Detection of Mtb and NTM: preclinical validation of a new asymmetric PCR-binary deoxyribozyme sensor assay.

Tuberculosis (TB) and infectious diseases caused by non-tuberculous mycobacteria (NTM) are global concerns. The development of a rapid and accurate diagnostic method, capable of detecting and identifying different mycobacteria species, is crucial. We propose a molecular approach, the BiDz-TB/NTM, based on the use of binary deoxyribozyme (BiDz) sensors for the detection of Mycobacterium tuberculosis (Mtb) and NTM of clinical interest. A panel of DNA samples was used to evaluate Mtb-BiDz, Mycobacterium abscessus/Mycobacterium chelonae-BiDz, Mycobacterium avium-BiDz, Mycobacterium intracellulare/Mycobacterium chimaera-BiDz, and Mycobacterium kansasii-BiDz sensors in terms of specificity, sensitivity, accuracy, and limit of detection. The BiDz sensors were designed to hybridize specifically with the genetic signatures of the target species. To obtain the BiDz sensor targets, amplification of a fragment containing the hypervariable region 2 of the 16S rRNA was performed, under asymmetric PCR conditions using the reverse primer designed based on linear-after-the-exponential principles. The BiDz-TB/NTM was able to correctly identify 99.6% of the samples, with 100% sensitivity and 0.99 accuracy. The individual values of specificity, sensitivity, and accuracy, obtained for each BiDz sensor, satisfied the recommendations for new diagnostic methods, with sensitivity of 100%, specificity and accuracy ranging from 98% to 100% and from 0.98 to 1.0, respectively. The limit of detection of BiDz sensors ranged from 12 genome copies (Mtb-BiDz) to 2,110 genome copies (Mkan-BiDz). The BiDz-TB/NTM platform would be able to generate results rapidly, allowing the implementation of the appropriate therapeutic regimen and, consequently, the reduction of morbidity and mortality of patients.IMPORTANCEThis article describes the development and evaluation of a new molecular platform for accurate, sensitive, and specific detection and identification of Mycobacterium tuberculosis and other mycobacteria of clinical importance. Based on BiDz sensor technology, this assay prototype is amenable to implementation at the point of care. Our data demonstrate the feasibility of combining the species specificity of BiDz sensors with the sensitivity afforded by asymmetric PCR amplification of target sequences. Preclinical validation of this assay on a large panel of clinical samples supports the further development of this diagnostic tool for the molecular detection of pathogenic mycobacteria.

Non-tuberculous mycobacteria hybridisation profiles in the GenoType MTBDRplus assay: experience from a diagnostic routine of a high-throughput laboratory.

Introduction. Disease caused by non-tuberculous mycobacteria (NTM) is an emergent problem. Because NTM pulmonary disease and tuberculosis (TB) have similar clinical presentations, many cases of NTM may be misdiagnosed as TB before laboratory identification of the NTM species.Hypothesis/Gap Statement. Clinical laboratories should always perform differentiation between Mycobacterium tuberculosis complex (MTBC) and NTM to guide patients' correct treatment.Aim. To describe the characteristics and to identify mycobacterial isolates presumptively classified as MTBC by macroscopic characteristics in culture media that tested negative in GenoType MTBDRplus.Methodology. All cultures from February 2019 to December 2021 showing MTBC macroscopic characteristics were processed by GenoType MTBDRplus. MTBC-negative cultures underwent species identification by immunochromatography, line probe assays and PRA-hsp65. Patients' data were obtained from Brazilian surveillance systems.Results. Only 479 (3.1%) of 15 696 isolates presumptively identified as MTBC were not confirmed by GenoType MTBDRplus and were then subjected to identification. A total of 344 isolates were shown to be NTM, of which 309 (64.5%) and 35 (7.3%) were identified to the species and genus levels, respectively. Of the 204 NTM isolates with MTBC characteristics, the most frequent species were M. fortuitum (n=52, 25.5%), M. abscessus complex (MABC; n=27, 13.2%) and M. avium complex (MAC; n=26, 12.7%). Regarding the GenoType MTBDRplus results from NTM isolates, there were diverse hybridisation profiles with rpoB gene's different wild-type (WT) probes. Seventy-six (16.1%) of the 473 patients were classified as having NTM disease, the most frequent being MAC (n=15, 19.7%), MABC (n=13, 17.1%), M. kansasii (n=10, 13.2%) and M. fortuitum (n=6, 7.9%).Conclusion. Because the signs and symptoms of pulmonary TB are similar to those of pulmonary mycobacteriosis and treatment regimens for TB and NTM are different, identifying the disease-causing species is paramount to indicate the correct management. Thus, in the laboratory routine, when an isolate presumptively classified as MTBC is MTBC-negative, it is still essential to perform subsequent identification.

2,3,5-triphenyl tetrazolium chloride as colorimetric indicator for drug susceptibility testing against nontuberculous mycobacteria.

This study aimed to propose and evaluate a drug susceptibility testing (DST) using the 2,3,5-triphenyl tetrazolium chloride (TTC) as a colorimetric indicator against Mycobacterium abscessus complex (MABC), M. avium complex (MAC), and M. kansasii strains, main nontuberculous mycobacteria (NTM) of clinical relevance. Our results demonstrated that the assay using TTC and the broth microdilution method recommended by the Clinical and Laboratory Standards Institute had essential agreement above 91%, 92%, and 100%, for drugs tested against MABC, MAC, and M. kansasii strains, respectively. Categorical agreement above 91% was obtained for most drugs tested against MABC, except to cefoxitin (76.5%). For drugs tested against MAC and M. kansasii, categorical agreement above 92% and 100% was observed, respectively. TTC showed to be a promising colorimetric indicator of growth to be used in DST for NTM, allowing an easier reading of results.

Cost analysis of GenoType® MTBDRplus and GenoType® MTBDRsl at the State Laboratory of São Paulo, Brazil.

BACKGROUND: We aimed to evaluate the costs of GenoType® MTBDRplus and MTBDRsl incurred during the diagnosis of first- and second-line drug-resistant tuberculosis (TB) in São Paulo, Brazil. METHODS: Mean and activity-based costs of GenoType® were calculated in a referral laboratory for TB in Brazil. RESULTS: The mean cost value and activity-based cost of GenoType® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. CONCLUSIONS: The cost of GenoType® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

Development of multiplex real-time PCR for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.

Introduction. The M. abscessus molecular identification and its drug-resistance profile are important to choose the correct therapy.Aim. This work developed a multiplex real-time PCR (mqPCR) for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.Methodology. Isolates received by Adolfo Lutz Institute from 2010 to 2012, identified by PCR restriction enzyme analysis of a fragment of the hsp65 gene (PRA-hsp65) as M. abscessus type 1 (n=135) and 2 (n=71) were used. Drug susceptibility test (DST) for CLA were performed with reading on days 3 and 14. Subespecies identification by hsp65 and rpoB genes sequencing and erm(41) and rrl genes for mutation detection and primer design were performed. erm(41) gene deletion was detected by conventional PCR. Primers and probes were designed for five detections: erm(41) gene full size and with deletion; erm(41) gene T28 and C28; rrl gene A2058.Results. In total, 191/206 (92.7 %) isolates were concordant by all methods and 13/206 (6.3 %) were concordant only between molecular methods. Two isolates (1.0 %) were discordant by mqPCR compared to rrl gene sequencing. The mqPCR obtained 204/206 (99.0 %) isolates in agreement with the gold standard, with sensitivity and specificity of 98 and 100 %, respectively, considering the gold standard method and 92 and 93 % regarding DST.Conclusion. The mqPCR developed by us proved to be an easy-to-apply tool, minimizing time, errors and contamination.

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.

Cost analysis of GenoType® MTBDRplus and GenoType® MTBDRsl at the State Laboratory of São Paulo, Brazil

ABSTRACT Background: We aimed to evaluate the costs of GenoType® MTBDRplus and MTBDRsl incurred during the diagnosis of first- and second-line drug-resistant tuberculosis (TB) in São Paulo, Brazil. Methods: Mean and activity-based costs of GenoType® were calculated in a referral laboratory for TB in Brazil. Results: The mean cost value and activity-based cost of GenoType® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. Conclusions: The cost of GenoType® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

Epidemiological profile and follow-up of patients with pulmonary disease by non-tuberculous mycobacteria in Baixada Santista area, São Paulo.

OBJECTIVE: To present the frequency and species diversity of non-tuberculous mycobacteria, estimate the prevalence of non-tuberculous mycobacterial pulmonary disease, describe the epidemiological profile, and determine the follow-up of patients with non-tuberculous mycobacterial pulmonary disease living in a region with a high burden of tuberculosis. METHODS: This a retrospective cohort observational study using data records obtained from the Instituto Adolfo Lutz - Santos and from the São Paulo Sistema de Vigilância de Tuberculose do Estado de São Paulo in the period between 2000 and 2009. The studied variables were: socio-demographic characteristics, current and past history of tuberculosis, aspects related to diagnosis, and treatment and associated diseases. RESULTS: We included 319 non-tuberculous mycobacteria isolates in the study, corresponding to 257 patients. The species Mycobacterium kansasii (28.5%) and Mycobacterium fortuitum (16.6%) presented the higher occurrence. In 10.9% (24) of the patients, there was a criterion for confirming a case of pulmonary disease due to non-tuberculous mycobacteria. In relation to gender and age, male and individuals over 50 years old were the most frequent. Considering the confirmed cases, 47.8% had a past history of tuberculosis. CONCLUSION: The lack of information about the cases is evident, since pulmonary disease due to non-tuberculous mycobacteria is not mandatory. The therapeutic regimen according to the identified species is fundamental for success in combating the infections caused by non-tuberculous mycobacteria. Besides that, information about the regional epidemiology of pulmonary disease caused by non-tuberculous mycobacteria and the search for associations with other comorbidities are important to establish the correct treatment. In order to improve surveillance of pulmonary diseases by non-tuberculous mycobacteria, we suggest the implantation of a sentinel surveillance and of population-based studies.

Tuberculosis/COVID-19 co-infection detected in a single sputum sample using a rapid molecular test.

Tuberculosis (TB) and COVID-19 affect the lungs and are transmitted mainly by aerosols or particles of saliva from infected persons. Clinical similarities between diseases can affect correct diagnosis. Individuals belonging to the population deprived of liberty (PDL) are at increased risk of contagion due to precarious sanitary conditions and overcrowded environments. A variety of specimens may be suitable for the diagnosis of COVID-19, using molecular diagnostic techniques; however, there is little data on the analysis of sputum samples with the Xpert Xpress SARS-CoV-2® for the diagnosis of COVID-19, especially in this population group. The present study reports a case of TB and COVID-19 co-infection detected in sputum from an individual belonging to the PDL. For the detection, it used the GeneXpert platform (Cepheid, USA). Mycobacterium tuberculosis complex (MTC) was detected using the Xpert MTB/RIF Ultra® cartridge and SARS-CoV-2 was detected using the Xpert Xpress SARS-CoV-2® cartridge. The genes IS6110 and IS1081 were detected within 80 min indicating the presence of MTC, with no mutations related to resistance to rifampicin. The SARS-CoV-2 E and N2 genes were detected within 45 min. The result was confirmed by RT-qPCR with detection of E, N, and RdRP/S genes in the sputum and nasopharyngeal (NP) specimens. Rapid diagnoses that allow the identification and differentiation of such diseases are important for adequate epidemiological surveillance, isolation of infected individuals, and interruption of the transmission chain. Using the GeneXpert platform, specimens can be tested as soon as they are received, without the need for prior preparation. The US Food and Drug Administration has issued emergency authorization for the use of the Cepheid Xpert Xpress SARS-CoV-2 for the rapid detection of SARS-CoV-2 using specimens from a NP or nasal wash/aspirate. The case presented here gains an innovation with the use of the sputum to COVID-19 diagnosis.

Intradermal comparative cervical tuberculin test in the diagnosis of caprine tuberculosis.

Tuberculosis remains one of the most important infectious diseases with well-known zoonotic nature that affect humans, wildlife, and domestic animals, including goats. Nonetheless, no intradermal tuberculin test has been standardized for caprine diagnosis of tuberculosis. The present study investigated the intradermal comparative cervical tuberculin test (ICCTT) in the diagnosis of tuberculosis among 60 goats from farms with history of tuberculosis. The cutoff applied to goats was based on a study where goats had been experimentally infected with Mycobacterium bovis and Mycobacterium avium. Clinical examination, bacteriological culture, and histopathological staining were assessed to the diagnosis. Isolates compatible with mycobacteria were subjected for molecular diagnosis based on gyrB-restriction fragment length polymorphism (RFLP) analysis and PCR restriction-enzyme analysis (PRA) of hsp65 gene by BstEII and HaeIII, namely PRA-hsp65 assay. From all goats, 60% (n = 36/60), 3.3% (n = 2/60), and 36.7% (n = 22/60) showed positive, inconclusive, and negative reactions, respectively. Out of 36 goats with ICCTT positive, 75% (n = 27/36) had isolation of mycobacteria and were detected M. bovis by gyrB-RFLP. Molecular diagnosis and histopathological findings compatible with tuberculosis showed 86.1% (n = 31/36) concordance with the ICCTT. When compared ICCTT with M. bovis isolation, gyrB-RFLP, and histopathology, the better arithmetic means of sensitivity and specificity were 2.5 mm for ICCTT compared with M. bovis isolation and gyrB-RFLP, and 4.55 mm when compared with histopathology. Both receiver operating characteristic (ROC) curves presented statistical significance (P < 0.001). The identification of other mycobacteria, e.g., M. kansasii, M. flavescens, M. avium, M. florentinum, M. lentiflavum, M. simiae, and Corynebacterium pseudotuberculosis, not influenced positive results in ICCTT. The concordance between bacteriological, histopathological, and molecular identification with ICCTT findings indicate that the tuberculin test may be used as a valuable tool for diagnosis of caprine tuberculosis and reinforce the importance of association of methods to diagnostic of the disease from animal origin.

Epidemiological profile and follow-up of patients with pulmonary disease by non-tuberculous mycobacteria in Baixada Santista area, São Paulo

ABSTRACT Objective To present the frequency and species diversity of non-tuberculous mycobacteria, estimate the prevalence of non-tuberculous mycobacterial pulmonary disease, describe the epidemiological profile, and determine the follow-up of patients with non-tuberculous mycobacterial pulmonary disease living in a region with a high burden of tuberculosis. Methods This a retrospective cohort observational study using data records obtained from the Instituto Adolfo Lutz - Santos and from the São Paulo Sistema de Vigilância de Tuberculose do Estado de São Paulo in the period between 2000 and 2009. The studied variables were: socio-demographic characteristics, current and past history of tuberculosis, aspects related to diagnosis, and treatment and associated diseases. Results We included 319 non-tuberculous mycobacteria isolates in the study, corresponding to 257 patients. The species Mycobacterium kansasii (28.5%) and Mycobacterium fortuitum (16.6%) presented the higher occurrence. In 10.9% (24) of the patients, there was a criterion for confirming a case of pulmonary disease due to non-tuberculous mycobacteria. In relation to gender and age, male and individuals over 50 years old were the most frequent. Considering the confirmed cases, 47.8% had a past history of tuberculosis. Conclusion The lack of information about the cases is evident, since pulmonary disease due to non-tuberculous mycobacteria is not mandatory. The therapeutic regimen according to the identified species is fundamental for success in combating the infections caused by non-tuberculous mycobacteria. Besides that, information about the regional epidemiology of pulmonary disease caused by non-tuberculous mycobacteria and the search for associations with other comorbidities are important to establish the correct treatment. In order to improve surveillance of pulmonary diseases by non-tuberculous mycobacteria, we suggest the implantation of a sentinel surveillance and of population-based studies.

Evaluation of drug susceptibility in nontuberculous mycobacteria using the SLOMYCO and RAPMYCO sensititre plates.

Background: Non-tuberculous Mycobacteria (NTM) cause different forms of diseases. According to recent guideline by ATS/ERS/ESCMID/IDSA, drug susceptibility test (DST) is an important requirement to choose adequate treatment. The minimum inhibitory concentration (MIC) test is the recommended method. Sensititre SLOMYCO and RAPMYCO commercial panels were developed to perform mycobacteria DST easier. However, there are only two comparative studies between SLOMYCO and the MIC method and none for the RAPMYCO panel. The present study aimed to evaluate the Sensititre SLOMYCO and RAPMYCO plates in determining drug susceptibility compared to the gold standard method (MIC). Methods: The tests were carried out with clinical isolates received in the diagnostic routine of the Tuberculosis Laboratory at Institute Adolfo Lutz from the most frequent species in the state of São Paulo, Brazil. Reference strains were tested for repeatability and reproducibility analyses. MIC and Sensititre plates readings were compared with and without resazurin stain. Agreement between results was defined as MIC within the same dilution or dilution variation resulting the same category in both tests. Results were classified by categorical errors. Results: The RAPMYCO panel had 100% agreement for the drugs amikacin, doxycycline, ciprofloxacin and trimethoprim/sulfamethoxazole, 83.3% for clarithromycin and moxifloxacin and 60% for cefoxitin. The SLOMYCO panel had 80% agreement for amikacin and moxifloxacin and 60% for clarithromycin, rifabutin, rifampicin and ciprofloxacin. The repeatability and reproducibility with RAPMYCO and SLOMYCO plates showed a high level of agreement for the drugs tested, being higher with the use of resazurin. However, an evaluation on routine condition is needed. Conclusions: The present study found that the fewer steps in the tests with Sensititre plates and reading with resazurin allow its use with greater safety and efficiency in the laboratory routine. The results presented here will facilitate the execution of a validation for complete incorporation of Sensititre plates into a diagnostic routine.

A laboratory-based analysis of rapidly growing mycobacteria in Northwest Paulista, Sao Paulo, Brazil.

Background: Rapidly growing mycobacteria (RGM) are a group of nontuberculous mycobacteria (NTM) implicated in difficult-to-treat pulmonary and extrapulmonary diseases, possibly associated with invasive procedures and failures in sterilization of materials and equipment. Methods: We report our experience with the laboratory identification of RGM in a routine work and give an overview of the RGM isolated in our setting. Laboratorial data from all RGM mycobacterial isolates received at Adolfo Lutz Institute of São José do Rio Preto were analyzed from January 2000 to December 2015. Results: Five hundred and seventy-nine isolates were identified with NTM, of which 193 were RGM, which affected 113 patients. Among the 113 patients, the female gender was more frequent (55%) and the average age was 50 years. Pulmonary samples were the most frequent (79%), and 54.9% of the cases were isolated from sputum. Twelve different species were found and the most identified were group Mycobacterium abscessus and Mycobacterium fortuitum, making up 77.9% of all identified RGM. The most frequent comorbidities were smoking (n = 21), alcoholism (n = 12), and human immunodeficiency virus (n = 16). Drug susceptibility test was performed for nine patients and all showed susceptibility to amikacin and seven resistances to doxycycline. Conclusions: This study showed the experience of mycobacterial diagnosis in a routine laboratory, revealing that failure to meet the bacteriological criteria generates losses in the establishment of cases of RGM and consequently its correct treatment.

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.

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.