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.

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.

Two Novel Mutations Associated with Polymyxin-B Resistance in a Pandemic Lineage of Uropathogenic Escherichia coli of the Sequence Type 69.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are frequent pathogens worldwide, impacting on the morbidity and economic costs associated with antimicrobial treatment. OBJECTIVES: We report two novel mutations associated with polymyxin-B resistance in an UPEC isolate collected in 2019. METHODS: Isolate was submitted to antimicrobial susceptibility testing including broth microdilution for polymyxin B. Whole genome was sequenced and analyzed. RESULTS: Polymyxin-B total inhibition occurred at 16 mg/L (resistant). UPEC isolate was assigned to the phylogroup D, serotype O117:H4, and Sequence Type 69. mcr genes were not detected, but two novel mutations in the pmrA/basS (A80S) and pmrB/basR (D149N) genes were identified. CONCLUSIONS: The occurrence of non-mcr polymyxin resistance in E. coli from extraintestinal infections underscores the need of a continuous surveillance of this evolving pathogen.

High rates of Salmonella contamination in raw kibbe from commercial establishments: predominance of Salmonella Give.

Salmonella is one of the major causes of gastroenteritis worldwide in both humans and animals and one of the main agents involved in foodborne disease outbreaks. In this study, 70 raw kibbe samples from different commercial establishments were analyzed for Salmonella spp. The isolates were seroyped and tested for susceptibility to antimicrobial agents. Pulsed-field Gel Electrophoresis was carried out following the standard protocol of the PulseNet network. Fifteen (21.4%) samples were contaminated with Salmonella and S. Give was the prevalent serotype (46.7%). Similarity of 96.3% was observed among the S. Give isolates (n = 7), which indicates the possible spread of the same clone in the analyzed commercial establishments. S. Rissen and S. Typhimurium showed antimicrobial resistance. The detection of a significant percentage of contamination in raw kibbe and of the resistant strains indicates the risk that the consumption of this dish may represent.

Hypervirulent Klebsiella pneumoniae as Unexpected Cause of Fatal Outbreak in Captive Marmosets, Brazil.

After the sudden death of captive marmosets in São Paulo, Brazil, we conducted a histologic and microbiologic study. We found hyperacute septicemia caused by hypermucoviscous sequence type 86 K2 Klebsiella pneumoniae. We implemented prophylactic antimicrobial therapy, selected dedicated staff for marmoset interactions, and sanitized the animals' fruit to successfully control this outbreak.

Genomic Characterization of a Clinical NDM-1-Producing Klebsiella michiganensis from Brazil.

Public health faces daily challenges due to increasing reports of pathogenic microorganisms with new antimicrobial resistance. Klebsiella michiganensis, an emerging pathogen, poses difficulty in its identification using conventional techniques. This study presents the first documented case of NDM-1-producing K. michiganensis in Brazil, identified as the new ST418. Initially, the isolate from a tracheal secretion was misidentified as K. oxytoca. However, accurate identification was achieved through ANI analyses. Whole-genome sequencing was conducted to characterize the genetic context of the resistance genes, to identify virulence factors, and to construct a phylogenetic tree. The blaNDM-1 gene was found to be harbored on an IncFIB plasmid approximately 112 kb in length, which was transferable in conjugation assays. The detection of carbapenem resistance genes in this species highlights the importance of public health vigilance, as it may serve as a reservoir and disseminator of significant resistance genes.

High levels of multidrug-resistant isolates of genetically similar Salmonella 1,4, [5],12:I:- from Brazil between 1983 and 2020.

Introduction. Salmonella 1,4, [5],12:i:- strains with different antimicrobial resistance profiles have been associated with foodborne disease outbreaks in several countries. In Brazil, S. 1,4, [5],12:i:- was identified as one of the most prevalent serovars in São Paulo State during 2004-2020.Gap Statement. However, few studies have characterized this serovar in Brazil.Aim. This study aimed to determine the antimicrobial resistance profiles of S. 1,4, [5],12:i:- strains isolated from different sources in Southeast Brazil and compare their genetic diversity.Methodology. We analysed 113 S. 1,4, [5],12:i:- strains isolated from humans (n=99), animals (n=7), food (n=5) and the environment (n=2) between 1983 and 2020. Susceptibility testing against 13 antimicrobials was performed using the disc diffusion method for all the strains. Plasmid resistance genes and mutations in the quinolone resistance-determining regions were identified in phenotypically fluoroquinolone-resistant strains. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for all strains and multilocus sequence typing (MLST) for 40 selected strains.Results. Of the 113 strains, 54.87 % were resistant to at least one antimicrobial. The highest resistance rates were observed against ampicillin (51.33 %), nalidixic acid (39.82 %) and tetracycline (38.05 %). Additionally, 39 (34.51 %) strains were classified as multidrug-resistant (MDR). Nine fluoroquinolone-resistant strains exhibited the gyrA mutation (Ser96→Tyr96) and contained the qnrB gene. The 113 strains were grouped into two clusters using ERIC-PCR, and most of strains were present in one cluster, with a genetic similarity of ≥80 %. Finally, 40 strains were typed as ST19 using MLST.Conclusion. The prevalence of MDR strains is alarming because antimicrobial treatment against these strains may lead to therapeutic failure. Furthermore, the ERIC-PCR and MLST results suggested that most strains belonged to one main cluster. Thus, a prevalent subtype of Salmonella 1,4, [5],12:i:- strains has probably been circulating among different sources in São Paulo, Brazil, over decades.

Polymyxin Resistance in Salmonella: Exploring Mutations and Genetic Determinants of Non-Human Isolates.

Until 2015, polymyxin resistance was primarily attributed to chromosomal mutations. However, with the first report of mobile colistin resistance (mcr-1) in commensal Escherichia coli from food animals in China, the landscape has changed. To evaluate the presence of polymyxin resistance in Salmonella spp., a drop screening test for colistin and polymyxin B was carried out on 1156 isolates of non-human origin (animals, food, and the environment), received in Brazil, between 2016 and 2021. Subsequently, 210 isolates with resistant results in the drop test were subjected to the gold-standard test (broth microdilution) for both colistin and polymyxin B. Whole-genome sequencing (WGS) of 102 resistant isolates was performed for a comprehensive analysis of associated genes. Surprisingly, none of the isolates resistant to colistin in the drop test harbored any of the mcr variants (mcr-1 to mcr-10). WGS identified that the most common mutations were found in pmrA (n= 22; T89S) and pmrB (n = 24; M15T, G73S, V74I, I83A, A111V). Other resistance determinants were also detected, such as the aac(6')-Iaa gene in 72 isolates, while others carried beta-lactamase genes (blaTEM-1blaCTX-M-2, blaCMY-2). Additionally, genes associated with fluoroquinolone resistance (qnrB19, qnrS1, oqxA/B) were detected in 11 isolates. Colistin and polymyxin B resistance were identified among Salmonella from non-human sources, but not associated with the mcr genes. Furthermore, the already-described mutations associated with polymyxin resistance were detected in only a small number of isolates, underscoring the need to explore and characterize unknown genes that contribute to resistance.

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.

Virulence potential of Salmonella 1,4, [5],12:i:- strains isolated during decades from different sources in the Southeast region of Brazil.

Salmonella 1,4, [5],12:i:- is one of the most prevalent serovars associated with gastroenteritis in several countries, including Brazil. However, few studies have analyzed the virulence potential of this variant in this country. Therefore, this study aimed to characterize S. 1,4, [5],12:i:- strains isolated in Southeast Brazil. To this end, 113 S. 1,4, [5],12:i:- strains isolated from different sources between 1983 and 2020 were analyzed. For all strains, the frequencies of 11 virulence genes were investigated using PCR and the molecular typing was performed using pulsed-field gel electrophoresis (PFGE). Furthermore, 40 strains isolated from human and non-human sources were characterized by survival under acid and oxidative stress, and virulence analysis in Galleria mellonella was performed for 20 selected strains. All virulence genes were detected in more than 91% of the strains. The studied strains were grouped into four clusters using PFGE. Most strains were present in one cluster, named PFGE-A, with a genetic similarity of ≥ 79.5%. All 40 strains survived acid stress after 10 min and 1 h of exposure. Under oxidative stress, all 40 strains survived after 10 min, and 36 survived after 1 h of exposure. In the G. mellonella assay, nine isolates from non-human sources and six isolates from human showed high-to-intermediate virulence profiles. In conclusion, the pathogenic potential of the strains studied was corroborated by the high frequency of all the virulence genes identified. The PFGE results suggested that most strains belonged to one main cluster that has been prevailing in the São Paulo State, Brazil. The S. 1,4, [5],12:i:- strains isolated from human and non-human sources successfully survived the unfavorable conditions in the human gastrointestinal tract. Finally, strains isolated from non-human sources showed a higher proportion of isolates with high to intermediate virulence profiles in G. mellonella than in human isolates, suggesting a possible difference between isolates from different origins.

Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal.

OBJECTIVES: This study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. METHODS: Bacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. RESULTS: Isolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. CONCLUSIONS: Not surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

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.

Whole genome sequencing analyses revealed that Salmonella enterica serovar Dublin strains from Brazil belonged to two predominant clades.

Salmonella Dublin is a cattle-associated serovar sporadically causing disease in humans. S. Dublin strains isolated in Brazil and in other countries were analyzed to determine their phylogenetic relationships, the presence of genes, plasmids, genomic regions related to virulence and antimicrobial resistance genes repertoire, using WGS analyses. Illumina was used to sequence the genome of 112 S. Dublin strains isolated in Brazil from humans (n = 82) and animals (n = 30) between 1983 and 2016. Furthermore, 87 strains from other countries were analyzed. WGSNP analysis revealed three different clades, in which the strains from Brazil belonged to two clades, A and C. Most of the genes and genomic regions searched varied among the strains studied. The siderophore genes iroB and iroC were exclusively found in strains from Brazil and pegD gene, related to fimbrial adherence determinants, were positive in 124 strains from clades A and B but absent in all the strains from clade C (n = 71). Eleven plasmid replicons were found in the strains from Brazil, and nine were exclusively found in strains from other countries. The antimicrobial resistance genes mdsA and mdsB, that encode an efflux pump, were found in all the strains studied. The strains from Brazil carried other resistance genes, such as tet(A) (n = 11), tet(B) (n = 4) and tet(C) (n = 4), blaTEM-1 (n = 4), catA1 (n = 1), aadA1 (n = 1), and sul1 (n = 1). In conclusion, S. Dublin strains isolated in Brazil presented some few unique genes not found in strains from other countries and were allocated into two distinct clades with strains of human and animal origin epidemiologically related. This fact stresses the zoonotic potential of S. Dublin circulating in Brazil for more than 30 years.

Salmonella enterica serotypes from human and nonhuman sources in Sao Paulo State, Brazil, 2004-2020.

Salmonellosis ranks among the most frequently reported zoonosis worldwide and is often associated with foodborne outbreaks. Since the 1950s, the distribution of Salmonella serotypes in Sao Paulo State, Brazil, has been documented and periodically reported. In this study, we updated the data on the distribution of Salmonella serotypes received in our reference laboratory, isolated from human infections and nonhuman sources, from 2004 to 2020. In that period, a total of 9,014 Salmonella isolates were analyzed, of which 3,553 (39.4%) were recovered from human samples, mainly of stool (65%) and blood (25.6%), and 5,461 (60.6%) were isolated from nonhuman origins, such as animals (47.2%), food (27.7%) and animal environments (18.6%). In human isolates, a total of 104 serotypes were identified and the most frequent ones were Enteritidis, Typhimurium, S . I. 4,[5],12:i:-, Dublin and Typhi. A consistent reduction of the Enteritidis proportion was observed over the years. Among the 156 serotypes identified in isolates with nonhuman origins, Enteritidis, Mbandaka, Typhimurium, Agona and Anatum were ranked as the top five Salmonella serotypes; in more recent years, S . Heidelberg has increased in frequency. Although with different proportions, the top 10 prevalent serotypes were identified in both human and nonhuman origins, underscoring the role of animals, food products and environment as reservoirs of Salmonella with potential to cause human salmonellosis.

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.

Genomic characterization of a multi-drug resistant, CTX-M-65-producing clinical isolate of Salmonella Infantis isolated in Brazil.

A multi-drug resistant, CTX-M-65 producing Salmonella Infantis was identified from a patient in Brazil. Whole genome sequencing followed by hybrid assembly (short and long reads) indicated the presence of blaCTX-M-65 in a pESI-like megaplasmid in this ST32 isolate and phylogenetic analysis showed high similarity with IncFIB S. Infantis isolates from food and poultry in the USA.

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.