Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii.

Objectives: To investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain. Methods: A. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT-PCR. Results: In isolate pairs, disruption of adeS or adeN by ISs increased adeB or adeJ expression and conferred increased resistance to at least three antimicrobial classes, respectively. The insertion of ISAba1 in adeN was observed in more than 30% of tested isolates and was the most prevalent IS. Furthermore, the insertion of ISAba125 and ISAba27 into adeN was observed for the first time in A. baumannii isolates. Besides ISs, several different mutations were observed in adeN (e.g. deletions and premature stop codons), all of which led to increased tigecycline MICs. Moreover, several amino acid substitutions were detected in AdeRS, AdeN and AdeL. Of note, the substitutions D21V, G25S and D26N in AdeR were found in multiple sequences and suggest a mutational hotspot. Conclusions: This study provides an insight into the different mechanisms associated with tigecycline resistance using a genomic approach and points out the importance of considering adeRS and adeN as markers for tigecycline-resistant A. baumannii isolates.

Effects of Saline, an Ambient Acidic Environment, and Sodium Salicylate on OXA-Mediated Carbapenem Resistance in Acinetobacter baumannii.

Different physiological conditions, such as NaCl, low pH, and sodium salicylate, have been shown to affect antibiotic resistance determinants in Acinetobacter baumannii isolates. Therefore, the aim of this study was to investigate the effects of NaCl, sodium salicylate, and low pH on the susceptibility of A. baumannii to carbapenem. We cloned genes encoding oxacillinases (OXA) of different subclasses, with their associated promoters, from carbapenem-resistant A. baumannii isolates into the same vector and transferred them to the A. baumannii reference strains ATCC 19606 and ATCC 17978. Carbapenem MICs were determined at least in triplicate by agar dilution under standard conditions, as well as in the presence of 200 mM NaCl or 16 mM sodium salicylate, or at pH 5.8. OXA-58-like gene expression was determined by reverse transcription-quantitative PCR (qRT-PCR). Under some experimental conditions, significant MIC reductions were shown for some transformants but not for others. Only in one instance were all transformants harboring the same OXA affected by the same condition: at pH 5.8, the imipenem and meropenem MICs for strains expressing OXA-58-like enzymes decreased from a resistant level (32 to 64 mg/liter) to an intermediate-susceptible level (8 mg/liter). However, blaOXA-58-like gene expression remained the same. MICs for both wild-type reference strains were not affected by the conditions tested. Our results indicate that the effects of the experimental conditions tested on OXA in vivo are mostly strain dependent. MICs were not reduced to wild-type levels, suggesting that the conditions tested do not lead to complete OXA inhibition in the bacterial cell.

Characterization of blaOXA-143 variants in Acinetobacter baumannii and Acinetobacter pittii.

The acquired carbapenem-hydrolyzing oxacillinase (OXA) OXA-143 has thus far been detected only in Acinetobacter baumannii isolates from Brazil. The aim of this study was to characterize three OXA-143 variants: OXA-231 and OXA-253 from carbapenem-resistant A. baumannii isolates and OXA-255 in a carbapenem-susceptible Acinetobacter pittii isolate originating from Brazil, Honduras, and the United States, respectively. The 5' rapid amplification of cDNA ends (RACE) technique identified the same transcription initiation site for all blaOXA-143-like genes and revealed differences in the putative promoter regions. However, all cloned OXA-143 variants conferred carbapenem resistance on A. baumannii ATCC 17978 and OXA-255 conferred carbapenem resistance on A. pittii SH024, which was correlated with blaOXA-255 gene expression. This is the first description of OXA-143-like outside A. baumannii. Detection of OXA-143-like in the United States and Honduras indicates its dissemination through the American continent.

OXA-235, a novel class D ß-lactamase involved in resistance to carbapenems in Acinetobacter baumannii.

We investigated the mechanism of carbapenem resistance in 10 Acinetobacter baumannii strains isolated from the United States and Mexico between 2005 and 2009. The detection of known metallo-ß-lactamase or carbapenem-hydrolyzing oxacillinase (OXA) genes by PCR was negative. The presence of plasmid-encoded carbapenem resistance genes was investigated by transformation of A. baumannii ATCC 17978. Shotgun cloning experiments and sequencing were performed, followed by the expression of a novel ß-lactamase in A. baumannii. Three novel OXA enzymes were identified, OXA-235 in 8 isolates and the amino acid variants OXA-236 (Glu173-Val) and OXA-237 (Asp208-Gly) in 1 isolate each. The deduced amino acid sequences shared 85% identity with OXA-134, 54% to 57% identities with the acquired OXA-23, OXA-24, OXA-58, and OXA-143, and 56% identity with the intrinsic OXA-51 and, thus, represent a novel subclass of OXA. The expression of OXA-235 in A. baumannii led to reduced carbapenem susceptibility, while cephalosporin MICs were unaffected. Genetic analysis revealed that blaOXA-235, blaOXA-236, and blaOXA-237 were bracketed between two ISAba1 insertion sequences. In addition, the presence of these acquired ß-lactamase genes might result from a transposition-mediated mechanism. This highlights the propensity of A. baumannii to acquire multiple carbapenem resistance determinants.

Conversion of OXA-66 into OXA-82 in clinical Acinetobacter baumannii isolates and association with altered carbapenem susceptibility.

OBJECTIVES: Three clinical Acinetobacter baumannii isolates (A-C) were isolated from three separate patients during an outbreak in a hospital in Krakow, Poland. Isolate A was recovered first and was susceptible to carbapenems, whereas isolates B and C were resistant. The aim of this study was to investigate the differences in carbapenem susceptibility in these outbreak-related isolates. METHODS: Clonal relatedness was determined using rep-PCR-based DiversiLab. The bla(OXA-51-like) genes and their upstream regions were sequenced. Expression of the genes encoding OXA-51-like and the three major porins CarO, OprD-like and 33-36 kDa Omp were investigated by semi-quantitative RT-PCR. Comparison of outer membrane protein (OMP) profiles was performed using SDS-PAGE. ISAba1-bla(OXA-82) was cloned into the shuttle vector pWH1266 and transferred into A. baumannii ATCC 17978. RESULTS: The isolates were identical by rep-PCR and clustered with international clonal lineage 2. Sequencing of bla(OXA-51-like) revealed a conversion of OXA-66 (isolate A) into OXA-82 (isolates B and C). bla(OXA-82) was also associated with ISAba1. Expression analysis revealed overexpression of bla(OXA-82). There was no difference in OMP expression between the isolates. ISAba1-bla(OXA-82) conferred carbapenem resistance in ATCC 17978. CONCLUSIONS: Carbapenem resistance in outbreak-related isolates was mediated by conversion of OXA-66 into OXA-82 and its subsequent overexpression. This further highlights the genome plasticity of A. baumannii, leading to carbapenem resistance.

Detection of intrinsic blaOXA-51-like by multiplex PCR on its own is not reliable for the identification of Acinetobacter baumannii.

Three clinical A. baumannii isolates Ab-508, Ab-511, and Ab-653 were recovered from South Africa, South Korea, and Turkey, respectively. Multiplex PCR to detect OXA-type carbapenemases showed atypical blaOXA-51-like amplification products. The aim of this study was to investigate the background of changes in blaOXA-51-like PCR products. Isolates were confirmed as A. baumannii using gyrB multiplex and rpoB sequencing and were epidemiologically unrelated by rep-PCR-based DiversiLab. Sequencing of blaOXA-51-like revealed an insertion of ISAba15 in blaOXA-66 (isolate Ab-511) and an insertion of the novel ISAba19 in blaOXA-78 (isolates Ab-508 and Ab-653). Detection of the intrinsic blaOXA-51-like by OXA-multiplex PCR should not be considered a fully reliable method for identification of A. baumannii when used without an additional independent method. Other species identification methods such as gyrB multiplex PCR and rpoB sequencing should be used to reliably identify A. baumannii.

Association between ß-lactamase-encoding bla(OXA-51) variants and DiversiLab rep-PCR-based typing of Acinetobacter baumannii isolates.

This study investigated the correlation between bla(OXA-51) variants and Acinetobacter baumannii worldwide clonal lineages 1 to 8 (WW1 to -8). The bla(OXA-51-like) genes of 102 A. baumannii isolates were sequenced. Using DiversiLab repetitive-sequence-based PCR (rep-PCR) typing, 92 of these isolates had previously been assigned to WW1 to -8 and 10 were unclustered. Clustering of DNA sequences was performed using the neighbor-joining method and the Jukes-Cantor phylogenetic correction. bla(OXA-51) variants were in good correlation with DiversiLab-defined clonal lineages. Sequence-based typing of bla(OXA-51) variants has the potential to be applied for epidemiologic characterization of A. baumannii and to identify worldwide clonal lineages 1 to 8.

Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007.

OBJECTIVES: To investigate the genetic environment of the metallo-ß-lactamase gene bla(NDM-1) in an Acinetobacter baumannii isolated in 2007 in a German hospital. METHODS: Antimicrobial susceptibility testing was performed and resistance genes were characterized by PCR amplification and sequencing. Transferability of ß-lactam resistance was tested by broth mating assays and transformation of plasmids. The genetic background of bla(NDM-1) was analysed by primer walking. Typing of the A. baumannii strain was performed by repetitive extragenic palindromic sequence-based PCR (rep-PCR) using the DiversiLab system. RESULTS: The multidrug-resistant A. baumannii isolate harboured ß-lactamase genes bla(NDM-1) and intrinsic bla(OXA-64), but without the insertion sequence ISAba1 often located upstream. Transfer of carbapenem resistance by conjugation and transformation failed. Hybridization of isolated plasmid DNA with bla(NDM) probes was not successful. Shotgun cloning of whole genomic DNA and sequence analyses revealed that bla(NDM-1) was located between two insertion elements of ISAba125. Furthermore, this bla(NDM-1)-containing transposon structure was integrated into a chromosomal gene encoding a putative A. baumannii major facilitator superfamily (MFS) metabolite/H+ symporter. CONCLUSIONS: The metallo-ß-lactamase gene bla(NDM-1) in this A. baumannii strain was integrated in the chromosome on a new transposon structure composed of two copies of insertion sequence ISAba125. The variability of the genetic environment of bla(NDM-1) likely facilitates the rapid dissemination of this gene within many Gram-negative bacterial species.

Development of a rapid diagnostic assay based on magnetic bead purification of OXA-ß-lactamase mRNA.

AIM: To countermeasure the global spread of ß-lactamases, we developed a rapid molecular test for the highly variable OXA-ß-lactamases that allows minimizing the time to effective treatment. METHODS: OXA-mRNA was specifically enriched from total RNA using group-specific biotinylated DNA probes and streptavidin-coated magnetic beads. Phylogenetic OXA groups were distinguished by PCR product size. RESULTS: This mRNA fishing method is highly sensitive, yielding specific results from as little as 1 ng total RNA. It enables discrimination of OXA-extended substrate spectrum ß-lactamases and carbapenemases and the semi-quantitative detection of highly expressed ISAba1-controlled variants. CONCLUSION: Targeting mRNA with specific probes on magnetic beads will allow for adaptation to automated systems, such as point-of-care diagnostics.

Worldwide dissemination of acquired carbapenem-hydrolysing class D ß-lactamases in Acinetobacter spp. other than Acinetobacter baumannii.

The aim of this study was to identify acquired OXA-type carbapenemases in Acinetobacter spp. other than Acinetobacter baumannii. From a total of 453 carbapenem-susceptible and -resistant Acinetobacter isolates collected worldwide, 23 were positive for blaOXA genes by multiplex PCR. These isolates were identified as Acinetobacter pittii (n=18), Acinetobacter nosocomialis (n=2), Acinetobacter junii (n=1) and Acinetobacter genomic species 14TU/13BJ (n=2). The blaOXA genes and associated insertion sequence (IS) elements were sequenced by primer walking. In 11 of these isolates, sequencing of the PCR products revealed that they were false-positive for blaOXA. The remaining 12 isolates, originating from Europe, Asia, South America, North America and South Africa, harboured OXA-23 (n=4), OXA-58 (n=5), OXA-40-like (n=1) and OXA-143-like (n=1); one A. pittii isolate harboured both OXA-23 and OXA-58. IS elements were associated with blaOXA in 10 isolates. OXA multiplex PCR showed a high degree of false-positive results (47.8%), indicating that detection of blaOXA in non-baumanniiAcinetobacter spp. should be confirmed using additional methods.