RESUMO
Bacillus cereus sensu lato strains (B. cereus group) are widely distributed in nature and have received interest for decades due to their importance in insect pest management, food production and their positive and negative repercussions in human health. Consideration of practical uses such as virulence, physiology, morphology, or ill-defined features have been applied to describe and classify species of the group. However, current comparative studies have exposed inconsistencies between evolutionary relatedness and biological significance among genomospecies of the B. cereus group. Here, the combined analyses of core-based phylogeny and all versus all Average Nucleotide Identity values based on 2116 strains were conducted to update the genomospecies circumscriptions within B. cereus group. These analyses suggested the existence of 57 genomospecies, 37 of which are novel, thus indicating that the taxonomic identities of more than 39% of the analyzed strains should be revised or updated. In addition, we found that whole-genome in silico analyses were suitable to differentiate genomospecies such as B. anthracis, B. cereus and B. thuringiensis. The prevalence of toxin and virulence factors coding genes in each of the genomospecies of the B. cereus group was also examined, using phylogeny-aware methods at wide-genome scale. Remarkably, Cry and emetic toxins, commonly assumed to be associated with B. thuringiensis and emetic B. paranthracis, respectively, did not show a positive correlation with those genomospecies. On the other hand, anthrax-like toxin and capsule-biosynthesis coding genes were positively correlated with B. anthracis genomospecies, despite not being present in all strains, and with presumably non-pathogenic genomospecies. Hence, despite these features have been so far considered relevant for industrial or medical classification of related species of the B. cereus group, they were inappropriate for their circumscription. In this study, genomospecies of the group were accurately affiliated and representative strains defined, generating a rational framework that will allow comparative analysis in epidemiological or ecological studies. Based on this classification the role of specific markers such as Type VII secretion system, cytolysin, bacillolysin, and siderophores such as petrobactin were pointed out for further analysis.
Assuntos
Bacillus anthracis , Bacillus , Bacillus cereus/genética , Humanos , Fenótipo , FilogeniaRESUMO
Several Acinetobacter spp. act as opportunistic pathogens causing healthcare-associated infections worldwide, and in this respect their ability to resist antimicrobial compounds has certainly boosted up their global propagation. Acinetobacter clinical strains have demonstrated a remarkable ability to evolve and become resistant to almost all available drugs in the antimicrobial arsenal, including the last-resort carbapenem ß-lactams. The dissemination of antimicrobial resistant genes (ARG), heavy metals-detoxification systems and other traits such as virulence factors is facilitated by mobile genetic elements (MGE) through horizontal gene transfer. Among them, plasmids have been shown to play a critical role in this genus. Despite the continuous increase of Acinetobacter plasmid sequences present in databases, there are no reports describing the basic traits carried by these MGE. To fill this gap, a broad analysis of the Acinetobacter plasmidome was performed. A search for Acinetobacter complete plasmids indicated that 905 sequences have been deposited in the NCBI-GenBank public database, of which 492 are harbored by Acinetobacter baumannii strains. Plasmid-classification schemes based on Rep proteins homology have so far described 23 different groups for A. baumannii (GR1-23), and 16 Acinetobacter Rep3 Groups (AR3G1-16) for the complete genus. Acinetobacter plasmids size ranges from 1.3 to 400 kb. Interestingly, widespread plasmids which are < 20 kb make up 56% of the total present in members of this genus. This led to the proposal of Acinetobacter plasmid assignation to two groups according to their size (< 20 kb and > 20 kb). Usually, smaller plasmids are not self-transmissible, and thereby employ alternative mechanisms of dissemination. For instance, a subgroup of < 20 kb-plasmids belonging to the pRAY-family, lack a rep gene, but encode a relaxase enabling their mobilization by conjugative plasmids. Other subgroup, including small GR2 Acinetobacter plasmids, does not encode a relaxase gene. However, they could still be mobilized by conjugative plasmids which recognize an oriT region carried by these small plasmids. Also, these < 20 kb-plasmids usually carry accessory genes bordered by XerC/D-recombinases recognition sites which have been hypothesized to mediate plasmid plasticity. Conversely, many cases of larger plasmids are self-transmissible and might encode virulence factors and their regulators, thus controlling strain pathogenicity. The ARGs carried by the > 20 kb-plasmids are usually encoded within other MGEs such as transposons, or as part of integrons. It has been recently noted that some of the > 20 kb-plasmids are derived from excised phages, and thus dubbed as phage-like plasmids. All in all, the plethora of plasmids found in strains of this genus and the multiple strategies promoting their evolution and dissemination have certainly contributed to survival of the Acinetobacter members in different habitats, including the clinical environment.
Assuntos
Acinetobacter baumannii/genética , DNA Bacteriano/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Análise de Sequência de DNARESUMO
Acinetobacter baumannii (Aba) is an emerging opportunistic pathogen associated to nosocomial infections. The rapid increase in multidrug resistance (MDR) among Aba strains underscores the urgency of understanding how this pathogen evolves in the clinical environment. We conducted here a whole-genome sequence comparative analysis of three phylogenetically and epidemiologically related MDR Aba strains from Argentinean hospitals, assigned to the CC104O/CC15P clonal complex. While the Ab244 strain was carbapenem-susceptible, Ab242 and Ab825, isolated after the introduction of carbapenem therapy, displayed resistance to these last resource ß-lactams. We found a high chromosomal synteny among the three strains, but significant differences at their accessory genomes. Most importantly, carbapenem resistance in Ab242 and Ab825 was attributed to the acquisition of a Rep_3 family plasmid carrying a blaOXA-58 gene. Other differences involved a genomic island carrying resistance to toxic compounds and a Tn10 element exclusive to Ab244 and Ab825, respectively. Also remarkably, 44 insertion sequences (ISs) were uncovered in Ab825, in contrast with the 14 and 11 detected in Ab242 and Ab244, respectively. Moreover, Ab825 showed a higher killing capacity as compared to the other two strains in the Galleria mellonella infection model. A search for virulence and persistence determinants indicated the loss or IS-mediated interruption of genes encoding many surface-exposed macromolecules in Ab825, suggesting that these events are responsible for its higher relative virulence. The comparative genomic analyses of the CC104O/CC15P strains conducted here revealed the contribution of acquired mobile genetic elements such as ISs and plasmids to the adaptation of A. baumannii to the clinical setting.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/classificação , Farmacorresistência Bacteriana , Plasmídeos/genética , Sequenciamento Completo do Genoma/métodos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Adaptação Fisiológica , Aminoglicosídeos/farmacologia , Animais , Argentina , Composição de Bases , Carbenicilina/farmacologia , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Genômica , Humanos , Filogenia , SinteniaRESUMO
Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.
Assuntos
Acinetobacter/genética , Plasmídeos , Infecções por Acinetobacter/genética , Infecções por Acinetobacter/microbiologia , Proteínas de Bactérias/genética , Biologia Computacional , DNA Bacteriano , Feminino , Genoma Bacteriano , Humanos , Pessoa de Meia-Idade , Filogenia , Homologia de SequênciaRESUMO
Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii as the species of greatest concern worldwide due to its multi-drug resistance and recent appearance of hyper-virulent strains in the clinical setting. Acinetobacter colonization of the environment and the host is associated with a multitude of factors which remain poorly characterized. Among them, the secretion systems (SS) encoded by Acinetobacter species confer adaptive advantages depending on the niche occupied. Different SS have been characterized in this group of microorganisms, including T6SS used by several Acinetobacter species to outcompete other bacteria and in some A. baumannii strains for Galleria mellonella colonization. Therefore, to better understand the distribution of the T6SS in this genus we carried out an in-depth comparative genomic analysis of the T6SS in 191 sequenced strains. To this end, we analyzed the gene content, sequence similarity, synteny and operon structure of each T6SS loci. The presence of a single conserved T6SS-main cluster (T6SS-1), with two different genetic organizations, was detected in the genomes of several ecologically diverse species. Furthermore, a second main cluster (T6SS-2) was detected in a subgroup of 3 species of environmental origin. Detailed analysis also showed an impressive genetic versatility in T6SS-associated islands, carrying VgrG, PAAR and putative toxin-encoding genes. This in silico study represents the first detailed intra-species comparative analysis of T6SS-associated genes in the Acinetobacter genus, that should contribute to the future experimental characterization of T6SS proteins and effectors.
RESUMO
Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D ß-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the first evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra-molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.
RESUMO
Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.
Assuntos
Carboxiliases/genética , Enterococcus faecalis/enzimologia , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carboxiliases/isolamento & purificação , Carboxiliases/metabolismo , Ácido Cítrico/metabolismo , Citoplasma/enzimologia , Enterococcus faecalis/genética , Enterococcus faecalis/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Ácido Oxaloacético/metabolismo , Subunidades Proteicas , Proteínas Recombinantes , Deleção de Sequência , TransgenesRESUMO
We report the draft genome sequence of Enterococcus mundtii CRL1656, which was isolated from the stripping milk of a clinically healthy adult Holstein dairy cow from a dairy farm of the northwestern region of Tucumán (Argentina). The 3.10-Mb genome sequence consists of 450 large contigs and contains 2,741 predicted protein-coding genes.
Assuntos
Enterococcus/classificação , Enterococcus/genética , Genoma Bacteriano , Animais , Argentina/epidemiologia , Bovinos , Feminino , Mastite Bovina/epidemiologia , Mastite Bovina/microbiologia , Leite/microbiologia , Dados de Sequência MolecularRESUMO
Diacetyl and acetoin are pyruvate-derived metabolites excreted by many micro-organisms, and are important in their physiology. Although generation of these four-carbon (C4) compounds in Enterococcus faecalis is a well-documented phenotype, little is known about the gene regulation of their biosynthetic pathway and the physiological role of the pathway in this bacterium. In this work, we identified the genes involved in C4 compound biosynthesis in Ent. faecalis and report their transcriptional analysis. These genes are part of the alsSD bicistronic operon, which encodes α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD). Our studies showed that alsSD operon transcription levels are maximal during the exponential phase of growth, decreasing thereafter. Furthermore, we found that this transcription is enhanced upon addition of pyruvate to the growth medium. In order to study the functional role of the alsSD operon, an isogenic alsSD mutant strain was constructed. This strain lost its capacity to generate C4 compounds, confirming the role of alsSD genes in this metabolic pathway. In contrast to the wild-type strain, the alsSD-deficient strain was unable to grow in LB medium supplemented with pyruvate at an initial pH of 4.5. This dramatic reduction in growth parameters for the mutant strain was simultaneously accompanied by the inability to alkalinize the internal and external medium under these conditions. In sum, these results suggest that the decarboxylation reactions related to the C4 biosynthetic pathway give enterococcal cells a competitive advantage during pyruvate metabolism at low pH.