/ml). The highest MIC was 5-fluorocytosine (range MIC 2­32µg/ml), and ampho-tericin B (range MIC 0.25­2µg/ml). In conclusion, the present study expanded the epidemiological diseasedatabase and described for the first timeF. nubicaandR. similisas chromoblastomycosis agents in theBrazilian Amazon region. Our results confirmed the importance of using molecular methods to identify themelanized fungi and stimulate the recognition of the disease in other places where no cases have beenreported.

Caracterização molecular e fenotípica de amostras bacterianas pertencentes ao complexo Acinetobacter calcoaceticus-Acinetobacter baumannii / Molecular and phenotypic characterization of Acinetobacter calcoaceticus-Acinetobacter baumannii isolates

Nos últimos 30 anos, Acinetobacter tornou-se um dos patógenos de maior preocupação clínica pela falta de terapias eficazes em virtude do fenótipo de multirresistência frequentemente apresentado. Dentre as espécies do gênero Acinetobacter, A. baumannii, A. genoespécie 3 e A. genoespécie 13TU são as mais comumente encontradas a partir de amostras biológicas. Estas espécies ao lado de A. calcoaceticus constituem o complexo A. calcoaceticus-A. baumannii (ACB). Este estudo propõe um esquema composto de duas PCRs para a identificação das espécies de interesse médico que fazem parte do complexo ACB. O método é simples, rápido e, além de identificar as espécies, permite pesquisar a presença de genes de resistência. Foram identificadas 515 amostras do complexo ACB, isoladas de pacientes no período de janeiro de 2005 a dezembro de 2010. A identificação das espécies do complexo ACB foi realizada por esquema composto de duas reações de PCR. Foram avaliados os perfis de sensibilidade por disco difusão e a pesquisa da presença dos genes blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, blaVIM, blaSIM, blaSPM e blaGIM foi realizada por PCR utilizando-se iniciadores específicos. No grupo de amostras estudas, 82,5% são A. baumannii (425), 11,5% A. genoespécie 13TU (59) e 6,0% A. genoespécie 3 (30), sendo A. baumannii mais isolado em pacientes internados em UTIs (p=0,0407) e A. genoespécie 13TU mais isolado em pacientes de outros ambientes hospitalares (p=0,0204). A. baumannii apresentou menor sensibilidade a todos os antimicrobianos quando comparado com A. genoespécie 13TU e A. genoespécie. 3 (p<0,05). Foi possível observar ao longo do período estudado o aumento significativo da resistência aos carbapenêmicos e da sensibilidade a gentamicina por A. baumannii entre os isolados de pacientes de UTIs (p<0.05). Nenhum dos genes codificadores para metalo-lactamases foi detectado nas amostras estudadas Dentre os cepas resistentes aos carbapenêmico...

The genus Acinetobacter has emerged as one of the most troublesome pathogens for health care institutions globally. Its clinical significance, especially over the last 15 years, has been driven by its remarkable ability to up regulate or acquire resistance determinants, making it one of the organisms threatening the current antibiotic era. A. baumannii, A. 3 and A. 13TU are the most commonly species found from biological samples. These species beside A. calcoaceticus are very closely related and difficult to distinguish from each other by phenotypic properties. Therefore, it has been proposed to refer to these species as the A.calcoaceticus-A. baumannii complex(ACB). In the period from 2005 to 2009, the most frequent bacterial isolates among the nosocomial infection at the HU-USP was ACB (18%). Due to the frequency with which species are involved in ACB outbreaks of infection in the HU-USP and the emergency clinic because of expression of the phenotype of resistance to several classes of antibiotics, this study aimed to identify and characterize the species of complex ACB by molecular methods, to study their mechanisms of resistance and to characterize the different clones from patients admitted to different hospital areas. Furthermore, the ability to characterize biofilm formation, adhesion to different cell lines as well as the mechanisms of cell-cell communication were analyzed. From the ACB complex, 515 samples were identified, isolated from patients from January 2005 to December 2010. The identification of clinical species of the ACB was performed by molecular methods that were developed and validated for identification of Acinetobacter sp. include two reactions of PCR. The profiles of sensibility were evaluated by disc diffusion and the detection of the presence of genes blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, blaVIM, blaSIM, blaGIM, and blaSPM were performed using specific primers. Molecular typing was performed using...