Dynamic state of plasmid genomic architectures resulting from XerC/D-mediated site-specific recombination in Acinetobacter baumannii Rep_3 superfamily resistance plasmids carrying blaOXA-58 - and TnaphA6-resistance modules.

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.

Pseudomonas putida group species as reservoirs of mobilizable Tn402-like class 1 integrons carrying blaVIM-2 metallo-ß-lactamase genes.

The Pseudomonas putida group (P. putida G) is composed of at least 21 species associated with a wide range of environments, including the clinical setting. Here, we characterized 13 carbapenem-resistant P. putida G clinical isolates bearing class 1 integrons/transposons (class 1 In/Tn) carrying blaVIM-2 metallo-ß-lactamase gene cassettes obtained from hospitals of Argentina. Multilocus sequencing (MLSA) and phylogenetic analyses based on 16S rDNA, gyrB and rpoD sequences distinguished 7 species among them. blaVIM-2 was found in three different cassette arrays: In41 (blaVIM-2-aacA4), In899 (only blaVIM-2), and In528 (dfrB1-aacA4-blaVIM-2). In41 and In899 were associated with complete tniABQC transposition modules and IRi/IRt boundaries characteristic of the Tn5053/Tn402 transposons, which were designated Tn6335 and Tn6336, respectively. The class 1 In/Tn element carrying In528, however, exhibited a defective tni module bearing only the tniC (transposase) gene, associated with a complete IS6100 bounded with two oppositely-oriented IRt end regions. In some P. putida G isolates including P. asiatica, P. juntendi, P. putida G/II, and P. putida G/V, Tn6335/Tn6336 were carried by pLD209-type conjugative plasmids capable of self-mobilization to P. aeruginosa or Escherichia coli. In other isolates of P. asiatica, P. putida G/II, and P. monteiliieilii, however, these blaVIM-2-containing class 1 In/Tn elements were found inserted into the res regions preceding the tnpR (resolvase) gene of particular Tn21 subgroup members of Tn3 transposons. The overall results reinforce the notion of P. putida G members as blaVIM-2 reservoirs, and shed light on the mechanisms of dissemination of carbapenem resistance genes to other pathogenic bacteria in the clinical setting.

What do we know about plasmids carried by members of the Acinetobacter genus?

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.

Acquisition of plasmids conferring carbapenem and aminoglycoside resistance and loss of surface-exposed macromolecule structures as strategies for the adaptation of Acinetobacter baumannii CC104O/CC15P strains to the clinical setting.

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.

Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii.

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.

The Acinetobacter Outer Membrane Contains Multiple Specific Channels for Carbapenem ß-Lactams as Revealed by Kinetic Characterization Analyses of Imipenem Permeation into Acinetobacter baylyi Cells.

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.

Draft Genome Sequence of Acinetobacter bereziniae HPC229, a Carbapenem-Resistant Clinical Strain from Argentina Harboring blaNDM-1.

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.

Polyclonal outbreak of bacteremia caused by Burkholderia cepacia complex and the presumptive role of ultrasound gel.

A nosocomial polyclonal outbreak associated to bacteremia caused by different Burkholderia cepacia complex (BCC) species and clones is reported. Molecular characterization identified Burkholderia stabilis, Burkholderia contaminans, and Burkholderia ambifaria among BCC isolates obtained from patients in neonatal and adult intensive care units. BCC was also isolated from an intrinsically contaminated ultrasound gel, which constituted the presumptive BCC source. Prior BCC outbreak related to contaminated ultrasound gels have been described in the setting of transrectal prostate biopsy. Outbreak caused strains and/or clones of BCC have been reported, probably because BCC are commonly found in the natural environment; most BCC species are biofilm producers, and different species may contaminate an environmental source. The finding of multiple species or clones during the analysis of nosocomial BCC cases might not be enough to reject an outbreak from a common source.

Differential Role of the T6SS in Acinetobacter baumannii Virulence.

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

The complete nucleotide sequence of the carbapenem resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain reveals a chimeric design formed by modules derived from both environmental and clinical bacteria.

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.

Spreading of AbaR-type genomic islands in multidrug resistance Acinetobacter baumannii strains belonging to different clonal complexes.

In order to determine the occurrence of AbaR-type genomic island in multidrug resistant Acinetobacter baumannii (MDRAb) strains circulating in Argentina, Uruguay, and Chile, we studied 51 MDRAb isolates recovered from several hospitals over 30 years. AbaR-type genomic resistance islands were found in 36 MDRAb isolates since 1986 till now. MLST technique allowed us to identify the presence of four different Clonal Complexes (109, 104, 119, 113) among the positive AbaR-type island positive strains. This is the first description of AbaR-type islands in the CC104 and CC113 that are the most widespread Clonal Complexes in Argentina. In addition, PCR mapping exposed different arrays to those previously described, evidencing the plasticity of this island. Our results evidence a widespread distribution of the AbaR-type genomic islands along the time in the MDRAb population, including the epidemic global clone 1 (GC1) as well as different clonal complexes to those already described in the literature.

Horizontal gene transfer and assortative recombination within the Acinetobacter baumannii clinical population provide genetic diversity at the single carO gene, encoding a major outer membrane protein channel.

We described previously the presence in Acinetobacter baumannii of a novel outer membrane (OM) protein, CarO, which functions as an L-ornithine OM channel and whose loss was concomitant with increased carbapenem resistance among clonally related nosocomial isolates of this opportunistic pathogen. Here, we describe the existence of extensive genetic diversity at the carO gene within the A. baumannii clinical population. The systematic analysis of carO sequences from A. baumannii isolates obtained from public hospitals in Argentina revealed the existence of four highly polymorphic carO variants among them. Sequence polymorphism between the different A. baumannii CarO variants was concentrated in three well-defined protein regions that superimposed mostly to predicted surface-exposed loops. Polymorphism among A. baumannii CarO variants was manifested in differential electrophoretic mobilities, antigenic properties, abilities to form stable oligomeric structures, and l-ornithine influx abilities through the A. baumannii OM under in vivo conditions. Incongruence between the phylogenies of the clinical A. baumannii isolates analyzed and those of the carO variants they harbor suggests the existence of assortative (entire-gene) carO recombinational exchange within the A. baumannii population. Exchange of carO variants possessing differential characteristics mediated by horizontal gene transfer may constitute an A. baumannii population strategy to survive radically changing environmental conditions, such as the leap from inanimate sources to human hosts and vice versa, persistence in a compromised host, and/or survival in health care facilities.

ISAba825, a functional insertion sequence modulating genomic plasticity and bla(OXA-58) expression in Acinetobacter baumannii.

ISAba825, an insertion sequence found inactivating Acinetobacter baumannii carO, was tagged with a kanamycin (Kn) resistance cassette. ISAba825::Kn effectively transposed in A. baumannii, showing preference for short, AT-enriched target sequences, generating 6- to 9-bp target duplications. Additionally, we detected the presence of ISAba825 upstream of a plasmid-borne bla(OXA-58) gene, generating a hybrid promoter largely enhancing its expression and leading to carbapenem resistance. Overall, a role for ISAba825 in carbapenem resistance modulation in A. baumannii is proposed.

Secretion of GOB metallo-beta-lactamase in Escherichia coli depends strictly on the cooperation between the cytoplasmic DnaK chaperone system and the Sec machinery: completion of folding and Zn(II) ion acquisition occur in the bacterial periplasm.

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes produced by many clinically relevant gram-negative pathogens that can hydrolyze most beta-lactam antibiotics. MbetaLs are synthesized in the bacterial cytoplasm as precursors and are secreted into the periplasm. Here, we report that the biogenesis process of the recently characterized MbetaL GOB-18 demands cooperation between a main chaperone system of the bacterial cytoplasm, DnaK, and the Sec secretion machinery. Using the expression of the complete gob-18 gene from the gram-negative opportunistic pathogen Elizabethkingia meningoseptica in Escherichia coli as a model system, we found that the precursor of this metalloenzyme is secreted by the Sec pathway and reduces cell susceptibility to different beta-lactam antibiotics. Moreover, acting with different J proteins such as cytoplasmic DnaJ and membrane-associated DjlA as cochaperones, DnaK plays an essential role in the cytoplasmic transit of the GOB-18 precursor to the Sec translocon. Our studies also revealed a less relevant role, that of assisting in GOB-18 secretion, for trigger factor, while no significant functions were found for other main cytoplasmic chaperones such as SecB or GroEL/ES. The overall findings indicate that the biogenesis of GOB-18 involves cytoplasmic interaction of the precursor protein mainly with DnaK, secretion by the Sec system, and final folding and incorporation of Zn(II) ions into the bacterial periplasm.

A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers.

OBJECTIVES: The dissemination of metallo and serine carbapenem-hydrolysing beta-lactamases among Gram-negative nosocomial bacteria represents an acute problem worldwide. Here, we present a rapid and sensitive assay for the characterization of carbapenemase producers to aid in infection control and prevention. METHODS: The assay involves a rapid disruption of bacterial isolates with silicon dioxide microbeads, followed by the testing in cell-free extracts of hydrolytic activity towards various beta-lactams including two carbapenems (imipenem and meropenem) and a cephalosporin (ceftazidime). A parallel testing of the effects of selective beta-lactamase inhibitors such as EDTA and clavulanic acid allows differentiation of metallo carbapenemases from serine carbapenemases, and also clavulanic-acid-sensitive from -resistant enzymes among the latter. RESULTS: The efficiency of bacterial disruption using silicon dioxide microbeads was identical to that of ultrasonic treatment. The subsequent microbiological assay aimed to evaluate both substrate specificity and inhibitor profile of carbapenem-hydrolysing enzymes present in the extracts and allowed an accurate differentiation of A, B and D types, as judged by the analysis of 24 well-characterized clinical strains that included metallo-beta-lactamase producers (i.e. VIM-, IMP- and SPM-type Pseudomonas producers; an L1 Stenotrophomonas maltophilia producer; and a GOB-18 Elizabethkingia meningoseptica producer) as well as serine carbapenemase producers (i.e. an SME-type Serratia marcescens producer, a GES-2 Pseudomonas aeruginosa producer, Klebsiella pneumoniae and Citrobacter freundii KPC-2 producers and OXA-type Acinetobacter baumannii producers). CONCLUSIONS: We have developed a convenient microbiological assay aimed to more accurately and in a short time characterize carbapenem-hydrolysing enzymes produced by Gram-negative bacteria. The assay possesses broad applicability in the clinical setting.

Biochemical characterization of metallo-beta-lactamase VIM-11 from a Pseudomonas aeruginosa clinical strain.

A detailed biochemical characterization of the Pseudomonas aeruginosa VIM-11 metallo-beta-lactamase (MbetaL) is reported. The only substitution differentiating VIM-11 from VIM-2 (N165S) promoted a slightly improved catalytic efficiency of the former on 3 out of 12 substrates, notably the bulky cephalosporins. Thus, MbetaL-mediated resistance also may be modulated by remote mutations.

CarO, an Acinetobacter baumannii outer membrane protein involved in carbapenem resistance, is essential for L-ornithine uptake.

We previously associated the emergence of carbapenem resistance in Acinetobacter baumannii with the loss of an outer membrane (OM) protein designated CarO. CarO was found essential for L-ornithine uptake: CarO-deficient strains were specifically impaired to grow only on L-ornithine, and failed to incorporate L-[(14)C] ornithine from the medium. L-arginine, and histidine and lysine to a lower extent, could effectively compete for L-[(14)C] ornithine uptake. L-ornithine also reduced A. baumannii sensitivity to imipenem, suggesting that both compounds compete for uptake. The overall results indicate that CarO participates in the selective uptake of L-ornithine, carbapenems, and other basic amino acids in A. baumannii.

The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site.

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.