Acinetobacter baumannii: An Emerging Multidrug-resistant Threat

Thomas D Gootz; Andrea Marra

Disclosures

Expert Rev Anti Infect Ther. 2008;6(3):309-325. 

In This Article

Resistance to Aminoglycosides

Aminoglycoside resistance is common in clinical isolates of acinetobacters. As with other Gram-negative bacilli, the major mechanism of resistance to these agents is due to the dissemination of genes encoding aminoglycoside-modifying enzymes.[81] Rapid molecular typing methods are available, including multiplex PCR, to detect specific genes from all three modifying enzyme groups (phosphotransferases, acetyltransferases and nucleotidyltransferases).[120] Some PCR methods based on detection of the structural gene of these enzymes are now used for epidemiologic typing of A. baumannii,[121] replacing the older standard methods of resistance-gene identification by phenotypic analysis using MICs.[122] Some evidence exists that aminoglycoside resistance can also occur by methylation of the 16S ribosomal RNA, which is a common mechanism found in many bacterial species.

Porins are outer membrane proteins that form water-filled pores, allowing the access of nutrients and antibiotics into the cell. Acinetobacters, similar to other MDR, Gram-negative bacteria, can easily downregulate porin expression in the presence of antibiotic selective pressure without acquiring new genetic material.

Compared with the porins of the Enterobacteriaceae and P. aeruginosa, little is known about porins of acinetobacters. Reports describing antibiotic accumulation found that the coefficient of permeability for cephalosporins into P. aeruginosa was two- to sevenfold greater than that measured for Acinetobacter.[123] As discussed in a recent review,[124] the relatively low permeability of Acinetobacter to antibiotics may be due to either a reduced number of porins in the outer membrane or their poor diffusion characteristics for small molecules. There have been at least five putative porins detected in acinetobacters. The best characterized is the 35,636-Da heat-modifiable protein (HMP-AB). This 346-amino acid porin is in the same family as OmpA of E. coli.[125] Unlike the functional trimeric OmpF porin in E. coli, the HMP-AB forms a monomeric protein in the outer membrane of A. baumannii. The N-terminal domain of HMP-AB spans the outer membrane, while the C-terminal end appears to attach to peptidoglycan.[124,125] The HMP-AB and OmpA porins are referred to as 'slow porins' since small molecules, such as antibiotics, are poorly permeable across the outer membrane, thereby serving to increase the intrinsic resistance of A. baumannii to antibiotics.

The second most characterized putative porin in acinetobacters is the 29-kDa CarO protein.[124] The exact porin properties of CarO are unclear and, in at least one study, evidence was presented that CarO was associated with a second 25-kDa protein, designated Omp25.[126] Both proteins were cloned and overexpressed in E. coli and the components incorporated into artificial lipid bilayers. These studies suggested that only the CarO monomeric protein formed a porin channel and that it demonstrated selectivity for cationic molecules.[126] Given that CarO and Omp25 are associated in the outer membrane of A. baumannii and are difficult to resolve by polyacrylamide gel electrophoresis methodology, care should be taken when trying to make correlations between the absence of CarO protein in outer membrane preparations as a causative factor in antibiotic resistance. Data from the same study using isolated CarO failed to demonstrate binding of imipenem, questioning whether or not this protein, free or associated with Omp25, actually functions as a porin for this carbapenem.[126] This interpretation is in conflict with another study that found a loss of the 29-kDa CarO in outer membranes isolated from imipenem- and meropenem-resistant A. baumannii.[127] Many of the resistant isolates examined in this study had a 975-bp insertion within the 3'-end of the carO gene. This fragment possessed several sequence characteristics of an IS element, including a 7-bp duplication (TAACGAT) at the site of insertion, bound by a perfect 17-bp inverted repeat.[127] This IS, designated ISAba825, was present in two to three copies in the genome of the carbapenem-resistant A. baumannii. The G plus C content of the IS was lower than that of other genomic regions of these strains, suggesting that it was acquired from another species. The use of such IS fragments to inactivate porin genes is not a new finding and is somewhat a common mechanism by which other MDR species, such as K. pneumoniae, respond to antibiotic selective pressure.[128]

Loss of other putative porins of A. baumannii have been implicated in resistance to carbapenems, either alone or in combination with efflux systems or carbapenemase genes, such as blaOXA.[95,129,130]

Efflux of structurally unrelated classes of antibiotics from MDR, Gram-negative bacilli is now established as an important mechanism of resistance. The intrinsic efflux systems identified to date in A. baumannii are summarized in Table 1 . The most extensively studied of these is the AdeABC efflux operon that is under regulation by a two-component signal transduction system.[124,131,132,133] This pump belongs to the family of resistance-nodulation-division (RND) pumps, which utilize the energy derived from proton motive force. The AdeABC efflux pump has been implicated in the enhanced resistance to aminoglycosides, ß-lactams, chloramphenicol, tetracyclines and fluoroquinolones.[124] Regulation of the pump may be influenced by the external environment of the cell, since the adeS (sensor) and adeR (regulator) genes are located immediately upstream of the three-pump structural genes adeA, adeB and adeC. The regulatory genes are transcribed in the opposite direction from the pump-protein genes, which together form an operon.[131,132] In this system, AdeS serves as a cell surface-sensor for histidine kinase, which phosphorylates the AdeR-response-regulator protein. Insertional mutagenesis was used to sequentially inactivate these genes in order to further define their roles in pump expression and function. Structural studies also indicated that AdeA functions as a cytoplasmic membrane protein, while AdeB functions as the RND pump protein that accomplishes the efflux of substrate.[132] The third pump protein, AdeC, is homologous to the OprM outer membrane pore protein, which, in this system, is nonessential, since other outer membrane-associated proteins can compensate for its loss. The adeC gene is independently regulated by a 5' upstream hairpin-loop structure that can minimize expression of the adeC gene independently from the adeA-adeB transcript. Insertional inactivation of the AdeB pump protein (in A. baumannii BM4542) lowers MICs to gentamicin and kanamycin 48- and eightfold, respectively.[132] Similar studies suggest that loss of AdeB lowers MICs at least twofold for aminoglycosides, ceftazidime, tetracycline and fluoroquinolones.[131] Spontaneous mutations identified in the kinase region of adeS, or in the adeR gene, confer constitutive expression of the pump, usually resulting in higher levels of resistance to several antibiotics.

Recent studies indicate that the AdeABC efflux pump is implicated in decreased susceptibility to the new antimicrobial, tigecycline. Constitutive expression of adeABC, achieved through insertion of an ISAba1 element into the adeS sensor gene, raised tigecycline MICs to 4 µg/ml in an A. baumannii clinical isolate.[134] Another study examining tigecycline resistance in two clinical isolates of A. baumannii using real-time PCR indicated that resistant strains had a 40- to 54-fold increase in adeB mRNA compared with tigecycline-susceptible A. baumannii strains.[135] The investigators also found that serial transfer of susceptible strains (MICs of 2 µg/ml) to increasing concentrations of tigecycline, resulted in elevated MICs (up to 24 µg/ml). This increase in MIC was accompanied by similar increases in the MICs for minocycline, aminoglycosides and chloramphenicol.[135] The mutations in the adeABC pump responsible for tigecycline resistance were not able to be determined in this study. Interestingly, there have been several studies where clinical resistance has developed rapidly in patients receiving tigecycline, leading to post-therapy isolates with MICs of 4-16 µg/ml.[136,137] A recent study from Israel also illustrates the existence of highly tigecycline-resistant (MIC90 of 32 µg/ml) MDR A. baumannii, respresenting 19 different pulsotypes.[138] Such studies illustrate the clinical relevance of the AdeABC efflux pump in conferring antibiotic resistance in this species to new antimicrobial agents that initially appear to have promise against this organism.

The Tet(A), Tet(B) and AbeM efflux pumps ( Table 1 ) have also been detected in A. baumannii.[124] The Tet(A) pump confers resistance to tetracycline, while Tet(B) confers resistance to tetracycline and minocycline, although neither confers resistance to the new glycylcyclines.[124] These tet genes are often encoded on mobile transposons and integrons, explaining their extensive dissemination in clinical isolates of this species.[139] The AbeM pump belongs to the MATE family of transporters and its presence has been implicated in raising MICs to norfloxacin, ofloxacin, ciprofloxacin and gentamicin, although its role in conferring multidrug resistance has not been widely studied.[124]

As with the Mex efflux pumps found in P. aerguinosa that play an important role in conferring antibiotic resistance, it is clear that the intrinsic efflux pumps found in A. baumannii also play an important and often overlooked role in antibiotic resistance, particularly in the presence of other intrinsic or acquired resistance mechanisms.

Resistance to fluoroquinolones in acinetobacters most commonly results from sequencial accumulation of point mutations in the topoisomerase gyrA and parC genes.[81] As in the case of other antibiotics, porin deletions and increased efflux can also contribute to resistance.[8] A typical surveillance study examining quinolone-resistant outbreaks and sporadic strains of A. baumannii collected in Europe and the USA were examined by pulsed-field gel electrophoresis and by analysis of their gyrA and parC gene sequences.[140] The MIC90s of the collection against ciprofloxacin, levofloxacin and moxifloxacin were 64, 8 and 16 µg/ml, respectively. Mutations in the quinolone resistance-determining region of gyrA were detected in 33% of the strains and double mutations in both gyrA and parC genes were detected in 17% of these isolates.[140] Mutations were identified more frequently in A. baumannii outbreak isolates than in randomly collected, sporadic-resistant isolates. Substitutions of Ser-83 in GyrA were sufficient to confer ciprofloxacin MICs of at least 4 µg/ml. Double mutants with substitutions in ParC at Ser-80 to Leu or Phe, or in Gly-84 to Lys, had elevated ciprofloxacin MICs of at least 32 µg/ml. While other mutations have been described in gyrA of A. baumannii, the commonly encountered sequential mutations in gyrA and parC correlated best with high-level resistance to all available fluoroquinolones, as occurs in many other species of Gram-negative bacilli.

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