Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log 10 SARS-CoV-2 genome copies (GC) L −1 ), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log 10 SARS-CoV-2 GC L −1 , ranging from 5.49 ± 0.02 log 10 GC L −1 (27th November 2019) to 6.68 ± 0.02 log 10 GC L −1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

We analysed human sewage located in Florianopolis (Santa Catalina, Brazil) from late October until the Brazil lockdown on early March. We detected SARS-CoV-2 in two samples collected independently on 27th November 2019 (5.49 ± 0.02 log genome copies/L). Subsequent samplings were positive until 4th March 2020 (coinciding with the first COVID-19 case reported in Santa Catalina), with a SARS-CoV-2 RNA increase of one log (6.68 ± 0.02 log genome copies/L). Our results show that SARS-CoV-2 has been circulating in Brazil since late November 2019, much earlier than the first reported case in the Americas (21st January 2020, USA).

Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and β-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35 , ompk36 , and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. β-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35 . The presence of bla KPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A bla KPC–2 -bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

Objectives Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is imperative internationally, but only eight (22.9%) countries in the WHO Region of the Americas reported complete AMR data to the WHO Global Gonococcal Antimicrobial Surveillance Program (WHO GASP) in 2016. Genomic studies are ideal for enhanced understanding of gonococcal populations, including the spread of AMR strains. To elucidate the circulating gonococcal lineages/sublineages, including their AMR determinants, and the baseline genomic diversity among gonococcal strains in Brazil, we conducted WGS on 548 isolates obtained in 2015–16 across all five macroregions in Brazil. Methods A total of 548 gonococcal isolates cultured across Brazil in 2015–16 were genome sequenced. AMR was determined using agar dilution and/or Etest. Genome sequences of isolates from Argentina (n = 158) and the 2016 WHO reference strains (n = 14) were included in the analysis. Results We found 302, 68 and 214 different NG-MAST, MLST and NG-STAR STs, respectively. The phylogenomic analysis identified one main antimicrobial-susceptible lineage and one AMR lineage, which was divided into two sublineages with different AMR profiles. Determination of NG-STAR networks of clonal complexes was shown as a new and valuable molecular epidemiological analysis. Several novel mosaic mtrD (and mtrR and mtrE) variants associated with azithromycin resistance were identified. Conclusions We describe the first genomic baseline data to support the Brazilian GASP. The high prevalence of resistance to ciprofloxacin, tetracycline and benzylpenicillin, and the high number of isolates with mosaic penA and azithromycin resistance mutations, should prompt continued and strengthened AMR surveillance, including WGS, of N. gonorrhoeae in Brazil.

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoB in RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Plasmid-mediated polymyxin resistance has become a global health concern, not only because its dissemination has occurred drastically but also because it has begun to be reported in multidrug-resistant (MDR) pathogens. We hereby report microbiological and genomic characteristics of two mcr-1.1-positive polymyxin-resistant Escherichia coli isolates identified for the first time in community patients, in Santa Catarina, Southern Brazil. E. coli strains belonging to ST206 and ST354 and the resistome analysis revealed the presence of clinically important genes responsible for MDR profile. Interestingly, in both polymyxin-resistant E. coli strains, mcr-1.1 genes were carried by IncX4 plasmids, responsible for the worldwide dissemination of mcr-type genes. In this regard, plasmid backbones were almost identical to the first IncX4 plasmid reported in Brazil and sharing more than 99.9% identity to IncX4 plasmids from China, also lacking the ISApl1 insertion sequence upstream of mcr-1. In conclusion, these data confirm the presence of international ST206 and ST354 carrying mcr-1.1 genes and that the IncX4 plasmids have been key vectors contributing to the endemic status of mcr-1.1-positive polymyxin-resistant E. coli in Brazil. Also, we described the first known clinical isolate with the mrc1.1 gene in Santa Catarina state, Brazil, showing that plasmid-mediated polymyxin resistance has been affecting humans earlier than has been known so far.

Mycobacterium tuberculosis (M.tb), the pathogen responsible for tuberculosis (TB) poses as the major cause of death among infectious diseases. The knowledge about the molecular diversity of M.tb enables the implementation of more effective surveillance and control measures and, nowadays, Whole Genome Sequencing (WGS) holds the potential to produce high-resolution epidemiological data in a high-throughput manner. Florianópolis, the state capital of Santa Catarina (SC) in south Brazil, shows a high TB incidence (46.0/100,000). Here we carried out a WGS-based evaluation of the M.tb strain diversity, drug-resistance and ongoing transmission in the capital metropolitan region. Resistance to isoniazid, rifampicin, streptomycin was identified respectively in 4.0% (n = 6), 2.0% (n = 3) and 1.3% (n = 2) of the 151 studied strains by WGS. Besides, resistance to pyrazinamide and ethambutol was detected in 0.7% (n = 1) and reistance to ethionamide and fluoroquinolone (FQ) in 1.3% (n = 2), while a single (0.7%) multidrug-resistant (MDR) strain was identified. SNP-based typing classified all isolates into M.tb Lineage 4, with high proportion of sublineages LAM (60.3%), T (16.4%) and Haarlem (7.9%). The average core-genome distance between isolates was 420.3 SNPs, with 43.7% of all isolates grouped across 22 genomic clusters thereby showing the presence of important ongoing TB transmission events. Most clusters were geographically distributed across the study setting which highlights the need for an urgent interruption of these large transmission chains. The data conveyed by this study shows the presence of important and uncontrolled TB transmission in the metropolitan area and provides precise data to support TB control measures in this region.

Objectives To (i) describe the nationwide antimicrobial susceptibility of Neisseria gonorrhoeae (NG) isolates cultured across Brazil in 2018–20 and compare it with NG antimicrobial resistance data from 2015–16, and (ii) present epidemiological data of the corresponding gonorrhoea patients in 2018–20. Methods Twelve representative sentinel sites cultured NG isolates from men with urethral discharge. Susceptibility to eight antimicrobials was examined using agar dilution method, according to WHO standards. The consenting participants were invited to provide epidemiological data. Results In total, 633 NG isolates (one isolate per participant) were analysed, and 449 (70.9%) questionnaires were answered. Heterosexual (68.2%) and homosexual (23.1%) sexual orientations were common, and most prevalent types of unprotected sexual intercourse were vaginal insertive (69.9%), oral giving (56.6%) and anal insertive (47.4%). The levels of in vitro NG resistance to ciprofloxacin, tetracycline, benzylpenicillin, azithromycin, cefixime, gentamicin, spectinomycin and ceftriaxone were 67.3%, 40.0%, 25.7%, 10.6%, 0.3%, 0%, 0% and 0%, respectively. Compliance with the recommended first-line ceftriaxone 500 mg plus azithromycin 1 g therapy was high (90.9%). Conclusions Compared with 2015–16, ciprofloxacin resistance has remained high and azithromycin and cefixime resistance rates have increased in Brazil. Resistance remained lacking to ceftriaxone, gentamicin and spectinomycin, which all are gonorrhoea treatment options. The increasing azithromycin resistance in Brazil and internationally may threaten the future use of azithromycin in dual regimens for treatment of gonorrhoea. Consequently, continued and enhanced quality-assured surveillance of gonococcal AMR, and ideally also treatment failures and including WGS, is imperative in Brazil and worldwide.