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OBJECTIVE: Despite the increasing reports of blaNDM in Enterobacterales in Brazil, comprehensive whole genome sequencing (WGS) data remain scarce. To address this knowledge gap, our study focuses on the characterization of the genome of an New Delhi Metallo-ß-lactamase (NDM)-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae (KQPN) clinical strain isolated in Brazil. METHODS: The antimicrobial susceptibility profile of the A-73.113 strain was performed by agar dilution or broth microdilution following the Brazilian Antimicrobial Susceptibility Testing Committee/European Committee on Antimicrobial Susceptibility Testing recommendations. WGS was performed using the Illumina® NextSeq platform and the generated reads were assembled using the SPAdes software. The sequences obtained were submitted to the bioinformatics pipelines to determine the sequence type, resistome, plasmidome, and virulome. RESULTS: The A-73.113 strain was identified as KQPN and was susceptible to polymyxins (MICs, ≤0.25 µg/mL), tigecycline (MIC, 0.5 µg/mL), ciprofloxacin (MIC, 0.5 µg/mL), and levofloxacin (MIC, 1 µg/mL). WGS analysis revealed the presence of genes conferring resistance to ß-lactams (blaNDM-1, blaCTX-M-15, blaOXA-9, blaOKP-A-5, blaTEM-1), aminoglycosides [aph(3')-VI, aadA1, aac(6')-Ib], and fluoroquinolones (oqxAB, qnrS1, aac(6')-Ib-cr]. Additionally, the presence of the plasmid replicons Col(pHAD28), IncFIA(HI1), IncFIB(K) (pCAV1099-114), IncFIB(pQil), and IncFII(K), as well as virulence-encoding genes fimABCDEFGHIK (type 1 fimbria), pilW (type IV pili), iutA (aerobactin), entABCDEFS/fepABCDG/fes (Ent siderophores), iroE (salmochelin), and allABCDRS (allantoin utilization) was verified. Furthermore, we found that the A-73.113 strain belongs to ST1040. CONCLUSIONS: Here we report the genomic characteristics of an NDM-1-producing KQPN ST1040 strain isolated from blood cultures in Brazil. These data will enhance our comprehension of how this species contributes to the acquisition and dissemination of blaNDM-1 in Brazilian nosocomial settings.

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Hematopoietic stem cell transplant (HSCT) recipients are at -increased risk for severe COVID-19. The aim of this study was to evaluate the burden of COVID-19 in a cohort of HSCT recipients. This retrospective study evaluated a cohort of adult hospitalized HSCT recipients diagnosed with COVID-19 in two large hospitals in São Paulo, Brazil post-HSCT, from January 2020 to June 2022. The primary outcome was all-cause mortality. Of 49 cases, 63.2% were male with a median age of 47 years. Allogeneic-HSCT (51.2%) and autologous-HSCT (48.9%) patients were included. The median time from HSCT to COVID-19 diagnosis was 398 days (IQR: 1211-134), with 22 (44.8%) cases occurring within 12 months of transplantation. Most cases occurred during the first year of the pandemic, in non-vaccinated patients (n=35; 71.4%). Most patients developed severe (24.4%) or critical (40.8%) disease; 67.3% received some medication for COVID-19, primarily corticosteroids (53.0%). The probable invasive aspergillosis prevalence was 10.2%. All-cause mortality was 40.8%, 51.4% in non-vaccinated patients and 14.2% in patients who received at least one dose of the vaccine. In the multiple regression analyses, the variables mechanical ventilation (OR: 101.01; 95% CI: 8.205 - 1,242.93; p = 0.003) and chest CT involvement at diagnosis ≥50% (OR: 26.61; 95% CI: 1.06 - 664.26; p = 0.04) remained associated with all-cause mortality. Thus, HSCT recipients with COVID-19 experienced high mortality, highlighting the need for full vaccination and infection prevention measures.

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Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

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Bacterial bloodstream infections (BSI) are a common threat among patients with haematological malignancies (HM) and hematopoietic stem cell transplant recipients (HSCT). The purpose of this research was to describe clinical and microbiological aspects of BSI caused by carbapenem-resistant Klebsiella pneumoniae (CRKp) and assess risk factors associated with 30-day mortality in a 10-year cohort of haematological patients. A total of 65 CRKp-BSI episodes occurring in HM patients and HSCT recipients and CRKp-BSI between January 2010 and December 2019 were retrospectively studied. Acute leukemias were the most frequently observed underlying disease (87.7%) and 18 patients (27.7%) received HSCT. Mucosal barrier injury in the gastrointestinal tract was the primary cause of bacteremia (86.1%). Also, 14 individuals (21.6%) had an Invasive Fungal Disease (IFD) throughout the episode. Regarding treatment, in 31 patients (47.7%) empirical therapy was deemed appropriate, whereas 33 (50.8%) patients received a combination therapy. Microbiological data revealed that the majority of isolates (53-58%) had the Polymyxin B co-resistance phenotype, while amikacin resistance was less common (16 samples, or 24.7%). The mortality rates at 14 and 30 days were 32.3% and 36.9%, respectively. In a multivariate Cox regression analysis, prompt appropriate antibiotic administration within three days was associated with a better outcome (Adjusted Hazard Ratio [aHR]: 0.33; 95% Confidence Interval [CI]: 0.14-0.76; p = 0.01), whereas hypotension at presentation (aHR: 3.88; 95% CI: 1.40-10.74; p = 0.01) and concurrent IFD (aHR: 2.97; 95% CI: 1.20-7.37; p = 0.02) were independently associated with death within 30 days. Additionally, a favorable correlation between combination therapy and overall survival was found (aHR: 0.18; 95%CI: 0.06-0.56; p = 0.002). In conclusion, 30-day mortality CRKp-BSI was elevated and most of the isolates were polymyxin B resistant. Early appropriate antimicrobial treatment and the use of combination therapy were linked to a better outcome.

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Although it has been hypothesized that the acquisition of plasmids—especially those bearing virulence factors and antimicrobial resistance genes—increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from − 4.7 to 5.2% per generation. This suggests that there are few biological barriers—or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model—against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.

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ABSTRACT Hematopoietic stem cell transplant (HSCT) recipients are at -increased risk for severe COVID-19. The aim of this study was to evaluate the burden of COVID-19 in a cohort of HSCT recipients. This retrospective study evaluated a cohort of adult hospitalized HSCT recipients diagnosed with COVID-19 in two large hospitals in São Paulo, Brazil post-HSCT, from January 2020 to June 2022. The primary outcome was all-cause mortality. Of 49 cases, 63.2% were male with a median age of 47 years. Allogeneic-HSCT (51.2%) and autologous-HSCT (48.9%) patients were included. The median time from HSCT to COVID-19 diagnosis was 398 days (IQR: 1211-134), with 22 (44.8%) cases occurring within 12 months of transplantation. Most cases occurred during the first year of the pandemic, in non-vaccinated patients (n=35; 71.4%). Most patients developed severe (24.4%) or critical (40.8%) disease; 67.3% received some medication for COVID-19, primarily corticosteroids (53.0%). The probable invasive aspergillosis prevalence was 10.2%. All-cause mortality was 40.8%, 51.4% in non-vaccinated patients and 14.2% in patients who received at least one dose of the vaccine. In the multiple regression analyses, the variables mechanical ventilation (OR: 101.01; 95% CI: 8.205 - 1,242.93; p = 0.003) and chest CT involvement at diagnosis ≥50% (OR: 26.61; 95% CI: 1.06 - 664.26; p = 0.04) remained associated with all-cause mortality. Thus, HSCT recipients with COVID-19 experienced high mortality, highlighting the need for full vaccination and infection prevention measures.

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Specific virulence factors that likely influence C. acnes invasion into deep tissues remain to be elucidated. Herein, we describe the frequency of C. acnes identification in deep tissue specimens of patients undergoing clean shoulder surgery and assess its phenotypic and genetic traits associated with virulence and antibiotic resistance patterns, compared with isolates from the skin of healthy volunteers. Multiple deep tissue specimens from the bone fragments, tendons, and bursa of 84 otherwise healthy patients undergoing primary clean-open and arthroscopic shoulder surgeries were aseptically collected. The overall yield of tissue sample cultures was 21.5% (55/255), with 11.8% (30/255) identified as C. acnes in 27.3% (23/84) of patients. Antibiotic resistance rates were low, with most strains expressing susceptibility to first-line antibiotics, while a few were resistant to penicillin and rifampicin. Phylotypes IB (73.3%) and II (23.3%) were predominant in deep tissue samples. Genomic analysis demonstrated differences in the pangenome of the isolates from the same clade. Even though strains displayed a range of pathogenic markers, such as biofilm formation, patients did not evolve to infection during the 1-year follow-up. This suggests that the presence of polyclonal C. acnes in multiple deep tissue samples does not necessarily indicate infection.

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Serratia marcescens is a Gram-negative bacterium presenting intrinsic resistance to polymyxins that has emerged as an important human pathogen. Although previous studies reported the occurrence of multidrug-resistance (MDR) S. marcescens isolates in the nosocomial settings, herein, we described isolates of this extensively drug-resistant (XDR) species recovered from stool samples of food-producing animals in the Brazilian Amazon region. Three carbapenem-resistant S. marcescens strains were recovered from stool samples of poultry and cattle. Genetic similarity analysis showed that these strains belonged to the same clone. Whole-genome sequencing of a representative strain (SMA412) revealed a resistome composed of genes encoding resistance to ß-lactams [blaKPC-2, blaSRT-2], aminoglycosides [aac(6')-Ib3, aac(6')-Ic, aph(3')-VIa], quinolones [aac(6')-Ib-cr], sulfonamides [sul2], and tetracyclines [tet(41)]. In addition, the analysis of the virulome demonstrated the presence of important genes involved in the pathogenicity of this species (lipBCD, pigP, flhC, flhD, phlA, shlA, and shlB). Our data demonstrate that food-animal production can act as reservoirs for MDR and virulent strains of S. marcescens.

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This study aimed to characterize a Klebsiella pneumoniae strain (KP411) recovered from the stool samples of poultry (Gallus gallus) in the Brazilian Amazon Region. The whole-genome sequencing of KP411 revealed the presence of an important arsenal of antimicrobial resistance genes to ß-lactams (blaCTX-M-14, blaTEM-1B, blaKPC-2, blaSVH-11), aminoglycosides [aph(3″)- Ib, aph(6)-Id, aph(3')-Ia], sulfonamides (sul1, sul2), quinolones (oqxAB), fosfomycin (fosAKP), and macrolides [mph(A)]. Furthermore, our analyses revealed that the KP411 strain belongs to the ST258 clonal lineage, which is one of the main epidemic clones responsible for the dissemination of KPC-2 worldwide. Our data suggest that food-producing animals may act as reservoirs of multidrug-resistant K. pneumoniae belonging to the ST258 clone, and, consequently, contribute to their dissemination to humans and the environment.

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The epidemiology of antimicrobial resistance (AMR) is complex, with multiple interfaces (human-animal-environment). In this context, One Health surveillance is essential for understanding the distribution of microorganisms and antimicrobial resistance genes (ARGs). This report describes a multicentric study undertaken to evaluate the bacterial communities and resistomes of food-producing animals (cattle, poultry, and swine) and healthy humans sampled simultaneously from five Brazilian regions. Metagenomic analysis showed that a total of 21,029 unique species were identified in 107 rectal swabs collected from distinct hosts, the highest numbers of which belonged to the domain Bacteria, mainly Ruminiclostridium spp. and Bacteroides spp., and the order Enterobacterales. We detected 405 ARGs for 12 distinct antimicrobial classes. Genes encoding antibiotic-modifying enzymes were the most frequent, followed by genes related to target alteration and efflux systems. Interestingly, carbapenemase-encoding genes such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1 were identified in distinct hosts. Our results revealed that, in general, the bacterial communities from humans were present in isolated clusters, except for the Northeastern region, where an overlap of the bacterial species from humans and food-producing animals was observed. Additionally, a large resistome was observed among all analyzed hosts, with emphasis on the presence of carbapenemase-encoding genes not previously reported in Latin America. IMPORTANCE Humans and food production animals have been reported to be important reservoirs of antimicrobial resistance (AMR) genes (ARGs). The frequency of these multidrug-resistant (MDR) bacteria tends to be higher in low- and middle-income countries (LMICs), due mainly to a lack of public health policies. Although studies on AMR in humans or animals have been carried out in Brazil, this is the first multicenter study that simultaneously collected rectal swabs from humans and food-producing animals for metagenomics. Our results indicate high microbial diversity among all analyzed hosts, and several ARGs for different antimicrobial classes were also found. As far as we know, we have detected for the first time ARGs encoding carbapenemases, such as blaAIM-1, blaCAM-1, blaGIM-2, and blaHMB-1, in Latin America. Thus, our results support the importance of metagenomics as a tool to track the colonization of food-producing animals and humans by antimicrobial-resistant bacteria. In addition, a network surveillance system called GUARANI, created for this study, is ready to be expanded and to collect additional data.

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