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.

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.

Genomic analysis and antimicrobial activity of ß-lactam/ß-lactamase inhibitors and other agents against KPC-producing Klebsiella pneumoniae clinical isolates from Brazilian hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are highly disseminated worldwide, and isolates co-resistant to other antimicrobial agents pose a threat to effective antimicrobial therapy. Therefore, evaluation of novel antimicrobial drugs is needed to identify potential treatments with better outcomes. We evaluated the in vitro activity of novel antimicrobial drugs/combinations against 97 KPC-producing Klebsiella pneumoniae isolates recovered from different hospitals in Brazil during 2021-2022. Clonality, resistance and virulence genes were detected by whole-genome sequencing. The majority of the isolates (54.6%) were classified as extensively drug resistant or multidrug resistant (44.3%); one isolate showed a pandrug resistance phenotype. The most active antimicrobial agents were meropenem-vaborbactam, cefiderocol, and ceftazidime-avibactam, with sensitivities higher than 90%; resistance to ceftazidime-avibactam was associated with KPC-33 or KPC-44 variants. Colistin and polymyxin B were active against 58.6% of the isolates. The 97 isolates were distributed into 17 different sequence types, with a predominance of ST11 (37.4%). Although high in vitro susceptibility rates were detected for meropenem-vaborbactam and cefiderocol, only ceftazidime-avibactam is currently available in Brazil. Our findings showed limited susceptibility to antimicrobial drugs employed for infection treatment of carbapenem-resistant K. pneumoniae, underscoring the urgent need for stringent policies for antimicrobial stewardship to preserve the activity of such drugs.

Genomics and Antimicrobial Susceptibility of Clinical Pseudomonas aeruginosa Isolates from Hospitals in Brazil.

Pseudomonas aeruginosa, an opportunistic pathogen causing infections in immunocompromised patients, usually shows pronounced antimicrobial resistance. In recent years, the frequency of carbapenemases in P. aeruginosa has decreased, which allows use of new beta-lactams/combinations in antimicrobial therapy. Therefore, the in vitro evaluation of these drugs in contemporary isolates is warranted. We evaluated the antimicrobial susceptibility and genomic aspects of 119 clinical P. aeruginosa isolates from 24 different hospitals in Brazil in 2021-2022. Identification was performed via MALDI-TOF-MS, and antimicrobial susceptibility was identified through broth microdilution, gradient tests, or disk diffusion. Whole-genome sequencing was carried out using NextSeq equipment. The most active drug was cefiderocol (100%), followed by ceftazidime-avibactam (94.1%), ceftolozane-tazobactam (92.4%), and imipenem-relebactam (81.5%). Imipenem susceptibility was detected in 59 isolates (49.6%), and the most active aminoglycoside was tobramycin, to which 99 (83.2%) isolates were susceptible. Seventy-one different sequence types (STs) were detected, including twelve new STs described herein. The acquired resistance genes blaCTX-M-2 and blaKPC-2 were identified in ten (8.4%) and two (1.7%) isolates, respectively. Several virulence genes (exoSTUY, toxA, aprA, lasA/B, plcH) were also identified. We found that new antimicrobials are effective against the diverse P. aeruginosa population that has been circulating in Brazilian hospitals in recent years.

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.

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 β-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 β-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.

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.

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.

Clonal spread of ArmA- and OXA-23-coproducing Acinetobacter baumannii International Clone 2 in Brazil during the first wave of the COVID-19 pandemic.

Introduction. Carbapenem-resistant Acinetobacter baumannii (CRAB) is the primary pathogen causing hospital-acquired infections. The spread of CRAB is mainly driven by the dissemination of resistant clones, and in Latin America, International Clones IC-1 (also known as clonal complex CC1), IC-4 (CC15) and IC-5 (CC79) are the most prevalent.Gap Statement. There are no documented outbreaks of CRAB International Clone 2 (IC-2) reported in Brazil.Aim. To describe a large outbreak of CRAB caused by the uncommon IC-2 in a Brazilian COVID-19 hospital.Methodology. From May 2020 to May 2021, 224 patients infected or colonized with CRAB were identified in a single hospital; 92 % of them were also infected with SARS-CoV-2. From these patients, 137 isolates were recovered and subjected to antimicrobial susceptibility testing, PCR analysis and molecular typing. Whole-genome sequencing and downstream analysis were carried out on a representative isolate (the first available isolate).Results. In 76 % of the patients, a single OXA-23-producing CRAB IC-2 was identified. All the isolates were susceptible to polymyxin B, but highly resistant (>95 %) to aminoglycosides, fluoroquinolones and beta-lactams. Genomic analysis revealed that the representative isolate also carried the 16S rRNA Methylase ArmA, which was detected for the first time in this species in Brazil.Conclusion. We report the rapid spread of an emerging CRAB clone responsible for causing a large outbreak in a hospital in Brazil, a country with predominance of other CRAB clones. Continuous and prospective surveillance is warranted to evaluate the impact of this clone in Brazilian hospital settings.

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.

A clinical Pseudomonas chlororaphis isolate harboring a new blaVIM-2 borne integron, In2088, isolated from bloodstream infection in a newborn patient.

We report the isolation and genomic characterization of a VIM-2 producing Pseudomonas chlororaphis causing bloodstream infection in a newborn in Brazil. A new integron, In2088 (intI1-blaVIM-2-aacA7-aacA27-gcu241), was identified and the first P. chlororaphis genome from a clinical isolate was deposited in public databases.

Use of an immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex clinical isolates in routine diagnosis.

Accurate identification of Mycobacterium tuberculosis complex (MTBC) isolates is essential for tuberculosis (TB) control, especially in a high-burden country such as Brazil. Conventional identification methods are laborious and time-consuming, while rapid molecular methods are expensive and require skilled personnel and appropriate physical laboratory infrastructure. Immunochromatographic assays (ICAs) have been shown to provide a rapid and reliable TB diagnosis at a low cost. The use of the SD Bioline TB Ag MPT64 ICA (MPT64 assay) for rapid identification of MTBC clinical isolates in the routine diagnosis of a large-volume reference TB laboratory was evaluated. We analysed 375 isolates on solid and liquid media concurrently with conventional phenotypic methods, the PRA-hsp65 molecular technique and the MPT64 assay. The sensitivity, specificity and accuracy of the ICA were 97.7, 100 and 98.1 %, respectively. The MPT64 assay yielded rapid and accurate results, enabling the treatment to be initiated early and also impacting on TB control.

Mycobacterium tuberculosis e micobactérias não tuberculose em paciente com síndrome da imunodeficiência adquirida e câncer: relato de caso / Mycobacterium tuberculosis and non-tuberculous mycobacteria in a patient with acquired immunodeficiency syndrome and cancer: case report

O presente estudo relata o caso de um paciente de difícil tratamento com infecção mista por tuberculose (TB) e micobactérias não tuberculosas (MNT). O paciente é portador de HIV, câncer e outras doenças associadas. A TB foi elucidada em internação devido a quadro de hemoptise. No período da TB/MNT, a carga viral manteve-se indetectável e o Linfócito T CD4+ variou de 117 a 622 cél/mm3. Os principais sintomas foram febre, tosse, emagrecimento e sudorese. O exame de Raio-X mostrou suspeita de TB bilateral cavitária, a baciloscopia foi negativa e várias culturas apresentaram resultado positivo. As identificações dos isolados foram: Complexo Mycobacterium tuberculosis, Mycobacterium intracelullare/chimaera e M. fortuitum, isolados de amostras pulmonares. Iniciado tratamento para TB em outubro de 2015, atualmente tratando de MNT e mantendo cultura positiva com identificação de M. intracelullare. Os Testes de suscetibilidade aos fármacos para M. intracelullare mostraram resistência a Isoniazida, Rifampicina, Ciprofloxacina, Etambutol e Rifabutina. A terapia para a síndrome da imunodeficiência adquirida (AIDS) aumentou a sobrevida do paciente, trazendo novos desafios para o diagnóstico, controle de tratamento e cuidados da atenção básica para os pacientes com TB/micobacterioses/HIV. Este caso exemplifica que a decisão por um tratamento empírico pode ser uma escolha acertada em casos com clínica e imagem compatíveis e baciloscopia ou TRM negativos.

The present study is a case report of a difficult-to-treat patient with mixed tuberculosis (TB) and non-tuberculous mycobacteria (NTM) infection. The patient has HIV, cancer and other associated diseases. TB was elucidated upon hospitalization due to hemoptysis. In the TB/NTM period, the viral load remained undetectable and CD4 ranged from 622 to 117 cells/mm3. The main symptoms were fever, cough, weight loss and sweating. The X-ray examination showed suspicion of bilateral cavitary TB; the bacilloscopy was negative and several cultures presented positive results. The following isolates were identified: Mycobacterium tuberculosis complex, Mycobacterium intracelullare/chimaera and M. fortuitum, isolated from lung samples. The TB treatment was initiated in October 2015, currently treating NTM and maintaining positive culture with identification of M. intracelullare. Antimicrobial sensitivity tests for M. intracelullare showed resistance to Isoniazid, Rifampicin, Ciprofloxacin, Ethambutol and Rifabutin. Acquired Immune Deficiency Syndrome (AIDS) therapy has increased patient survival, bringing new challenges for diagnosis, treatment control, and basic care for TB/mycobacterial/HIV patients. This case exemplifies that the decision of an empirical treatment may be the correct choice in cases with compatible clinical and imaging tests and negative smear microscopy or molecular tests.

Pseudooutbreak of rapidly growing mycobacteria due to Mycobacterium abscessus subsp bolletii in a digestive and respiratory endoscopy unit caused by the same clone as that of a countrywide outbreak.

BACKGROUND: The nontuberculous mycobacteria (NTM) are widely spread. In Brazil, 2,520 cases of rapidly growing mycobacteria (RGM) infections after medical procedures were reported, with 5.4% of cases related to nonsurgical invasive procedures and with an occurrence of 1 clone (BRA100) of Mycobacterium abscessus subsp bolletii. OBJECTIVE: To describe a pseudooutbreak of M abscessus subsp bolletii in an endoscopy and bronchoscopy unit. METHODS: The alert for a pseudooutbreak was given when 3 patients, in the same week, had a positive bronchoalveolar lavage culture for M abscessus subsp bolletii. The patients had no symptoms/signs of mycobacterial infection; thus, contamination of bronchoscopes was suspected. Samples for culturing were collected from bronchoscopes, digestive endoscopes, automated disinfection machines, and the water supply. Clinical samples were identified by polymerase chain reaction restriction-enzyme analysis (PRA) of the hsp65 gene and their pulsed-field gel electrophoresis pattern was compared with environmental samples. RESULTS: The investigation demonstrated a contamination of bronchoscopes, digestive endoscopes, and disinfection machines. Molecular typing demonstrated that all strains belonged to the same clone (MAB01), identical to clone BRA100. DISCUSSION: Cross-transmission due to poor disinfection as well as resistance to glutaraldehyde may play roles in the spread of MAB01 M abscessus subsp bolletii, which may have a unique resistance to the environment and adaption to human hosts. However the water supply may have played a role. Attention is needed to ensure the quality of water used to rinse disinfected equipment.