Esther Zander

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

OBJECTIVES To investigate the genetic environment of the metallo-β-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 β-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 β-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-β-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.

OXA-235, a Novel Class D β-Lactamase Involved in Resistance to Carbapenems in Acinetobacter baumannii

ABSTRACT 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-β-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 β-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 β-lactamase genes might result from a transposition-mediated mechanism. This highlights the propensity of A. baumannii to acquire multiple carbapenem resistance determinants.

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

ABSTRACT 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.

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.

Worldwide dissemination of acquired carbapenem-hydrolysing class D β-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.

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.

Characterization of blaOXA-143 Variants in Acinetobacter baumannii and Acinetobacter pittii

ABSTRACT 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.

High incidence of pandrug-resistant Acinetobacter baumannii isolates collected from patients with ventilator-associated pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial

Abstract Objectives To investigate the molecular epidemiology, antimicrobial susceptibility and carbapenem resistance determinants of Acinetobacter baumannii isolates from respiratory tract samples of patients diagnosed with ventilator-associated pneumonia (VAP) who were enrolled in the MagicBullet clinical trial. Methods A. baumannii isolates were prospectively cultured from respiratory tract samples from 65 patients from 15 hospitals in Greece, Italy and Spain. Susceptibility testing was performed by broth microdilution. Carbapenem resistance determinants were identified by PCR and sequencing. Molecular epidemiology was investigated using rep-PCR (DiversiLab) and international clones (IC) were identified using our in-house database. Results Of 65 isolates, all but two isolates (97%) were resistant to imipenem and these were always associated with an acquired carbapenemase, OXA-23 (80%), OXA-40 (4.6%), OXA-58 (1.5%) or OXA-23/58 (1.5%). Resistance to colistin was 47.7%. Twenty-two isolates were XDR, and 20 isolates were pandrug-resistant (PDR). The majority of isolates clustered with IC2 (n = 54) with one major subtype comprising isolates from 12 hospitals in the three countries, which included 19 XDR and 16 PDR isolates. Conclusions Carbapenem resistance rates were very high in A. baumannii recovered from patients with VAP. Almost half of the isolates were colistin resistant, and 42 (64.6%) isolates were XDR or PDR. Rep-PCR confirmed IC2 is the predominant clonal lineage in Europe and suggests the presence of an epidemic XDR/PDR A. baumannii clone that has spread in Greece, Italy and Spain. These data highlight the difficulty in empirical treatment of patients with A. baumannii VAP in centres with a high prevalence of carbapenem-resistant A. baumannii.

Identification of a novel insertion sequence element associated with carbapenem resistance and the development of fluoroquinolone resistance in Acinetobacter radioresistens

Sir, Acinetobacter radioresistens, although sometimes isolated on the skin of healthy humans, rarely causes serious illness. In a recent study, A. radioresistens was isolated from blood and urine cultures, but made up ,1% of the total Acinetobacter spp. isolated, while other studies found a higher incidence of A. radioresistens when only looking at Acinetobacter bloodstream isolates, where bacteraemia is most often associated with indwelling devices. Compelling evidence suggests that the origin of OXA-23, the most commonly acquired oxacillinase in Acinetobacter baumannii, is A. radioresistens, to which this enzyme is intrinsic. In A. baumannii, blaOXA-23 mediates carbapenem resistance when overexpressed and this is caused by the insertion element ISAba1, which is located upstream where it provides a strong promoter; however, this has not been found in A. radioresistens, so the mechanism of blaOXA-23 mobilization is still not fully understood. Despite possessing OXA-23, carbapenem resistance is rarely described in A. radioresistens and has only been reported when the additional acquired carbapenemases IMP-1 and OXA-58 were present. In this study, we investigated carbapenem resistance and the development of fluoroquinolone resistance in two A. radioresistens isolates that were recovered from a patient 17 days apart. The primer sequences used in this investigation are shown in Table S1 (available as Supplementary data at JAC Online). The ISAcra1-blaOXA-23 nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank nucleotide sequence database under accession no. JQ326202. Isolate F1244 was isolated from a blood culture of a patient with a catheter-related bloodstream infection. Ciprofloxacin was used for treatment. Isolate A1474 was recovered 17 days later from a sputum sample. Species identification was confirmed by rpoB sequencing and the isolates were typed by rep-PCR (DiversiLab) as previously described. The isolates showed 98% similarity in their rep-PCR patterns and confirms their clonality. The only difference we found between the isolates was in their fluoroquinolone susceptibility: isolate F1244 was susceptible to ciprofloxacin, levofloxacin and moxifloxacin, whereas isolate A1474 was resistant (Table 1). Carbapenem MICs were investigated by Etest and showed that both isolates were carbapenem resistant (Table 1). The isolates were negative for other blaOXA genes often associated with Acinetobacter and we investigated the expression of the intrinsic blaOXA-23 by qRT–PCR. qRT–PCR was performed three times in triplicate using freshly prepared RNA and cDNA as previously described, with rpoB as a reference gene. This revealed that F1244 and A1474 expressed similar levels of blaOXA-23 and that it was overexpressed .100-fold when compared with the carbapenem-susceptible control strain A. radioresistens SH164 (data not shown). PCR to detect ISAba1 adjacent to blaOXA-23 proved negative. Analysis of the A. radioresistens genome sequence revealed putative O-sialoglycoproteinand ATPase-encoding genes flanking blaOXA-23. The primer pair OXA-23-up/OXA-23-down was designed to amplify and sequence the region between these flanking genes and revealed an 700 bp insertion in the 5′ region of blaOXA-23. Sequencing revealed a novel insertion element in a noncoding region 62 bp upstream of the blaOXA-23 start codon. This insertion element was submitted to the IS Database (http://www-is. biotoul.fr/) and termed ISAcra1. ISAcra1 is a 732 bp element that encodes a predicted 220 amino acid transposase that is flanked by 15 bp inverted repeats and a 7 bp target site duplication. The element belongs to the IS1595 family and the transposase showed 47% amino acid identity to ISFtu3 from Francisella tularensis. The blaOXA-23 gene and putative promoter region was amplified and cloned into shuttle plasmid pWH1266 and transformed into carbapenem-susceptible A. baumannii ATCC 17978 and A. radioresistens SH164, leading to carbapenem resistance (Table 1). We found a single nucleotide difference between the isolates in gyrA, leading to a Ser-83 Phe amino acid substitution in isolate A1474. In A. baumannii, Ser-83 in GyrA is the most commonly altered amino acid associated with low-level fluoroquinolone resistance (typically ciprofloxacin MICs of 4–32 mg/L). We did not detect any difference in parC between the two isolates. We have previously found efflux to be associated with the development of fluoroquinolone resistance. To test for an efflux phenotype, agar dilution MICs were performed with the following commonly effluxed substrates: tetracycline, chloramphenicol, rifampicin, gentamicin, erythromycin and the dye rhodamine 6-G. Agar dilution MICs did not show any difference in MICs between isolates F1244 and A1474 against these substrates, suggesting efflux was not selected (see Table S2 available as Supplementary data at JAC Online). In conclusion, this study demonstrates that carbapenem resistance was mediated through overexpression of the intrinsic blaOXA-23 and was associated with the novel ISAcra1. Fluoroquinolone resistance developed during ciprofloxacin therapy and was associated with a gyrA mutation. These data show for the first time the ability of A. radioresistens to develop fluoroquinolone resistance during therapy. Additionally, ISAcra1

Insertion sequence IS18 mediates overexpression of blaOXA-257 in a carbapenem-resistant Acinetobacter bereziniae isolate

Sir, Acinetobacter bereziniae, previously known as Acinetobacter genomic species 10, has been isolated primarily from clinical specimens and the hospital environment and more rarely from various other sources, including vegetables, soil and animals. Antibiotic resistance is rarely reported in this species. Over the last decade, carbapenem resistance in Acinetobacter spp., mainly Acinetobacter baumannii, has emerged as a threat in hospitals around the world. The most widespread mechanism resulting in carbapenem resistance in Acinetobacter spp. is mediated through carbapenemhydrolysing class D b-lactamases, also known as oxacillinases. The overexpression of blaOXA genes is often associated with insertion sequences (IS) located upstream and providing strong promoters. Carbapenem resistance in A. bereziniae has previously been associated with the metallo-b-lactamases IMP, SIM and VIM or overexpression of OXA-229, a variant of the intrinsic OXA-228-like, which was mediated by a mutated promoter. To date, OXA-228-like has not been associated with an IS. In the present study, we investigated a carbapenem-resistant Acinetobacter strain isolated from the bronchial secretions of a patient in Germany in 2012. Isolate KH243 was initially identified as Acinetobacter guillouiae by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry. However, rpoB sequencing revealed 100% similarity to the A. bereziniae type strain CIP 70.12 (accession no. DQ207475). By Etest, carbapenem MICs were 12 and .32 mg/L for imipenem and meropenem, respectively. Multiplex PCR for OXA subclasses that are associated with carbapenem resistance in Acinetobacter spp. (OXA-51, OXA-23, OXA-40, OXA-58, OXA-143 and OXA-235) was negative. Based on published A. bereziniae sequences, primers were designed to amplify and sequence the intrinsic blaOXA and its surrounding region from isolate KH243 (Table 1). PCR revealed an unexpected large amplicon of 2.1 kb. Sequencing of the purified PCR product by primer walking identified IS18 40 bp upstream of a novel blaOXA-228 variant, which was numbered blaOXA-257 by the Lahey b-lactamase database (http://www.lahey.org/Studies/) and was submitted to GenBank. OXA-257 possessed six amino acid differences compared with OXA-228. The IS18::blaOXA-257 nucleotide sequence reported in this paper has been submitted to EMBL/GenBank under accession number KC567681. The IS18 insertion element encoded a transposase that harboured eight amino acid changes compared with the IS18 sequence available in the IS database (http://www-is.biotoul.fr/). IS18 was flanked by a 3 bp target site duplication (TTT) and 26 bp imperfect inverted repeats. In Acinetobacter spp., IS are often located upstream of blaOXA genes and, by providing strong promoters, lead to overexpression of the OXA, resulting in carbapenem resistance. For example, the intrinsic blaOXA-51-like and the acquired blaOXA-58-like in A. baumannii are often associated with ISAba1 and ISAba3, respectively. IS18 has also been associated with blaOXA-58-like. 8 Other IS elements include ISAcra1, which was recently identified and overexpressed blaOXA-23 in a carbapenemresistant Acinetobacter radioresistens isolate. Two predicted promoters were found upstream of blaOXA-257 with both 235 boxes located within the right inverted repeat of IS18. One was a hybrid promoter based on those previously described in A. bereziniae isolates Nec (blaOXA-229) and Baz (blaOXA-228). 3 The 235 and 210 boxes were identical to the 235 box in Nec and the 210 box in Baz, respectively, and were