Emergence of zoonotic sporotrichosis in Brazil: a genomic epidemiology study.

BACKGROUND: Zoonotic sporotrichosis is a neglected fungal disease, whereby outbreaks are primarily driven by Sporothrix brasiliensis and linked to cat-to-human transmission. To understand the emergence and spread of sporotrichosis in Brazil, the epicentre of the current epidemic in South America, we aimed to conduct whole-genome sequencing (WGS) to describe the genomic epidemiology. METHODS: In this genomic epidemiology study, we included Sporothrix spp isolates from sporotrichosis cases from Brazil, Colombia, and the USA. We conducted WGS using Illumina NovaSeq on isolates collected by three laboratories in Brazil from humans and cats with sporotrichosis between 2013 and 2022. All isolates that were confirmed to be Sporothrix genus by internal transcribed spacer or beta-tubulin PCR sequencing were included in this study. We downloaded eight Sporothrix genome sequences from the National Center for Biotechnology Information (six from Brazil, two from Colombia). Three Sporothrix spp genome sequences from the USA were generated by the US Centers for Disease Control and Prevention as part of this study. We did phylogenetic analyses and correlated geographical and temporal case distribution with genotypic features of Sporothrix spp isolates. FINDINGS: 72 Sporothrix spp isolates from 55 human and 17 animal sporotrichosis cases were included: 67 (93%) were from Brazil, two (3%) from Colombia, and three (4%) from the USA. Cases spanned from 1999 to 2022. Most (61 [85%]) isolates were S brasiliensis, and all were reported from Brazil. Ten (14%) were Sporothrix schenckii and were reported from Brazil, USA, and Colombia. For S schenckii isolates, two distinct clades were observed wherein isolates clustered by geography. For S brasiliensis isolates, five clades separated by more than 100 000 single-nucleotide polymorphisms were observed. Among the five S brasiliensis clades, clades A and C contained isolates from both human and cat cases, and clade A contained isolates from six different states in Brazil. Compared with S brasiliensis isolates, larger genetic diversity was observed among S schenckii isolates from animal and human cases within a clade. INTERPRETATION: Our results suggest that the ongoing epidemic driven by S brasiliensis in Brazil represents several, independent emergence events followed by animal-to-animal and animal-to human transmission within and between Brazilian states. These results describe how S brasiliensis can emerge and spread within a country. FUNDING: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil; the São Paulo Research Foundation; Productivity in Research fellowships by the National Council for Scientific and Technological Development, and Ministry of Science and Technology of Brazil.

Large hospital-wide outbreak of Paenibacillus spp pseudobacteremia associated with contaminated nonsterile gloves.

We report a large, hospital-wide outbreak of pseudobacteremia by Paenibacillus spp. In total, 139 patients presented at least 1 positive blood culture during a 13-month period. Microbiological experiments indicated that contaminated nonsterile gloves were associated with the pseudobacteremia episodes. The outbreak was resolved by discontinuing the use of the involved brand.

Staphylococcus argenteus Infections, Brazil.

In 2015, two new species related to the Staphylococcus aureus were proposed. We describe five isolates of the new species Staphylococcus argenteus cultured from human cases of bacteremia and skin and soft tissue infections. This is the first report of S. argenteus, from South America, causing community-acquired and nosocomial infections.

A simple disk pre-diffusion test to predict in vitro aztreonam/avibactam activity against NDM-producing Klebsiella pneumoniae complex.

OBJECTIVES: The aim of this study was to describe a simple test to predict in vitro efficacy of aztreonam/avibactam (ATM-AVI) combination based on a pre-diffusion assay involving routinely available ceftazidime/avibactam (CAZ-AVI) and aztreonam (ATM) disks. METHODS: A total of 113 non-repetitive NDM-producing Klebsiella had the species identified by multiplex PCR. Minimum inhibitory concentrations (MICs) for ATM and ATM-AVI were determined by broth microdilution. For the combined disk pre-diffusion method, a disk containing ceftazidime 10 µg and avibactam 4 µg (CAZ-AVI) was applied on the surface of an uninoculated Mueller-Hinton agar plate. Following incubation for 2 h at 36°C, the disk was removed, the bacterial suspension was applied and a 30 µg ATM disk was placed precisely in the same position as the removed CAZ-AVI disk. Following incubation for 16-20 h, inhibition zone diameters were measured and correlated with ATM-AVI MICs. RESULTS: The distribution of species among the 113 isolates was 85 Klebsiella pneumoniae (75.2%), 19 Klebsiella quasipneumoniae (16.8%) and 9 Klebsiella variicola (8.0%). A total of 99 isolates had only blaNDM and 14 had both blaNDM and blaKPC genes. Regarding the isolates positive for blaNDM only, 38.4% were susceptible to ATM and 7.1% were susceptible, increased exposure. All isolates had ATM-AVI MICs of ≤1 mg/L, and the smallest inhibition zone diameter observed was 23 mm. CONCLUSION: Modified disk pre-diffusion can be used as a simple test to screen for ATM-AVI in vitro activity against Klebsiella, since ATM-AVI disks, gradient strips or microdilution panels are not commercially available.

Genotyping of paired KPC-producing Klebsiella pneumoniae isolates with and without divergent polymyxin B susceptibility profiles.

Polymyxins are still used mainly in treating infections caused by carbapenem-resistant Klebsiella pneumoniae worldwide. The most frequent mechanism of acquired resistance to polymyxins in Gram-negative bacilli is the occurrence of mutations in chromosomal genes regulating operons responsible for lipopolysaccharide modification. As we observed at Santa Casa de São Paulo hospital the occurrence of infections caused by isolates resistant to polymyxins in patients previously treated with this antimicrobial, and new infections caused by the same polymyxin-susceptible species, in this study, we aimed to determine the clonality of consecutive K. pneumoniae isolates from the same patients and characterize the molecular determinants of polymyxin resistance in paired or clonal isolates. A total of 24 pairs and one trio of K. pneumoniae isolates were included in this study. Species identification was achieved by mass spectrometry and multiplex PCR. Polymyxin B minimal inhibitory concentrations were determined by broth microdilution. Clonality was evaluated using pulsed-field gel electrophoresis. The presence of insertions in mgrB gene was tested by PCR, and mutations on pmrA, pmrB, phoP, and phoQ were evaluated by PCR and complete nucleotide sequencing. A fraction of 23.8% of strains resistant to polymyxin B had an insertion in mgrB. Amino acid substitution F204L in PmrB may be implicated in polymyxin resistance. Substitutions T246A and R256G in PmrB were not implicated in polymyxin resistance. In this study, polymyxin resistance after a first susceptible isolate was detected was most frequently due to an infection caused by a distinct clone.

How new molecular tools can help bugbusters: a Burkholderia cepacia complex outbreak investigation.

An outbreak of bloodstream infection (BSI) caused by members of the Burkholderia cepacia complex (Bcc) took place from March 2012 until April 2014 involving thirteen patients. AIM: To describe an outbreak investigation of BSI Bcc and showing how genetic sequencing tools contributed to confirm the hypothesis of extrinsic contamination proposed by an observational study. METHODS: The Infection Control Department revised and reinforced good practices of infusion therapy and catheter care, visits to affected wards, a case control study, and environmental screening based on the case-control findings. RESULTS: Data from the case-control study found an association of cases with central venous catheter (OR 1.36; CI 1.15-1.67) and intravenous cisatracurium use (OR 10.75; CI 1.67-68.89). Visits to the operatory block revealed problems related to the cold chain used for the preservation of thermolabile cisatracurium. We could not retrieve Bcc from environmental samples using classic microbiology. New samples from the same surfaces were obtained for genetic sequencing. Bcc was identified in the cooler box, refrigerator and reusable ice packages. CONCLUSION: Environmental screening using genetic sequencing proved to be a useful tool for confirming our hypothesis of extrinsic contamination raised by the case-control study.

The applicability of gene sequencing and MALDI-TOF to identify less common gram-negative rods (Advenella, Castellaniella, Kaistia, Pusillimonas and Sphingobacterium) from environmental isolates.

Our aim was to identify less common non-fermenting gram-negative rods during the bioremediation process. Five genera were found: Advenella, Castellaniella, Kaistia, Pusillimonas and Sphingobacterium, for a total of 15 isolates. Therefore, we evaluated the applicability of four methods currently available for bacteria identification: (1) conventional biochemical methods, (2) the VITEK®-2 system, (3) MALDI-TOF mass spectrometry and (4) 16S rRNA gene sequencing. The biochemical methods and the VITEK®-2 system were reliable only for the Sphingobacterium isolate and solely at the genus level. Both MALDI-TOF mass spectrometry platforms (Bruker and VITEK® MS) did not achieve reliable identification results for any of these genera. 16S rRNA gene sequencing identified eight isolates to the species level but not to the subspecies level, when applicable. The remaining seven isolates were reliably identified through 16S rRNA gene sequencing to the genus level only. Our findings suggest that the detection and identification of less common genera (and species) that appeared at certain moments during the bioremediation process can be a challenge to microbiologists considering the most used techniques. In addition, more studies are required to confirm our results.

Improved blood culture workflow for faster identification of KPC-producing Enterobacterales.

Carba-NP original report for blood cultures described the need of subculture and mechanical lysis before testing, reaching the turnaround time of approximately 4 hours for sample preparation. We tested 100 consecutive blood cultures positive for Gram-negative bacilli on the Gram stain from a large clinical laboratory. Bacterial pellets were prepared by centrifugation and submitted to Carba-NP and Blue-Carba tests and used further to prepare smears for Vitek MS. Results obtained with colonies grown on sheep blood agar using the same methodologies were used as the gold standard. Carbapenemase genes were confirmed by PCR and DNA sequencing. Vitek MS identified correctly 86% of the samples. Of note, 7% of the samples were incorrectly reported by the instrument as containing a single isolate. KPC-2 was the predominant carbapenemase detected. There was 100% concordance for both negative and positive results for Carba-NP. In contrast, for Blue-Carba the concordance for positive results was 92.8%, and 41% of strains negative for carbapenemases presented a yellowish color on control well turning the test non-interpretable. The turnaround time for sample preparation for preparing the pellet was 13 min, and no subculture or mechanical lysis is needed when detecting KPC production in Enterobacterales.

Antimicrobial resistance in Enterobacteriaceae in Brazil: focus on ß-lactams and polymyxins

ABSTRACT During the last 30 years there has been a dissemination of plasmid-mediated β-lactamases in Enterobacteriaceae in Brazil. Extended spectrum β-lactamases (ESBL) are widely disseminated in the hospital setting and are detected in a lower frequency in the community setting. Cefotaximases are the most frequently detected ESBL type and Klebsiella pneumoniae is the predominant species among ESBL producers. Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae became widely disseminated in Brazil during the last decade and KPC production is currently the most frequent resistance mechanism (96.2%) in carbapenem resistant K. pneumoniae. To date KPC-2 is the only variant reported in Brazil. Polymyxin B resistance in KPC-2-producing K. pneumoniae has come to an alarming rate of 27.1% in 2015 in São Paulo, the largest city in Brazil. New Delhi metallo-β-lactamase was detected in Brazil in 2013, has been reported in different Brazilian states but are not widely disseminated. Antimicrobial resistance in Enterobacteriaceae in Brazil is a very serious problem that needs urgent actions which includes both more strict adherence to infection control measures and more judicious use of antimicrobials.

Antimicrobial resistance in Enterobacteriaceae in Brazil: focus on ß-lactams and polymyxins.

During the last 30 years there has been a dissemination of plasmid-mediated ß-lactamases in Enterobacteriaceae in Brazil. Extended spectrum ß-lactamases (ESBL) are widely disseminated in the hospital setting and are detected in a lower frequency in the community setting. Cefotaximases are the most frequently detected ESBL type and Klebsiella pneumoniae is the predominant species among ESBL producers. Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae became widely disseminated in Brazil during the last decade and KPC production is currently the most frequent resistance mechanism (96.2%) in carbapenem resistant K. pneumoniae. To date KPC-2 is the only variant reported in Brazil. Polymyxin B resistance in KPC-2-producing K. pneumoniae has come to an alarming rate of 27.1% in 2015 in São Paulo, the largest city in Brazil. New Delhi metallo-ß-lactamase was detected in Brazil in 2013, has been reported in different Brazilian states but are not widely disseminated. Antimicrobial resistance in Enterobacteriaceae in Brazil is a very serious problem that needs urgent actions which includes both more strict adherence to infection control measures and more judicious use of antimicrobials.

Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion

Abstract Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2 μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125 μg/mL for both antimicrobials. The MIC90 were 4 μg/mL and 2 μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion.

Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2µg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125µg/mL for both antimicrobials. The MIC90 were 4µg/mL and 2µg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

Multilocus enzyme electrophoresis analysis of rapidly-growing mycobacteria: an alternative tool for identification and typing.

OBJECTIVES: Rapidly growing mycobacteria (RGM) have emerged as important pathogens in clinical settings, associated with esthetic procedures and postsurgical infections, pulmonary infections among cystic fibrosis patients, and other structural pulmonary diseases. Microorganisms belonging to Mycobacterium abscessus-Mycobacterium chelonae and to Mycobacterium fortuitum groups have frequently been associated with outbreaks and various epidemics. In the present study, RGM strains were characterized in order to investigate molecular markers based on proteomic analysis. METHODS: Multilocus enzyme electrophoresis (MLEE) was used for species identification and clonal analysis of RGM recovered from postsurgical wound infections during an epidemic. The study included 30M. abscessus subsp. bolletii clinical isolates, most belonging to the BRA100 clone (epidemic in Rio de Janeiro city), as well as 16 RGM ATCC reference strains. RESULTS: Molecular typing allowed the detection of diversity in the studied population and revealed species-specific isoenzymatic patterns. Additionally, the clonal relationship among M. abscessus subsp. bolletii outbreak isolates, as examined using MLEE, was markedly consistent. CONCLUSIONS: Isoenzymatic characterization was found to be a useful molecular tool to identify RGM species and to determine the relatedness among closely related M. abscessus subsp. bolletii isolates. This may be considered a powerful approach for epidemiological studies on RGM.

fosI Is a New Integron-Associated Gene Cassette Encoding Reduced Susceptibility to Fosfomycin.

In this work, we demonstrate that the fosI gene encodes a predicted small protein with 134 amino acids and determines reduced susceptibility to fosfomycin. It raised the MIC from 0.125 to 6 µg/ml when the pBRA100 plasmid was introduced into Escherichia coli TOP10 and to 16 µg/ml when the gene was cloned into the pBC_SK(-) vector and expressed in E. coli TOP10.

Frequency of Plasmid-Mediated AmpC ß-Lactamases in Escherichia coli Isolates from Urine Samples in São Paulo, Brazil.

Plasmid-mediated AmpC ß-lactamases (PMACBLs) in Enterobacteriaceae encode resistance to third-generation cephalosporins, and these can mediate carbapenem resistance when associated with porin loss. However, no standardized phenotypic method is available for detecting these enzymes in the clinical microbiology laboratory. Limited data are available concerning the frequency of PMACBLs in Enterobacteriaceae in Brazil. This study was conducted in response to an increased cefoxitin (CFO) resistance rate of 3.7% in Escherichia coli isolates from urine samples from patients with suspected urinary tract infections during 2010. We collected 2,266 E. coli isolates prospectively during January 2012. A total of 109 (4.8%) isolates were nonsusceptible to CFO. These strains were further examined using multiplex PCR for the presence of genes encoding PMACBLs and using inhibitor assays with CFO and ceftazidime (CAZ) disks with and without phenylboronic acid. Pulsed-field gel electrophoresis was used to evaluate clonal dissemination. Genes encoding PMACBLs were detected in 1.8% of the isolates from inpatients and 0.46% of isolates from outpatients. The most prevalent gene was blaCMY-2 and blaCMY-4 was also detected. The phenotypic analysis showed 100% sensitivity and specificity for CMY-2 and CMY-4 when CFO-resistant isolates with a minimum zone diameter difference of 5 mm for CAZ or CAZ and CFO were considered positive. Although most of the isolates were nonclonal, one clonal group with two isolates was observed. Thus, the most frequent PMACBL in E. coli from São Paulo, Brazil is CMY-2, and both clonal and plasmid-mediated dissemination occur.

Antimicrobial resistance in Enterobacteriaceae in Brazil: focus on -lactams and polymyxins

ABSTRACT During the last 30 years there has been a dissemination of plasmid-mediated -lactamases in Enterobacteriaceae in Brazil. Extended spectrum -lactamases (ESBL) are widely disseminated in the hospital setting and are detected in a lower frequency in the community setting. Cefotaximases are the most frequently detected ESBL type and Klebsiella pneumoniae is the predominant species among ESBL producers. Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae became widely disseminated in Brazil during the last decade and KPC production is currently the most frequent resistance mechanism (96.2%) in carbapenem resistant K. pneumoniae. To date KPC-2 is the only variant reported in Brazil. Polymyxin B resistance in KPC-2-producing K. pneumoniae has come to an alarming rate of 27.1% in 2015 in São Paulo, the largest city in Brazil. New Delhi metallo--lactamase was detected in Brazil in 2013, has been reported in different Brazilian states but are not widely disseminated. Antimicrobial resistance in Enterobacteriaceae in Brazil is a very serious problem that needs urgent actions which includes both more strict adherence to infection control measures and more judicious use of antimicrobials.

Characterization of Tn3000, a Transposon Responsible for blaNDM-1 Dissemination among Enterobacteriaceae in Brazil, Nepal, Morocco, and India.

In Enterobacteriaceae, the blaNDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world.