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 ICE. METHODS: All E. meningoseptica genomes from NCBI (n=87) were retrieved and annotated for resistome searching using the CARD database, and a phylogenic analysis was performed based on E. meningoseptica core genome. ICE was identified through comparative genomics with ICEs occurring in Elizabethkingia spp. RESULTS: Phylogenetic analysis showed E. meningoseptica genomes from six countries distributed in 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, the tet(X) and aadS genes were located in an ICE of 49 769 bp (ICEEmSQ101), which was completely obtained from the E. meningoseptica SQ101 genome. We also raised evidence that other 27 genomes also presented 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 basically in a single Chinese lineage. We identified that several E. meningoseptica genomes harbored an ICE that mobilized the tet(X) gene and exhibited characteristics similar to the ICEs of other Flavobacteria, which would favor their transmission in this bacteria 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.

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.

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.

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.

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.

Global Genomic Epidemiology of Escherichia coli (ExPEC) ST38 Lineage Revealed a Virulome Associated with Human Infections.

BACKGROUND: Most of the extraintestinal human infections worldwide are caused by specific extraintestinal pathogenic Escherichia coli (ExPEC) lineages, which also present a zoonotic character. One of these lineages belongs to ST38, a high-risk globally disseminated ExPEC. To get insights on the aspects of the global ST38 epidemiology and evolution as a multidrug-resistant and pathogenic lineage concerning the three axes of the One Health concept (humans, animals, and natural environments), this study performed a global phylogenomic analysis on ST38 genomes. METHODS: A phylogenetic reconstruction based on 376 ST38 genomes recovered from environments, humans, livestock, and wild and domestic animals in all continents throughout three decades was performed. The global information concerning the ST38 resistome and virulome was also approached by in silico analyses. RESULTS: In general, the phylogenomic analyses corroborated the zoonotic character of the ExPEC ST38, since clonal strains were recovered from both animal and human sources distributed worldwide. Moreover, our findings revealed that, independent of host sources and geographic origin, the genomes were distributed in two major clades (Clades 1 and 2). However, the ST38 accessory genome was not strictly associated with clades and sub-clades, as found for the type 2 T3SS ETT2 that was evenly distributed throughout Clades 1 and 2. Of note was the presence of the Yersinia pestis-like high-pathogenicity island (HPI) exclusively in the major Clade 2, in which prevails most of the genomes from human origin recovered worldwide (2000 to 2020). CONCLUSIONS: This evidence corroborates the HPI association with successful E. coli ST38 establishment in human infections.

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.

Diversity and distribution of Type VI Secretion System gene clusters in bacterial plasmids.

Type VI Secretion System (T6SS) is a nanomolecular apparatus that allows the delivery of effector molecules through the cell envelope of a donor bacterium to prokaryotic and/or eukaryotic cells, playing a role in the bacterial competition, virulence, and host interaction. T6SS is patchily distributed in bacterial genomes, suggesting an association with horizontal gene transfer (HGT). In fact, T6SS gene loci are eventually found within genomic islands (GIs), and there are some reports in plasmids and integrative and conjugative elements (ICEs). The impact that T6SS may have on bacteria fitness and the lack of evidence on its spread mechanism led us to question whether plasmids could represent a key mechanism in the spread of T6SS in bacteria. Therefore, we performed an in-silico analysis to reveal the association between T6SS and plasmids. T6SS was mined on 30,660 plasmids from NCBI based on the presence of at least six T6SS core proteins. T6SS was identified in 330 plasmids, all belonging to the same type (T6SSi), mainly in Proteobacteria (328/330), particularly in Rhizobium and Ralstonia. Interestingly, most genomes carrying T6SS-harboring plasmids did not encode T6SS in their chromosomes, and, in general, chromosomal and plasmid T6SSs did not form separate clades.

Multidrug-resistant Mycolicibacterium fortuitum infection in a companion cat (Felis silvestris catus) in Brazil.

Mycolicibacterium fortuitum is a fast-growing bacterium and an opportunistic pathogen implicated in human and animal infections. Here we report the first case and genetic characterization of a strain of M. fortuitum isolated from skin lesions of a companion cat with atypical cutaneous mycobacteriosis in Brazil. In addition, the genome of this strain was sequenced, representing the first genome of this opportunistic pathogen isolated from an animal infection. The in silico and in vitro analysis regarding antibiotic resistance of this strain showed an intrinsic multiresistance antibiotic profile. However, this strain showed sensitivity to amikacin and ciprofloxacin, and the cat was treated long-term with these drugs.

Conjugative transfer of naturally occurring plasmid in Mycolicibacterium sp.

Conjugation is considered the main horizontal gene transfer mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard about this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids, and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.

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.

Emergence of a VIM-2-producing extensively drug-resistant (XDR) Pseudomonas aeruginosa ST309 in South America: a comparative genomic analysis.

Pseudomonas aeruginosa is considered a top priority pathogen associated with elevated morbidity and mortality. Worldwide outbreaks have been associated with a few high-risk epidemic P. aeruginosa lineages. However, the biological features involved in the persistence and spread of such lineages in clinical settings remain to be unravelled. This study reports the emergence of an extensively drug-resistant (XDR) sequence type 309 (ST309) P. aeruginosa in South America (Brazil), specifically in the Amazon region. Genomic analyses were performed with 42 complete and draft ST309 genomes, giving insights into its epidemiology, resistome and mobilome. A heterogeneous distribution of acquired antimicrobial resistance genes among ST309 genomes was observed, which included blaVIM-2, blaIMP-15 and qnrVC1, all associated with class 1 integrons. Mobilome mining showed the presence of integrative and conjugative elements (ICEs), transposons and genomic islands (GIs) harbouring a huge arsenal of heavy metal resistance determinants, which probably provided adaptive advantages to the ST309 lineage.