Genomic Characterization of a Clinical NDM-1-Producing Klebsiella michiganensis from Brazil.

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.

Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal.

OBJECTIVES: This study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. METHODS: Bacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. RESULTS: Isolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. CONCLUSIONS: Not surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

Whole genome sequence analysis of the first reported isolate of Salmonella Agona carrying blaCTX-M-55 gene in Brazil

This study analyzes the genomic findings of the first report of Salmonella isolate carrying the blaCTX-M-55 gene, recovered from a bacteremic patient from Brazil. A bacterial isolate positive for the blaCTX-M-55 gene was submitted to antimicrobial susceptibility testing by disk diffusion and epsilometric test. Whole genome sequencing was performed using Illumina technology. Conjugation assay was performed; plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinION long reads sequencing. Isolate 288_18 was identified as sequence type ST13, resistant to ampicillin, cefotaxime, ceftazidime, cefepime, ceftriaxone, and aztreonam. A transferable IncFII plasmid sized approximately 67 kb was found to carry the blaTEM-1 and blaCTX-M-55 in a module consisting of IS26-blaTEM-1B-WbuC-blaCTX-M-55-IS26. In addition, an 117 kb IncI1plasmid was also identified in the 288_18 isolate, but without additional resistance genes. To the best of our knowledge, this is the first report of blaCTX-M-55 in Salmonella isolated from human infection in Brazil. The occurrence of blaCTX-M-55 in the IncFII epidemic plasmid in a relevant clinical human isolate of Salmonella Agona underscores the urgent need for enhanced and effective continuous surveillance for controlling its dissemination.

Implantação da rede laboratorial para realização do ensaio de liberação de interferon-gama (IGRA) para detecção de tuberculose latente no Estado de São Paulo: primeiros passos e desafios / Implementation of the laboratory network to carry out the interferon-gamma release assay (IGRA) to detect latent tuberculosis in the State of Sao Paulo: first steps and challenges

A tuberculose (TB) continua sendo um grande desafio para a saúde pública mundial e, para um controle eficiente, também é essencial identificar pessoas com tuberculose latente (ILTB). O ensaio de liberação de interferon-gama (IGRA), incorporado pelo SUS em 2021, permitirá ampliar o diagnóstico de ILTB, em complemento à prova tuberculínica. Para essa implantação, as coordenações do Programa Estadual e da Rede de Laboratórios de TB/SP iniciaram a identificação de executores do IGRA a partir da rede de laboratórios de TB e/ou CD4, para verificar possíveis barreiras para implantação do teste. Foram avaliados os insumos e os profissionais para execução do ensaio, a infraestrutura laboratorial e a disponibilidade de equipamentos. Dez laboratórios avaliaram amostras de sangue total com o kit QuantiFERON®-TB Gold Plus e relataram sua experiência quanto à logística de amostras, execução do ensaio e liberação de laudos. Para otimizar o exame, a coleta ocorreu em tubos heparinizados (sódio ou lítio). Foi sugerida a logística da rede de laboratórios de CD4, que foi utilizada por 20% dos laboratórios participantes, enquanto 50% optaram pelo agendamento. Não foram reportadas dificuldades na liberação de laudo. Dois laboratórios avaliaram o número de células T CD4+ prévio e no momento do IGRA, observando diferença em 10% dos pacientes, fator que pode ser relevante na análise do resultado. Ao todo, foram analisadas 383 amostras, 81 (21,1%) reagentes, 297 (77,5%) não reagentes e cinco (1,3%) indeterminados. Foi observada grande variação de positividade (3,6-50,0%) entre os laboratórios, provavelmente devido à população atendida. Apesar dos desafios encontrados, consideramos que a taxa média de positividade (~20%) sugere que a oferta do IGRA na rede pública possibilitará o aumento do diagnóstico de ILTB e melhor controle da TB.

Co-production of Classes A and B Carbapenemases BKC-1 and VIM-2 in a Clinical Pseudomonas Putida Group Isolate from Brazil.

Emergence of resistance to classical antimicrobial agents is a public health issue, especially in countries with high antimicrobial consumption rates. Carbapenems have been employed as first-choice option for empirical treatment complicated infections. However, in the last decades, frequency of carbapenemase-producing Gram-negative bacteria has rising, demanding the use of alternative antimicrobial agents. By sequencing the entire genomes with short and long reads technologies, we report the isolation and genomic characterization of a carbapenem-resistant Pseudomonas clinical isolate. The identification based on average nucleotide identity indicates a putative new species into the Pseudomonas putida Group, which carries both the blaBKC-1 and blaVIM-2 carbapenemase genes. The blaBKC-1 was found to be on a transferable IncQ plasmid backbone, whereas blaVIM-2 was found in a new integron, In2126 (intl1∆-blaVIM-2-aacA7-blaVIM-2∆-aacA27-3'CS), described in this study. Our findings indicate that co-occurrence of classes A and B carbapenemase enzymes underscores the evolving emergence of more complex antimicrobial resistance in opportunistic pathogens.

Genomic characterization of a multi-drug resistant, CTX-M-65-producing clinical isolate of Salmonella Infantis isolated in Brazil.

A multi-drug resistant, CTX-M-65 producing Salmonella Infantis was identified from a patient in Brazil. Whole genome sequencing followed by hybrid assembly (short and long reads) indicated the presence of blaCTX-M-65 in a pESI-like megaplasmid in this ST32 isolate and phylogenetic analysis showed high similarity with IncFIB S. Infantis isolates from food and poultry in the USA.

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.

Genomic and Clinical Characterization of IMP-1-Producing Multidrug-Resistant Acinetobacter bereziniae Isolates from Bloodstream Infections in a Brazilian Tertiary Hospital.

Acinetobacter baumannii is the main species of the Acinetobacter genus; however, non-baumannii Acinetobacter (NBA) species causing infections have been described for the past years, as well as antimicrobial resistance. In this study, we describe the occurrence of two multidrug-resistant (MDR) IMP-1-producing Acinetobacter bereziniae isolates recovered from bloodstream infections in different patients but in the same intensive care unit among 134 carbapenem-resistant Acinetobacter screened. Antimicrobial susceptibility testing revealed resistance to carbapenems, extended spectrum, and antipseudomonad cephalosporins, amikacin, and trimethoprim-sulfamethoxazole. Both A. bereziniae isolates shared the same ApaI-pulsed-field gel electrophoresis (PFGE) pattern. Whole-genome sequencing of both isolates revealed that blaIMP-1 was embedded into an In86 Class I integron carrying also sul1, aac(6')-31, and aadA genes. A new sequence type (ST1309 Pasteur) was deposited. The virulence genes lpxC and ompA, seen in A. baumannii, were detected in the A. bereziniae strains. Recognition of A. bereziniae causing invasive MDR infection underscores the role of NBA species as human pathogens especially in at-risk patients.

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

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Caracterização fenotípica e genotípica de cepas de Salmonella spp. e subtipagem molecular de plasmídeos carreando genes de beta-lactamases de espectro estendido e AmpC / Phenotypic and genotypic characterization of strains of Salmonella spp. and molecular subtyping of plasmids carrying extended-spectrum beta-lactamase and AmpC genes

Salmonella spp. são reconhecidas como um dos mais importantes patógenos que ocasionam doença entérica, sendo a terceira causa de morte entre as doenças transmitidas por alimentos (DTAs). A maior preocupação atual deve-se ao aumento da resistência aos beta-lactâmicos, e a produção de beta-lactamases mediadas por plasmídeos dentro do gênero. O objetivo deste trabalho foi caracterizar fenotipicamente e genotipicamente 44 isolados de Salmonella spp. produtores de beta lactamases além de, realizar a subtipagem dos plasmídeos de resistência aos beta lactâmicos de cepas de origem humana e não humana. Foi realizado o teste de sensibilidade aos antimicrobianos e a determinação da Concentração Inibitória Mínima (CIM). A PCR foi utilizada para a identificação dos genes blaESBL e blaAmpC, em seguida, confirmados por sequenciamento de Sanger. Para a subtipagem plasmídial dos isolados foram utilizadas as técnicas de PBRT e pMLST. Os resultados do teste de sensibilidade aos antimicrobianos mostrou que 97,7% dos isolados foram resistentes à cefotaxima, 86,3% resistentes à sulfonamida e tetraciclina, 81,9% resistentes ao ácido nalidíxico, 68,1% resistentes a ceftriaxona e 56,8% a estreptomicina. De acordo com a PCR, 24 isolados (54,5%) foram positivos para ESBLs (CTX-M), e 20 isolados (45,5%) positivos para beta-lactamase do tipo AmpC, CMY-2. A variante CTX-M-2 foi encontrada em 27,2% dos isolados, seguido da variante CTX-M-8 (22,7%). As variantes CTX-M-15 e CTX-M-65 foram detectadas em um isolado respectivamente. De acordo com a tipagem plasmidial foram encontrados os grupos de incompatibilidade IncI1/ST12 e IncA/C/ST2 em cepas positivas para blaCMY-2. Para as variantes de CTX-M foram detectados os grupos de incompatibilidade IncHI2 em cepas produtoras de CTX-M-2, IncI1, IncF e IncL/M identificados em cepas CTX-M-8, e dois isolados não tipáveis nas variantes CTX-M-15 e CTX-M-65. A análise do perfil genético dos isolados por PFGE sugerem a circulação de cepas geneticamente relacionadas entre fontes humanas e não humanas no sorotipo Muenchenn, e um perfil de similaridade de 82,3% no sorotipo Heidelberg, em cepas provenientes de fontes de origem e anos de isolamento diferentes. Os resultados encontrados neste estudo revelam o cenário atual da resistência aos beta-lactâmicos e reforçam a necessidade de intensificar o monitoramento de isolados portadores de plasmídeos epidêmicos carreadores de beta-lactamases (ESBL e AmpC) na clínica, ambiente e na cadeia produtiva. (AU)

Salmonella spp. are recognized as one of the most important pathogens that cause enteric disease, being the third leading cause of death among foodborne diseases. The greatest current concern should be the increase in resistance to beta-lactams and the production of plasmidmediated beta-lactamases within the genus. The objective of this work was to characterize phenotypically and genotypically 44 isolates of Salmonella spp. beta-lactamase producers, in addition to subtyping plasmids resistant to beta-lactams from human and non-human strains. The antimicrobial sensitivity test was performed and the Minimum Inhibitory Concentration (MIC) was determined. A PCR was used to identify the blaESBL and blaAmpC genes, followed by Sanger sequencing. For plasmid subtyping were used PBRT and pMLST techniques. The results of the antimicrobial sensitivity test showed that 97.7% of the strains were resistant to cefotaxime, 86.3% resistant to sulfonamide and tetracycline, 81.9% resistant to nalidic acid, 68.1% resistant to ceftriaxone and 56.8% streptomycin. According to the PCR, 24 (54.5%) were positive for ESBLs (CTX-M) and 20 (45.5%) positive for AmpC-type beta-lactamase, CMY-2. A CTX-M-2 variant was found in 27.2% of isolates followed by the CTX-M-8 variant (22.7%). The variants, CTX-M-15 and CTX-M-65 were detected in one isolate, respectively. According to plasmid typing, the incompatibility groups IncI1/ST12 and IncA/C/ST2 were found in positive strains for blaCMY-2. For the CTX-M variants, the IncHI2 incompatibility groups were detected in the CTX-M-2, IncI1, IncF and IncL/M were found in the CTX-M-8 strains, and CTX-M-15 and CTX-M-65 variants were not typeable. The analysis of the genetic profile by PFGE suggests the circulation of strains related between human and non-human sources in the Muenchen serotype, and a similarity index of 82.3% in the Heidelberg serotype, in strains from different sources and years. The results found in this study reveal the current scenario of resistance to betalactams and reinforce the need to intensify the monitoring of epidemic plasmids carrying beta-lactamases (ESBL and AmpC) in the clinic, environment and productive chain. (AU)

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.

Subtyping of plasmid-mediated quinolone resistance among Salmonella serotypes by whole genome sequencing.

Most known plasmids are identified by conferring virulence or antimicrobial resistance phenotypes and such characteristics aid in the success of the dispersion of different plasmid types between bacteria from different sources. This study aimed to perform the subtyping of the plasmid-mediated quinolone resistance, detected in Salmonella spp. A total of 34 Salmonella strains non-susceptible to ciprofloxacin were evaluated. Strains were selected based on the presence of PMQR determined by Polymerase Chain Reaction and further submitted to Next Generation Sequencing. Most of the strains presented the qnrB19 in small ColE-like plasmids and qnrB2 gene associated with IncN/ST5 plasmids also detected. Our results indicated the co-occurrence of PMQR and ESBLs in plasmids that are a lineage of epidemic plasmids circulating in Salmonella in which additional resistances were detected, highlighting the potential threat of resistance Salmonella to public health, particularly in infections in which antimicrobial therapy is needed.

Subtyping of plasmid-mediated quinolone resistance among Salmonella serotypes by whole genome sequencing

Most known plasmids are identified by conferring virulence or antimicrobial resistance phenotypes and such characteristics aid in the success of the dispersion of different plasmid types between bacteria from different sources. This study aimed to perform the subtyping of the plasmid-mediated quinolone resistance, detected in Salmonella spp. A total of 34 Salmonella strains non-susceptible to ciprofloxacin were evaluated. Strains were selected based on the presence of PMQR determined by Polymerase Chain Reaction and further submitted to Next Generation Sequencing. Most of the strains presented the qnrB19 in small ColE-like plasmids and qnrB2 gene associated with IncN/ST5 plasmids also detected. Our results indicated the co-occurrence of PMQR and ESBLs in plasmids that are a lineage of epidemic plasmids circulating in Salmonella in which additional resistances were detected, highlighting the potential threat of resistance Salmonella to public health, particularly in infections in which antimicrobial therapy is needed.

Subtyping of plasmid-mediated quinolone resistance among Salmonella serotypes by whole genome sequencing

Most known plasmids are identified by conferring virulence or antimicrobial resistance phenotypes and such characteristics aid in the success of the dispersion of different plasmid types between bacteria from different sources. This study aimed to perform the subtyping of the plasmid-mediated quinolone resistance, detected in Salmonella spp. A total of 34 Salmonella strains non-susceptible to ciprofloxacin were evaluated. Strains were selected based on the presence of PMQR determined by Polymerase Chain Reaction and further submitted to Next Generation Sequencing. Most of the strains presented the qnrB19 in small ColE-like plasmids and qnrB2 gene associated with IncN/ST5 plasmids also detected. Our results indicated the co-occurrence of PMQR and ESBLs in plasmids that are a lineage of epidemic plasmids circulating in Salmonella in which additional resistances were detected, highlighting the potential threat of resistance Salmonella to public health, particularly in infections in which antimicrobial therapy is needed.