RESUMO
The acquisition of bla OXA genes encoding different carbapenem-hydrolyzing class-D ß-lactamases (CHDL) represents a main determinant of carbapenem resistance in the nosocomial pathogen Acinetobacter baumannii. The blaOXA-58 gene, in particular, is generally embedded in similar resistance modules (RM) carried by plasmids unique to the Acinetobacter genus lacking self-transferability. The ample variations in the immediate genomic contexts in which blaOXA-58 -containing RMs are inserted among these plasmids, and the almost invariable presence at their borders of non-identical 28-bp sequences potentially recognized by the host XerC and XerD tyrosine recombinases (pXerC/D-like sites), suggested an involvement of these sites in the lateral mobilization of the gene structures they encircle. However, whether and how these pXerC/D sites participate in this process is only beginning to be understood. Here, we used a series of experimental approaches to analyze the contribution of pXerC/D-mediated site-specific recombination to the generation of structural diversity between resistance plasmids carrying pXerC/D-bounded bla OXA-58- and TnaphA6-containing RM harbored by two phylogenetically- and epidemiologically-closely related A. baumannii strains of our collection, Ab242 and Ab825, during adaptation to the hospital environment. Our analysis disclosed the existence of different bona fide pairs of recombinationally-active pXerC/D sites in these plasmids, some mediating reversible intramolecular inversions and others reversible plasmid fusions/resolutions. All of the identified recombinationally-active pairs shared identical GGTGTA sequences at the cr spacer separating the XerC- and XerD-binding regions. The fusion of two Ab825 plasmids mediated by a pair of recombinationally-active pXerC/D sites displaying sequence differences at the cr spacer could be inferred on the basis of sequence comparison analysis, but no evidence of reversibility could be obtained in this case. The reversible plasmid genome rearrangements mediated by recombinationally-active pairs of pXerC/D sites reported here probably represents an ancient mechanism of generating structural diversity in the Acinetobacter plasmid pool. This recursive process could facilitate a rapid adaptation of an eventual bacterial host to changing environments, and has certainly contributed to the evolution of Acinetobacter plasmids and the capture and dissemination of bla OXA-58 genes among Acinetobacter and non-Acinetobacter populations co-residing in the hospital niche.
RESUMO
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
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
The number and type of outer membrane (OM) channels responsible for carbapenem uptake in Acinetobacter are still not well defined. Here, we addressed these questions by using Acinetobacter baylyi as a model species and a combination of methodologies aimed to characterize OM channels in their original membrane environment. Kinetic and competition analyses of imipenem (IPM) uptake by A. baylyi whole cells allowed us to identify different carbapenem-specific OM uptake sites. Comparative analyses of IPM uptake by A. baylyi wild-type (WT) cells and ΔcarO mutants lacking CarO indicated that this OM protein provided a carbapenem uptake site displaying saturable kinetics and common binding sites for basic amino acids compatible with a specific channel. The kinetic analysis uncovered another carbapenem-specific channel displaying a somewhat lower affinity for IPM than that of CarO and, in addition, common binding sites for basic amino acids as determined by competition studies. The use of A. baylyi gene deletion mutants lacking OM proteins proposed to function in carbapenem uptake in Acinetobacter baumannii indicated that CarO and OprD/OccAB1 mutants displayed low but consistent reductions in susceptibility to different carbapenems, including IPM, meropenem, and ertapenem. These two mutants also showed impaired growth on l-Arg but not on other carbon sources, further supporting a role of CarO and OprD/OccAB1 in basic amino acid and carbapenem uptake. A multiple-carbapenem-channel scenario may provide clues to our understanding of the contribution of OM channel loss or mutation to the carbapenem-resistant phenotype evolved by pathogenic members of the Acinetobacter genus.
Assuntos
Acinetobacter/metabolismo , Aminoácidos Básicos/metabolismo , Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Imipenem/metabolismo , Porinas/deficiência , Acinetobacter/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Membrana Celular/química , Membrana Celular/metabolismo , Ertapenem , Evolução Molecular , Deleção de Genes , Expressão Gênica , Cinética , Meropeném , Porinas/genética , Tienamicinas/metabolismo , beta-Lactamas/metabolismoRESUMO
Metallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every ß-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions.
Assuntos
Farmacorresistência Bacteriana Múltipla , Flavobacteriaceae/enzimologia , Glutamina/química , Histidina/química , Zinco/química , beta-Lactamases/química , Antibacterianos/química , Antibacterianos/metabolismo , Carbapenêmicos/química , Carbapenêmicos/metabolismo , Domínio Catalítico , Cátions Bivalentes , Cefalosporinas/química , Cefalosporinas/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Flavobacteriaceae/química , Expressão Gênica , Glutamina/metabolismo , Histidina/metabolismo , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Penicilinas/química , Penicilinas/metabolismo , Periplasma/química , Periplasma/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
We report here the draft genome sequence of an NDM-1-producing Acinetobacter bereziniae clinical strain, HPC229. This strain harbors both plasmid and chromosomal resistance determinants toward different ß-lactams and aminoglycosides as well as several types of multidrug efflux pumps, most likely representing an adaptation strategy for survival under different environments.
RESUMO
The complete sequence of the carbapenem-resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain is presented. pLD209 is formed by 3 well-defined regions: an adaptability module encompassing a Tn402-like class 1 integron of clinical origin containing blaVIM-2 and aacA4 gene cassettes, partitioning and transfer modules, and a replication module derived from plasmids of environmental bacteria. pLD209 is thus a mosaic of modules originating in both the clinical and environmental (nonclinical) microbiota.
Assuntos
Carbapenêmicos/farmacologia , Pseudomonas putida/genética , Fatores R/genética , Sequência de Bases , Conjugação Genética/genética , Genes Bacterianos/genética , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Resistência beta-Lactâmica/genéticaRESUMO
The OmpW family consists of a ubiquitous group of small outer membrane (OM) ß-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hosts.