The outer membrane proteins responsible for the influx of carbapenem ␤-lactam antibiotics in the nonfermentative gram-negative pathogen Acinetobacter baumannii are still poorly characterized. Resistance to both imipenem and meropenem in multidrug-resistant clinical strains of A. baumannii is associated with the loss of a heat-modifiable 29-kDa outer membrane protein, designated CarO. The chromosomal locus containing the carO gene was cloned and characterized from different clinical isolates. Only one carO copy, present in a single transcriptional unit, was found in the A. baumannii genome. The carO gene encodes a polypeptide of 247 amino acid residues with a typical N-terminal signal sequence and a predicted transmembrane ␤-barrel topology. Its absence from different carbapenem-resistant clinical isolates of A. baumannii resulted from the disruption of carO by distinct insertion elements. The overall data thus support the notion that CarO participates in the influx of carbapenem antibiotics in A. baumannii. Moreover, database searches identified the presence of carO homologs only in species of the genera Acinetobacter, Moraxella, and Psychrobacter, disclosing the existence of a novel family of outer membrane proteins restricted to the family Moraxellaceae of the class ␥-Proteobacteria.The emergence of antibiotic resistance among both pathogenic and opportunistic microbes resident in hospitals represents a serious and recurrent problem for the treatment of infections (22). It generates a continuous demand for new antimicrobial agents, whose application feeds the undesired vicious circle of selection and dissemination of new patterns of antibiotic resistance. The otherwise bizarre but still elegant mechanisms that underlie some of these patterns of resistance demonstrate that the potential of microbes to challenge eradication attempts remains almost unexhausted (26). Yet, attempts to reduce the dissemination of rapidly evolving antibiotic-resistant pathogens are best based on a detailed knowledge of the causes that promote these patterns of resistance (26).The genus Acinetobacter, recently reassigned to the family Moraxellaceae in the class ␥-Proteobacteria (30), is constituted by gram-negative, pleomorphic aerobic species commonly isolated from many sources in the environment, including drinking and static water, soil, sewage, food, and the skin of humans and animals (5). Certain strains of a particular species of the genus, Acinetobacter baumannii, now account for a large percentage of nosocomial infections, including pneumonia, bacteremia, skin and wound infections, and urinary tract infections. These strains are almost invariably multidrug resistant, having successfully resisted eradication attempts by the use of penicillins, aminoglycosides, cephalosporins, and even fluoroquinolones (5). It is due to this outstanding ability to rapidly respond to the challenge of new antibiotics that the emerging resistance to carbapenems among nosocomial strains of A. baumannii represents a major concern (22).The molecular b...

Metallo-␤-lactamases (M␤Ls) are zinc-dependent enzymes able to hydrolyze and inactivate most ␤-lactam antibiotics. The large diversity of active site structures and metal content among M␤Ls from different sources has limited the design of a pan-M␤L inhibitor. Here we report the biochemical and biophysical characterization of a novel M␤L, 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 Asp 120 , His 121 , His 263 , and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear M␤Ls. Contrasting all other related M␤Ls, GOB-18 is fully active against a broad range of ␤-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of M␤Ls.The expression of ␤-lactam degrading enzymes (␤-lactamases) is the most common mechanism of antibiotic resistance among bacteria (1, 2). These enzymes have been grouped into four classes (A-D) according to sequence homology (3, 4). Class A, C, and D enzymes use an active site serine residue as a nucleophile, whereas class B lactamases (generically termed metallo-␤-lactamases, M␤Ls) 9 employ one or two Zn(II) ions to cleave the ␤-lactam ring.M␤Ls have particular importance in the clinical setting since they can hydrolyze a broader spectrum of ␤-lactam substrates than the serine-type enzymes and are resistant to most clinically employed inhibitors (5-11). The design of an efficient pan-M␤L inhibitor has been mostly limited by a striking diversity in the active site structures, catalytic features, and metal ion requirements for activity among different enzymes. Based on this heterogeneity, M␤Ls have been classified into three subclasses: B1, B2, and B3 (3, 6). Subclass B1 includes several chromosomally encoded enzymes such as BcII from Bacillus cereus (12-14), CcrA from Bacteroides fragilis (15-18), BlaB from Elizabethkingia meningoseptica (formerly, Chryseobacterium meningosepticum) (19), as well as the transferable VIM (20)-, IMP (21, 22)-, SPM (23, 24)-, and GIM-type enzymes. Subclass B2 includes the CphA (25, 26) and ImiS (27) lactamases from Aeromonas species. Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28 -30), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31) and GOB from E. meningoseptica (32), as well as from environmental bacteria such as CAU-1 from Caulobacter crescentus (33) and THIN-B from Janthinobacterium lividum (34).Molecular structures of M␤Ls from the three subclasses have been solved by x-ray crystallography (12,14,15,25,31). Comparison of the tertiary structure of enzymes belonging to the different subclasses reveals a common ␣␤/␤␣ sandwich fold, in which different insertions and deletions have resulted in different loop topologies and, ultimately, in different zinc coordination environments and metal site occupancies among B1, B2, and B3 en...

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 β-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.

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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.

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 L-ornithine uptake: CarO-deficient strains were specifically impaired to grow only on L L-ornithine, and failed to incorporate L L-[ 14 C] ornithine from the medium. L L-arginine, and histidine and lysine to a lower extent, could effectively compete for L L-[ 14 C] ornithine uptake. L 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 L-ornithine, carbapenems, and other basic amino acids in A. baumannii.

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.

Metallo-␤-lactamases (M␤Ls) are zinc-dependent enzymes produced by many clinically relevant gramnegative pathogens that can hydrolyze most ␤-lactam antibiotics. M␤Ls 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 M␤L 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 ␤-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.