Pittii Isolates Research Articles

Substitutions of Ser83Leu in GyrA and Ser80Leu in ParC Associated with Quinolone Resistance in Acinetobacter pittii.

To investigate the prevalence and the mechanism of quinolone-resistant Acinetobacter pittii, 634 Acinetobacter calcoaceticus-Acinetobacter baumannii complex isolates were collected throughout Zhejiang Province. Identification of isolates was conducted by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS), blaOXA-51-like gene, and partial RNA polymerase β-subunit (rpoB) amplification. Twenty-seven isolates of A. pittii were identified. Among the 634 isolates, A. baumannii, A. pittii, Acinetobacter nosocomialis, and A. calcoaceticus counted for 87.22%, 4.26%, 8.20%, and 0.32%, respectively. Antimicrobial susceptibility of nalidixic acid, ofloxacin, enoxacin, ciprofloxacin, lomefloxacin, levofloxacin, sparfloxacin, moxifloxacin, and gatifloxacin for 27 A. pittii were determined by the agar dilution method. Detection of quinolone-resistant determining regions of gyrA, gyrB, parC, and parE was performed for the A. pittii isolates. In addition, plasmid-mediated quinolone resistance (PMQR) determinants (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, qepA, oqxA, and oqxB) were investigated. All the 27 isolates demonstrated a higher minimum inhibitory concentration (MIC) to old quinolones than the new fluoroquinolones. No mutation in gyrA, gyrB, parC, or parE was detected in 20 ciprofloxacin-susceptible isolates. Seven ciprofloxacin-resistant A. pittii were identified with a Ser83Leu mutation in GyrA. Among them, six isolates with simultaneous Ser83Leu amino acid substitution in GyrA and Ser80Leu in ParC displayed higher MIC values against ciprofloxacin. Additionally, three were identified with a Met370Ile substitution in ParE, and two were detected with a Tyr317His mutation in ParE, which were reported for the first time. No PMQR determinants were identified in the 27 A. pittii isolates. In conclusion, mutations in chromosome play a major role in quinolone resistance in A. pittii, while resistance mechanisms mediated by plasmid have not been found. Ser83Leu substitution in GyrA and Ser80Leu substitution in ParC are associated with quinolone resistance in A. pittii. Whether Met370Ile and Tyr317His substitutions in ParE play a minor role requires further investigation.

Evaluation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based VITEK MS System for the Identification of Acinetobacter Species from Blood Cultures: Comparison with VITEK 2 and MicroScan Systems

BackgroundAcinetobacter species are the leading cause of bloodstream infection (BSI), but their correct identification is challenging. We evaluated the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based VITEK MS (bioMérieux, France), and two automated systems, VITEK 2 (bioMérieux) and MicroScan (Siemens, USA) for identification of Acinetobacter BSI isolates.MethodsA total of 187 BSI isolates recovered at a university hospital in Korea between 2010 and 2012 were analyzed. The identification results obtained using VITEK MS and two automated systems were compared with those of rpoB sequencing.ResultsOf 187 isolates analyzed, 176 were identified to the species level by rpoB sequencing: the Acinetobacter baumannii group (ABG; 101 A. baumannii, 43 A. nosocomialis, 10 A. pittii isolates) was most commonly identified (82.4%), followed by Acinetobacter genomic species 13BJ/14TU (5.3%), A. ursingii (2.1%), A. soli (2.1%), A. bereziniae (1.1%), and A. junii (1.1%). Correct identification rates to the species group (ABG) level or the species level was comparable among the three systems (VITEK MS, 90.3%; VITEK 2, 89.2%; MicroScan, 86.9%). However, VITEK MS generated fewer misidentifications (0.6%) than VITEK 2 (10.8%) and MicroScan (13.1%) (P<0.001). In addition, VITEK MS demonstrated higher specificity (100%) for discrimination between ABG and non-ABG isolates than the other systems (both, 31.8%) (P<0.001).ConclusionsThe VITEK MS system is superior to the VITEK 2 and MicroScan systems for identification of Acinetobacter BSI isolates, with fewer misidentifications and better discrimination between the ABG and non-ABG isolates.

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.

Characterization of a Novel Plasmid Type and Various Genetic Contexts of blaOXA-58 in Acinetobacter spp. from Multiple Cities in China

Background/ObjectiveSeveral studies have described the epidemiological distribution of bla OXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of bla OXA-58-carrying plasmids and the genetic context surrounding bla OXA-58 in Acinetobacter spp. in China.Methodology/Principal FindingsTwelve non-duplicated bla OXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of bla OXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of bla OXA-58-harboring plasmids. The genetic structure surrounding bla OXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype), three Acinetobacter nosocomialis isolates (belong to two pulsotypes) and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types). A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both bla OXA-58 and bla OXA-23. bla OXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in bla OXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of bla OXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15)-ΔISAba3-like element-bla OXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element-bla OXA-58 were carbapenem susceptible.ConclusionThe study revealed the unique features of bla OXA-58-carrying plasmids in Acinetobacter spp. in China, which were different from that of Acinetobacter spp. found in European countries. The diversity of the genetic contexts of bla OXA-58 contributed to various antibiotics resistance profiles.

Prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China

In order to assess the prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China, we conducted a polymerase chain reaction (PCR)-based surveillance of OXA-type β-lactamase gene clusters for a total of 2,880 Acinetobacter spp. isolates collected from 23 Chinese provinces. All isolates were tested for susceptibility to 12 antimicrobial agents and showed high rates of resistance to all these agents except minocycline. We also found that the vast majority of carbapenem-resistant Acinetobacter spp. were OXA-23-like-producing isolates, predominantly Acinetobacter baumannii isolates. Besides, bla OXA-58-like and bla OXA-24-like genes were detected in 32 and 11 isolates, respectively, involving many provinces throughout China. Furthermore, these two carbapenem-resistance determinants were located on transferable plasmids in most cases, indicating an emerging threat for both OXA-58-like- and OXA-24-like-producing Acinetobacter spp. isolates in China. Interestingly, a novel homologue of the bla OXA-143 gene was identified in a susceptible Acinetobacter pittii isolate. Overall, these observations suggest that the bla OXA-23-harboring A. baumannii isolates are the most frequent carbapenem-resistant Acinetobacter spp. in China, and the bla OXA-24-like and bla OXA-58-like genes have emerged as potential threats of hospital outbreaks of multidrug-resistant Acinetobacter spp.

Regional dissemination of Acinetobacter species harbouring metallo-β-lactamase genes in Japan

Metallo-β-lactamase (MBL) producers have been reported among the various Acinetobacter species worldwide. In this study, the epidemiology and molecular characteristics of carbapenemase-encoding genes and mobile elements were studied to analyse the regional dissemination of MBL genes in Acinetobacter species. From January 2001 to December 2006, 48 Acinetobacter isolates harbouring MBL genes identified from five hospitals in Kyoto and Shiga Prefecture, Japan were collected and analysed. The partial rpoB gene or the 16S-23S ribosomal RNA intergenic spacer region was sequenced to obtain a species-level identification. Molecular typing using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was performed. Twenty-five Acinetobacter pittii isolates were divided into eight PFGE types and five sequence types (STs) using MLST. Nine Acinetobacter bereziniae isolates belonged to five PFGE types. Five Acinetobacter nosocomialis isolates were divided into two PFGE types and two STs. Three unclassified Acinetobacter species isolates were divided into two PFGE types. Eighteen of the 25 A. pittii isolates belonged to ST119 and were identified from four hospitals. The blaIMP-19 gene was detected in 41 of 48 isolates, including all of the A. pittii ST119 isolates. The blaIMP-1 and blaIMP-11 genes were detected in four and three isolates, respectively. The MBL genes were all embedded within a class 1 integron as a gene cassette array: blaIMP-19-aac(6′)-31-blaOXA-21-aadA1, catB8-like/aacA4-blaIMP-1 and blaIMP-11. This study is the first report demonstrating the regional dissemination of MBL-producing Acinetobacter species. A. pittii ST119 harbouring blaIMP-19 was widely spread throughout the Kyoto-Shiga region.

Dissemination and characterization of NDM-1-producing Acinetobacter pittii in an intensive care unit in China

The sporadic emergence of New Delhi metallo-β -lactamase-1 (NDM-1)-producing Acinetobacter spp. has been reported in China; however, NDM-1-positive bacteria epidemics are rarely reported in intensive care units (ICUs) in China, or even in the world. During 15 months' surveillance Acinetobacter spp. isolated from patients, heathcare workers and surfaces of a Chinese ICU were screened for the blaNDM-1 gene. A total of 27 of 3114 Acinetobacter spp. strains were NDM-1 positive and identified as A. pittii with sequence type 63 (ST63) by multilocus sequence typing. Of the 27 NDM-1-positive A. pittii strains, 22 were isolated from the ICU surfaces and grouped into a major clone A using pulsed-field gel electrophoresis typing, while the other five strains isolated from the patients were classified into three clones (A, B and C). The blaNDM-1 gene was located on a 45-kb plasmid for all three A. pittii clones. The plasmid could be transferred to A. pittii and A. baumannii recipients at both 30 and 37°C but not to Escherichia coli J53. The plasmid could not be classified into any of the known plasmid incompatibility groups. The blaNDM-1 region in the plasmid was flanked by two insertion sequence elements, ISAba125 and ISAba11, and no other carbapenemase gene was present in this NDM-1-positive A. pittii isolate. Thus, we present the first report on the transmission and characterization of NDM-1-producing A. pittii in an ICU in China as well as a novel blaNDM-1 gene-bearing plasmid.

Diversity of Acinetobacter baumannii in Four French Military Hospitals, as Assessed by Multiple Locus Variable Number of Tandem Repeats Analysis

BackgroundInfections by A. calcoaceticus-A. baumannii (ACB) complex isolates represent a serious threat for wounded and burn patients. Three international multidrug-resistant (MDR) clones (EU clone I-III) are responsible for a large proportion of nosocomial infections with A. baumannii but other emerging strains with high epidemic potential also occur.Methodology/Principal FindingsWe automatized a Multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) protocol and used it to investigate the genetic diversity of 136 ACB isolates from four military hospitals and one childrens hospital. Acinetobacter sp other than baumannii isolates represented 22.6% (31/137) with a majority being A. pittii. The genotyping protocol designed for A.baumannii was also efficient to cluster A. pittii isolates. Fifty-five percent of A. baumannii isolates belonged to the two international clones I and II, and we identified new clones which members were found in the different hospitals. Analysis of two CRISPR-cas systems helped define two clonal complexes and provided phylogenetic information to help trace back their emergence.Conclusions/SignificanceThe increasing occurrence of A. baumannii infections in the hospital calls for measures to rapidly characterize the isolates and identify emerging clones. The automatized MLVA protocol can be the instrument for such surveys. In addition, the investigation of CRISPR/cas systems may give important keys to understand the evolution of some highly successful clonal complexes.

Characteristics of carbapenem-resistant Acinetobacter spp. other than Acinetobacter baumannii in South Korea

Although many studies have been performed on carbapenem-resistant Acinetobacter baumannii, only a few studies have addressed carbapenem resistance in Acinetobacter spp. other than A. baumannii (non- baumannii Acinetobacter). Amongst 287 Acinetobacter spp. isolates from patients with bacteraemia in a South Korean hospital collected between 2003 and 2010, 160 (55.7%) were non- baumannii Acinetobacter spp. Antimicrobial susceptibility testing was performed and the effect of efflux pump inhibitors was examined. Antimicrobial resistance genes were identified and pulsed-field gel electrophoresis (PFGE) analysis was performed. OprD expression was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), and CarO disruption was investigated by PCR. Seventeen non- baumannii Acinetobacter isolates (10.6%) were resistant to imipenem or meropenem, comprising eight Acinetobacter pittii (or Acinetobacter genomospecies 3), four Acinetobacter nosocomialis (or Acinetobacter genomospecies 13TU), two Acinetobacter genomospecies ‘close to 13TU’, two Acinetobacter bereziniae (or Acinetobacter genomospecies 10) and one Acinetobacter genomospecies 16. bla IMP-1 genes were detected in seven and two carbapenem-resistant A. pittii and A. bereziniae isolates, respectively. PFGE showed that A. pittii isolates carrying bla IMP-1 belonged to the same clone. In addition, bla SIM-1 and bla PER-1 genes were simultaneously identified in two A. nosocomialis isolates. In four isolates (one each of A. pittii, A. nosocomialis, Acinetobacter genomospecies ‘close to 13TU’ and Acinetobacter genomospecies 16), efflux pumps were implicated in the increase in carbapenem minimum inhibitory concentrations. No decreased expression of OprD was identified in any carbapenem-resistant non- baumannii Acinetobacter isolates, and disruption of carO was also not detected. Clonal spread of carbapenem-resistant A. pittii carrying bla IMP-1, which contributes to a high resistance rate in this species, was identified. The bla IMP-1 and bla SIM-1 genes were first identified in A. bereziniae and A. nosocomialis, respectively. Since no carbapenem resistance mechanisms could be identified, further efforts to find the resistance mechanism should be made.