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

Intrinsic ß-lactamases Found in

Clinical isolates of A. baumannii may encode a confusing array of ß-lactamases. Some of the genes encoding these are intrinsic to essentially all isolates of this species and are encoded on the chromosome, while the majority of the genes are acquired from other species or other Acinetobacter isolates, with the determinants encoded on mobile genetic elements. The literature pertaining to ß-lactamases in this organism is somewhat complicated by the lack of definitive molecular identification of the corresponding genes provided in some studies, the lack of organism speciation provided in other reports and the complicated ß-lactam resistance phenotype that occurs in isolates possessing multiple resistance determinants. An excellent review on this topic provides much-needed clarity for characterizing the many acquired ß-lactamase genes now found in this species.[69]

First, the intrinsic ß-lactamase genes found in A. baumannii will be considered. As illustrated in Table 1 , all isolates of A. baumanii have a chromosomally encoded ampC cephalosporinase gene analogous to the species-specific ampC gene found in Escherichia coli. Consensus reached from many studies indicates that this ampC gene is not inducible with ß-lactams and is normally expressed at low levels.[69,70,71,72,73] In addition, sequencing of the flanking regions around ampC has failed to identify the presence of ampR or ampD re-gulatory regions.[73]

Biochemical studies with the AmpC cephalosporinase purified from A. baumannii RYC 52763/97 measured the hydrolysis of cephaloridine and found that ceftazidime and cefotaxime were hydrolyzed 100-times as well, and that carbapenems were not substrates for this enzyme.[74] When this ampC gene was cloned into a plasmid vector, E. coli TG1 transformants were moderately resistant to cefotaxime and ceftazidime (MICs of 8-32 µg/ml).

More recent DNA sequence analysis and enzymatic characterization of ampC cephalosporinases from various Acinetobacter isolates have led to a more uniform designation of these enzymes, defining variants including ADC-1 to -7 (for Acinetobacter-derived cephalosporinases).[75] While there remains high identity among these variants, some differences in substrate utilization have been observed. The ADC-7 variant from A. baumannii (clone 9), when cloned into pET24a and transformed into E. coli BL21, resembled the cephalosporinase characterized in earlier studies in that it had a basic pI of 9.2 and utilized cephaloridine and cefoxitin as substrates but, unlike the enzyme characterized by Bou and Matine-Beltran,[74] it failed to hydrolyze cefotaxime or ceftazidime.[75] The Km of this cephalosporinase for ceftazidime (11.6 µM) was lower than that for the hydrolyzed substrate cephaloridine (45 µM), indicating that substrate affinity was not correlated with the rate of hydrolysis. In fact, relatively low Km values were reported (3.8-38 µM) for imipenem, sulopenem, meropenem and ertapenem, yet none of these agents were hydrolyzed and all were potent inhibitors of the ADC-7 enzyme.[75] Another variant, recently characterized from A. baylyi, ADC-8, shares less than 50% amino acid identity with the other ADC variants, yet it appears to confer a similar cephalosporin resistance phenotype in this species.[76]

In terms of conferring ß-lactam resistance, the kinetic properties of the ADC enzymes represent only part of the story. Acinetobacters have evolved an elegant method to overproduce the ADC cephalosporinases. Some strains of A. baumannii have aquired an insertion sequence (IS) integrated into the 5'-end of the ADC structural gene (ampC), which serves to upregulate the expression of the chromosomally encoded gene. In one study, 42 A. baumannii strains, including strains susceptible (MIC ≤ 8 µg/ml), intermediate (MIC 32 µg/ml) or highly resistant (MIC ≥ 256 µg/ml) to ceftazidime, were examined at the ampC-gene sequence, including regions adjacent to its 5'-end.[77] In all of the nonsusceptible isolates with ceftazidime MICs of at least 32 µg/ml, but in none of the susceptible isolates, a 1180-bp IS was detected immediately upstream of the ampC gene. The IS contained characteristic elements, including a direct repeat of 9 bp in the target sequence and an 11-bp inverted repeat at each end of the inserted fragment. In addition, -35 and -10 (TTGTTC and TATGAT) putative promoter sequences separated by 17 bp were found in the IS element, approximately 100 bp upstream of the ampC start codon.[76] That this upstream region of ampC acts as a strong promoter upregulating the gene was established using reverse transcription-PCR analysis, which demonstrated a marked increase in the amount of ampC mRNA in the ceftazidime-resistant strains compared with the susceptible ones.

Since the initial studies describing the acquisition of this IS element fused to the ADC gene, additional observations have demonstrated that this arrangement leads to the overproduction of cephalosporinase. Cloning and sequencing of this region from the A. baumannii strain, RAN, identified several putative promoters in the IS that could increase ampC expression.[78] The IS, now identified as 'ISAba1-like' once inserted ahead of the structural gene, resulted in a single nucleotide change in the ribosome binding site (TGAG to GGAG) in the blaAMPC gene.[79] Further analysis of the sequence from A. baumannii CLA-1 (GenBank accession number AY758396) indicated that the left inverted repeat sequence of the IS element was 9 bp upstream of the ATG start codon of ampC. Further genetic analysis of this sequence indicated that the change in the ribosome binding region was not responsible for the hypercephalosporinase phenotype in this strain, since constructs containing only this region did not confer the overexpression phenotype.[79] Unfortunately, direct measurements of the relative transcription levels resulting from different regions of the putative promoter sequences were not performed in this study.

Apparently, A. baumannii applies this elegant mechanism of ISAba1 for upregulating other resistance genes, such as the blaOXA-23 and blaOXA-27 carbapenemases and the sulII gene.[80] In studies with A. baumannii RAM, up to 13 copies of this IS element were found in the genome and this, as well as other IS elements, may add significantly to the evolutionary plasticity of the genome of this species. Interestingly, ISAba1 was not detected in the genomes of a limited number of P. aeruginosa and Enterobacteriaceae isolates examined in a parallel fashion.

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