Vibrio mimicus Lineage Carrying Cholera Toxin and Vibrio Pathogenicity Island, United States and China.

Vibrio mimicus bacteria have caused sporadic cases and outbreaks of cholera-like diarrhea throughout the world, but the association of lineages with such events is unexplored. Genomic analyses revealed V. mimicus lineages carrying the virulence factors cholera toxin and toxin coregulated pilus, one of which has persisted for decades in China and the United States.

Unveiling the genome of a high-risk pandrug-resistant Klebsiella pneumoniae emerging in the Brazilian Amazon Region, 2022.

BACKGROUND: Pandrug-resistant (PDR) Klebsiella pneumoniae has been reported sporadically in many countries and remains rare in Brazil. OBJECTIVES: This study unravelled the genetic determinants involved with the PDR background of a clinical ST11 K. pneumoniae recovered in the Brazilian Amazon Region, where K. pneumoniae genomic and epidemiological information is scarce. METHODS: Kp196 was submitted to the antimicrobial susceptibility test by the disk-diffusion method and minimum inhibitory concentration (MIC) determination. The whole genome sequencing was obtained and the sequence type was determined by core genome multilocus sequence typing (cgMLST). Its intrinsic and acquired resistome was assessed by Comprehensive Antibiotic Resistance Database (CARD) and comparison with wild-type genes. FINDINGS: The analyses revealed that Kp196 belonged to the pandemic ST11 and presented the PDR phenotype. Its acquired resistome was composed of a huge set of clinically relevant resistance determinants, including bla CTX-M-15 and bla NDM-1, all found in the vicinity of mobile platforms. Considering its intrinsic resistome, the multidrug resistance, especially to colistin, tigecycline and fluoroquinolones, was multifactorial and attributed to modifications (indels, missense mutations, and gene disruption) in several housekeeping genes (arnT/phoQ/mgrB/ramR/acrB/gyrA/parC/ompK35-36-37). The Kp196 intrinsic resistome was also observed in a ST11 environmental strain, although harbouring distinct acquired resistomes. CONCLUSIONS: An accumulation of different resistance mechanisms regarding the intrinsic resistome accounts for a more stable resistome, strongly contributing to the Kp196 PDR phenotype.

Mycolicibacterium fortuitum genomic epidemiology, resistome and virulome.

BACKGROUND: Mycolicibacterium fortuitum is an opportunistic pathogen associated with human and animal infection worldwide. Studies concerning this species are mainly represented by case reports, some of them addressing drug susceptibility with a focus on a specific geographic region, so there is a gap in relation to the global epidemiological scenario. OBJECTIVES: We aimed determine the global epidemiological scenario of M. fortuitum and analyse its traits associated with pathogenicity. METHODS: Based on publicly available genomes of M. fortuitum and a genome from Brazil (this study), we performed a genomic epidemiology analysis and in silico and in vitro characterisation of the resistome and virulome of this species. FINDINGS: Three main clusters were defined, one including isolates from the environment, human and animal infections recovered over nearly a century. An apparent intrinsic resistome comprises mechanisms associated with macrolides, beta-lactams, aminoglycosides and antitubercular drugs such as rifampin. Besides, the virulome presented Type VII secretion systems (T7SS), including ESX-1, ESX-3, ESX-4 and ESX-4-bis, some of which play a role on the virulence of Mycobacteriaceae species. MAIN CONCLUSIONS: Here, M. fortuitum was revealed as a reservoir of an expressive intrinsic resistome, as well as a virulome that may contribute to its success as a global opportunist pathogen.

Pyomelanin biosynthetic pathway in pigment-producer strains from the pandemic Acinetobacter baumannii IC-5.

BACKGROUND: Acinetobacter baumannii outbreaks have been associated with pandemic International Clones (ICs), but the virulence factors involved with their pathogenicity are sparsely understood. Pigment production has been linked with bacterial pathogenicity, however, this phenotype is rarely observed in A. baumannii. OBJECTIVES: This study aimed to characterise the reddish-brown pigment produced by A. baumannii strains, and to determine its biosynthetic pathway by genomic approaches. METHODS: Pigment characterisation and antimicrobial susceptibility were conducted by phenotypic tests. The clonal relationship was obtained by pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The genome of an A. baumannii was obtained for characterisation of genes involved with pigment production. FINDINGS: The pyomelanin was the pigment produced by A. baumannii. Strains were extensively drug resistant and belonged to the IC-5/ST79. The pyomelanin biosynthetic pathway was determined and presented a particular architecture concerning the peripheral (tyrB, phhB and hpd) and central (hmgB, hmgC and hmgR) metabolic pathway genes. The identification of a distant HmgA homologue, probably without dioxygenase activity, could explain pyomelanin production. Virulence determinants involved with adherence (csuA/BABCDE and a T5bSS-carrying genomic island), and iron uptake (basABCDEFGHIJ, bauABCDEF and barAB) were characterised. MAIN CONCLUSION: There is a biosynthetic pathway compatible with the pyomelanin production observed in persistent A. baumannii IC-5 strains.

Acinetobacter baumannii infections in Amazon Region driven by extensively drug resistant international clones, 2016-2018.

BACKGROUND: Acinetobacter baumannii is a leading cause of nosocomial infections. This species is characterised by the presence of pandemic lineages (International Clones) that present a broad antimicrobial resistance profile. OBJECTIVE: To perform the molecular epidemiology of carbapenem-resistant A. baumannii from a clinical setting in the Amazon Basin, and to characterise their antimicrobial resistance determinants. METHODS: The genetic relationship of carbapenem-resistant A. baumannii were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Class A, B and D ß-lactamase genes were screened by polymerase chain reaction (PCR) and sequencing. The antimicrobial susceptibility profile was obtained by Disc-diffusion method and minimum inhibitory concentration (MIC) determination. FINDINGS: All carbapenem-resistant A. baumannii strains belonged to three international clones, IC-1, IC-5 and IC-6, the latter recently reported by the first time in Brazil. The major determinant of carbapenem resistance in IC-1 and IC-5 strains was bla OXA-23, associated with ISAba1 and ISAba3, respectively, while IC-6 harboured the bla OXA-72. CONCLUSIONS: The A. baumannii epidemiology in Brazilian Amazon Region was unknown. It was demonstrated that A. baumannii XDR international clones were responsible for nosocomial infections in Boa Vista during 2016-2018, revealing that the epidemiological scenario of A. baumannii infections in Amazon Region resembles those from the cosmopolitan regions worldwide.

The invasive MenC cc103 lineage with penicillin reduced susceptibility persisting in Brazil.

Penicillin is the antibiotic of choice for the treatment of meningococcal infections, and mutations in penA gene are involved with reduced susceptibility (penI) emergence to this antibiotic. This study aimed to characterize the penA allelic diversity, their association with penI phenotype and distribution among prevalent meningococci serogroups in Brazil. The entire penA from 49 invasive strains of distinct serogroups circulating in Brazil for more than two decades were obtained by PCR and sequencing. Additionally, the penA from 22 publicly available complete Neisseria meningitidis genomes from Brazil were included in the study. The allelic diversity was determined and a genetic tree was built using the penA sequence alignment. The penicillin MIC was obtained by the E-Test method. In general, the identified penA alleles correlated with the observed penI phenotype. The canonical penA1 was the most prevalent allele, however, several altered penA were also identified in strains presenting increased penicillin MICs. It was identified a new penA amino acid position (residue 480) that possibly influence the penicillin MIC in some strains. Interestingly, the altered penA14 was found in penI invasive MenC cc103 strains spread in Brazil and persisting since 2011, indicating that the biological cost imposed by penI phenotype can be ameliorated by particular features present in this lineage, which represents an additional public health threat.

Complete plasmid sequence carrying type IV-like and type VII secretion systems from an atypical mycobacteria strain.

The genus Mycobacterium is highly diverse and ubiquitous in nature, comprehending fast- and slow-growing species with distinct impact in public health. The plasmid-mediated horizontal gene transfer represents one of the major events in bacteria evolution. Here, we report the complete sequence of a 160,489 bp circular plasmid (pCBMA213_2) from an atypical and fast-growing environmental mycobacteria. This is a unique plasmid, in comparison with the characterised mycobacteria plasmids, harboring a type IV-like and ESX-P2 type VII secretion systems. pCBMA213_2 can be further explored for evolutionary and conjugation studies as well as a tool to manipulate DNA within this bacteria genus.

Full characterization of the integrative and conjugative element carrying the metallo-ß-lactamase bla SPM-1 and bicyclomycin bcr1 resistance genes found in the pandemic Pseudomonas aeruginosa clone SP/ST277.

OBJECTIVES: This study aimed to characterize the genomic context of the bla SPM-1 gene in Brazilian strains belonging to the pandemic Pseudomonas aeruginosa clone SP/ST277. METHODS: WGS of clone SP/ST277 strains was performed using a Nextera paired-end library in an Illumina HiSeq 2500 sequencer. bla SPM-1 context was assessed by de novo assembly and gene prediction and annotation tools. bla SPM-1 was screened in P. aeruginosa genomes through BlastN, and comparative genomics were performed. RESULTS: The metallo-ß-lactamase bla SPM-1 has been disseminated by the pandemic Brazilian P. aeruginosa clone SP/ST277. In spite of its association with the CR4 element and with the Tn4371 element, the overall bla SPM-1 genomic context remains uncharacterized and its determination is valuable to understanding gene dispersion dynamics and the consequent emergence of carbapenem resistance. In this study, bla SPM-1 and its surrounding sequences (CR4-groEL-bla SPM-1-CR4-groEL) were found in the variable region of an ICE-like element resembling Tn4371 (where ICE stands for integrative and conjugative element). This element, named ICETn4371 6061, had 46 ORFs, including the bicyclomycin resistance bcr1 gene. An integrase gene and a set of conjugative transfer genes were identified. Gene content and order were shared with other Tn4371-ICEs, presenting remarkable amino acid identities. bla SPM-1 and surrounding sequences were missing in ICETn4371 6061 of PS600-MA, another isolate belonging to clone SP/ST277, indicating their mobilization. Eight/nine P. aeruginosa genomes assigned to clone SP/ST277, by in silico MLST, harboured bla SPM-1 inserted into ICETn4371 6061. CONCLUSIONS: The presence of bla SPM-1 in a Tn4371-ICE with intact integration/conjugation modules demonstrated that, besides gene dispersion by clonal expansion of the pandemic SP/ST277 lineage, bla SPM-1 may be spread through ICE conjugation.

Complete genome sequence of a clinical Bordetella pertussis isolate from Brazil.

There has been a resurgence in the number of pertussis cases in Brazil and around the world. Here, the genome of a clinical Bordetella pertussis strain (Bz181) that was recently isolated in Brazil is reported. Analysis of the virulence-associated genes defining the pre- and post-vaccination lineages revealed the presence of the prn2-ptxS1A-fim3B-ptxP3 allelic profile in Bz181, which is characteristic of the current pandemic lineage. A putative metallo-ß-lactamase gene presenting all of the conserved zinc-binding motifs that characterise the catalytic site was identified, in addition to a multidrug efflux pump of the RND family that could confer resistance to erythromycin, which is the antibiotic of choice for treating pertussis disease.

In-silico genomic characterization of Staphylococcus haemolyticus on a global scale: lineages, resistome, and virulome.

BACKGROUND: Staphylococcus haemolyticus belongs to the Coagulase-Negative Staphylococci (CoNS), exhibiting the highest levels of antibiotic resistance within this group of bacteria. This species has been increasingly implicated in nosocomial and animal infections worldwide, with a prevalence of methicillin-resistant Staphylococcus haemolyticus (MRSH). Most information about this organism comes from regional analyzes or with the absence of typing data, thus not revealing the real role of S. haemolyticus strains in world public health. METHODS: Here, we performed an enhanced global epidemiological analysis considering all available S. haemolyticus genomes from all continents, including genomes of nosocomial, environmental, and animal origin (n = 310). Furthermore, we added original genomic information from a clinical MRSH from the Brazilian Amazon region. The resistome and virulome of the genomes were associated with their mobilome, being inferred based on the presence of specific genes and databases such as CARD, VFDB, and PlasmidFinder, respectively. RESULTS: Phylogenetic analysis revealed three main groups, the main one covering most of the clinical clonal complex 3 (CC3) genomes in the world. The virulome of some genomes in this cluster showed the complete capsule operon (capA-capM). Importantly, this virulome trait could be associated with the mobilome, since the capsule operon, as well as a whole set of genes of the type VII secretion system, were observed in plasmids. In addition, the resistome of the main cluster (CC3) was larger, characterized mainly by the presence of the mecA gene, in addition to a set of other genes (aad, aac-aph, aph, erm), contrasting with the poor resistome of the other two clusters. Several insertion sequences were identified, some of them linked to specific clusters, and resistance genes, such as the rare cfrA (IS257). CONCLUSIONS: Therefore, successful lineages of CC3 S. haemolyticus causing human infections are widespread worldwide, raising concern about the impact of this scenario on public health.

Prevalence and characterization of an integrative and conjugative element carrying tet(X) gene in Elizabethkingia meningoseptica.

OBJECTIVES: To investigate the tet(X) gene, a determinant of tigecycline resistance, in the emerging pathogen Elizabethkingia meningoseptica and its association with an integrative and conjugative element (ICE). METHODS: All E. meningoseptica genomes from the National Center for Biotechnology Information (n = 87) were retrieved and annotated for resistome searches using the CARD database. A phylogenic analysis was performed based on the E. meningoseptica core genome. The ICE was identified through comparative genomics with other ICEs occurring in Elizabethkingia spp. RESULTS: Phylogenetic analysis revealed E. meningoseptica genomes from six countries distributed across different lineages, some of which persisted for years. The common resistome of these genomes included blaBlaB, blaCME, blaGOB, ranA/B, aadS, and catB (genes associated with resistance to ß-lactams, aminoglycosides, and chloramphenicol). Some genomes also presented additional resistance genes (dfrA, ereD, blaVEB, aadS, and tet(X)). Interestingly, tet(X) and aadS were located in an ICE of 49 769 bp (ICEEmSQ101), which was fully obtained from the E. meningoseptica SQ101 genome. We also showed evidence that the other 27 genomes harboured this ICE. The distribution of ICEEmSQ101, carrying tet(X), was restricted to a single Chinese lineage. CONCLUSIONS: The tet(X) gene is not prevalent in the species E. meningoseptica, as previously stated for the genus Elizabethkingia, since it is present only in a single Chinese lineage. We identified that several E. meningoseptica genomes harboured an ICE that mobilized the Elizabethkingia tet(X) gene and exhibited characteristics similar to the ICEs of other Flavobacteria, which would favour their transmission in this bacterial family.

In-depth analysis of Klebsiella aerogenes resistome, virulome and plasmidome worldwide.

Klebsiella aerogenes is an emergent pathogen associated with outbreaks of carbapenem-resistant strains. To date, studies focusing on K. aerogenes have been small-scale and/or geographically restricted. Here, we analyzed the epidemiology, resistome, virulome, and plasmidome of this species based on 561 genomes, spanning all continents. Furthermore, we sequenced four new strains from Brazil (mostly from the Amazon region). Dozens of STs occur worldwide, but the pandemic clones ST93 and ST4 have prevailed in several countries. Almost all genomes were clinical, however, most of them did not carry ESBL or carbapenemases, instead, they carried chromosomal alterations (omp36, ampD, ampG, ampR) associated with resistance to ß-lactams. Integrons were also identified, presenting gene cassettes not yet reported in this species (blaIMP, blaVIM, blaGES). Considering the virulence loci, the yersiniabactin and colibactin operons were found in the ICEKp10 element, which is disseminated in genomes of several STs, as well as an incomplete salmochelin cluster. In contrast, the aerobactin hypervirulence trait was observed only in one ST432 genome. Plasmids were common, mainly from the ColRNAI replicon, with some carrying resistance genes (mcr, blaTEM, blaNDM, blaIMP, blaKPC, blaVIM) and virulence genes (EAST1, senB). Interestingly, 172 genomes of different STs presented putative plasmids containing the colicin gene.

Three outbreak-causing Neisseria meningitidis serogroup C clones, Brazil(1.).

During 2003-2012, 8 clusters of meningococcal disease were identified in Rio de Janeiro State, Brazil, all caused by serogroup C Neisseria meningitidis. The isolates were assigned to 3 clonal complexes (cc): cc11, cc32, and cc103. These hyperinvasive disease lineages were associated with endemic disease, outbreaks, and high case-fatality rates.

Carbapenem-resistant Acinetobacter baumannii from Brazil: role of carO alleles expression and blaOXA-23 gene.

BACKGROUND: Carbapenems are the antibiotics of choice to treat infections caused by Acinetobacter baumannii, and resistance to this class can be determined by loss of membrane permeability and enzymatic mechanisms. Here, we analyzed the basis of carbapenem resistance in clinical A. baumannii isolates from different Brazilian regions. RESULTS: The analyses addressed the carbapenemase activity of OXA-23, CarO expression and alterations in its primary structure. Susceptibility test revealed that the strains presented the COS (Colistin-Only-Sensitive) profile. PCR and sequencing showed the presence of the chromosomally-encoded blaOXA-51 in all isolates. The majority of strains (53%) carried the carbapenemase blaOXA-23 gene associated with ISAba1. The Hodge test indicated that these strains are carbapenemase producers. PFGE revealed 14 genotypes among strains from Rio de Janeiro and Maranhão. The influence of carO on imipenem resistance was evaluated considering two aspects: the composition of the primary amino acid sequence; and the expression level of this porin. Sequencing and in silico analyses showed the occurrence of CarOa, CarOb and undefined CarO types, and Real Time RT-PCR revealed basal and reduced carO transcription levels among isolates. CONCLUSIONS: We concluded that, in general, for these Brazilian isolates, the major carbapenem resistance mechanism was due to OXA-23 carbapenemase activity and that loss of CarO porin plays a minor role in this phenotype. However, it was possible to associate the carO alleles and their expression with imipenem resistance. Therefore, these findings underline the complexity in addressing the role of different mechanisms in carbapenem resistance and highlight the possible influence of CarO type in this phenotype.

Persistence of a carbapenem-resistant Acinetobacter baumannii (CRAB) International Clone II (ST2/IC2) sub-lineage involved with outbreaks in two Brazilian clinical settings.

BACKGROUND: Acinetobacter baumannii international clone II (IC2) is a widespread pandemic clone, however, it is rarely described in South America. The present study reported an outbreak caused by XDR IC2 strains in a clinical setting in Rio de Janeiro in 2022. METHODS: Molecular epidemiology analysis was conducted with MLST to determine the clonal relationship and to assign a sequence type. The antimicrobial resistance profile of A. baumannii strains was assessed by the disk-diffusion method and MIC determination, and the presence of antibiotic resistance genes was determined by PCR and Sanger sequencing. The whole genome of one representative strain (AB91) was sequenced to prospect its resistome and virulome. RESULTS: The MLST revealed that all strains belonged to the ST2 (Pasteur scheme) that corresponded to the pandemic IC2 lineage. They presented the XDR phenotype, which was compatible with their resistome composed of several acquired resistance genes and altered housekeeping genes. Additionally, an expressive virulome was revealed in AB91 genome. Genomic comparison with the unique other available IC2 genome from Brazil revealed that outbreaks occurring during (São Paulo - 2020/2021) and after (Rio de Janeiro - 2022) COVID-19 pandemics were caused by the same IC2 lineage. CONCLUSIONS: This study suggests that the presence of a huge arsenal of resistance and virulence genes may have contributed to the persistence and the successful establishment of IC2 in Brazilian clinical settings during and after the COVID-19 pandemics in response to a series of events, such as the antibiotic overused during that period.

Outbreak of high-risk XDR CRAB of international clone 2 (IC2) in Rio Janeiro, Brazil.

OBJECTIVES: Among the high-risk clones of Acinetobacter baumannii, called international clones (ICs), IC2 represents the main lineage causing outbreaks worldwide. Despite the successful global spread of IC2, the occurrence of IC2 is rarely reported in Latin America. Here, we aimed to evaluate the susceptibility and genetic relatedness of isolates from a nosocomial outbreak in Rio de Janeiro/Brazil (2022) and perform genomic epidemiology analyses of the available genomes of A. baumannii. METHODS: Sixteen strains of A. baumannii were subjected to antimicrobial susceptibility tests and genome sequencing. These genomes were compared phylogenetically with other IC2 genomes from the NCBI database, and virulence and antibiotic resistance genes were searched. RESULTS: The 16 strains represented carbapenem-resistant A. baumannii (CRAB) with an extensively drug-resistant profile. In silico analysis established the relationship between the Brazilian CRAB genomes and IC2/ST2 genomes in the world. The Brazilian strains belonged to three sub-lineages, associated with genomes from countries in Europe, North America, and Asia. These sub-lineages presented three distinct capsules, KL7, KL9, and KL56. The Brazilian strains were characterised by the co-presence of blaOXA-23 and blaOXA-66, in addition to the genes APH(6), APH(3"), ANT(3"), AAC(6'), armA, and the efflux pumps adeABC and adeIJK. A large set of virulence genes was also identified: adeFGH/efflux pump; the siderophores barAB, basABCDFGHIJ, and bauBCDEF; lpxABCDLM/capsule; tssABCDEFGIKLM/T6SS; and pgaABCD/biofilm. CONCLUSION: Widespread extensively drug-resistant CRAB IC2/ST2 is currently causing outbreaks in clinical settings in southeastern Brazil. This is due to at least three sub-lineages characterised by an enormous apparatus of virulence and resistance to antibiotics, both intrinsic and mobile.

Functional characterization of a Cassette-specific promoter in the class 1 integron-associated qnrVC1 gene.

Integrons are natural expression vectors due to the presence of an intrinsic promoter (P(c)). Although rare, gene cassettes can harbor their own promoter. This study determined the functionality of an internal promoter in the qnrVC1 cassette whose presence was suggested by a level of transcription similar to that of the preceding cassette (aadA2) and confirmed by in silico analysis. Its functionality was determined by 5' rapid amplification of cDNA ends (RACE) and cloning into promoter-probe vectors. P(qnrVC) was found in the qnrVC cassette family, stressing its role in contributing to resistance manifestation.

Genomics of Klebsiella pneumoniae Species Complex Reveals the Circulation of High-Risk Multidrug-Resistant Pandemic Clones in Human, Animal, and Environmental Sources.

The Klebsiella species present a remarkable genetic and ecological diversity, being ubiquitous in nature. In particular, the Klebsiella pneumoniae species complex (KpSC) has emerged as a major public health threat in the world, being an interesting model to assess the risk posed by strains recovered from animals and the environment to humans. We therefore performed a genomic surveillance analysis of the KpSC using every public genome in Brazil, aiming to show their local and global relationships, and the connectivity of antibiotic resistance and virulence considering human, animal, and environmental sources. The 390 genomes from distinct sources encompassed the K. pneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella variicola subsp. variicola, Klebsiella variicola subsp. tropica, and Klebsiella grimontii species and subspecies. K. pneumoniae harbored dozens of antibiotic resistance genes, while most of the genomes belong to the high-risk pandemic CC258 occurring in humans, animals, and the environment. In K. pneumoniae ST11, a high prevalence of the virulence determinants yersiniabactin, colibactin, and T6SS was revealed in association with multi-drug resistance (MDR), including carbapenem resistance. A diversity of resistance genes is carried by plasmids, some shared between strains from different STs, regions, and sources. Therefore, here were revealed some factors driving the success of KpSC as a pathogen.

Integron Functionality and Genome Innovation: An Update on the Subtle and Smart Strategy of Integrase and Gene Cassette Expression Regulation.

Integrons are considered hot spots for bacterial evolution, since these platforms allow one-step genomic innovation by capturing and expressing genes that provide advantageous novelties, such as antibiotic resistance. The acquisition and shuffling of gene cassettes featured by integrons enable the population to rapidly respond to changing selective pressures. However, in order to avoid deleterious effects and fitness burden, the integron activity must be tightly controlled, which happens in an elegant and elaborate fashion, as discussed in detail in the present review. Here, we aimed to provide an up-to-date overview of the complex regulatory networks that permeate the expression and functionality of integrons at both transcriptional and translational levels. It was possible to compile strong shreds of evidence clearly proving that these versatile platforms include functions other than acquiring and expressing gene cassettes. The well-balanced mechanism of integron expression is intricately related with environmental signals, host cell physiology, fitness, and survival, ultimately leading to adaptation on the demand.