Genetic Characterization of Plasmid-Borne blaOXA-58 and blaOXA-72 in Acinetobacter pittii in Shaanxi, China.

BACKGROUND: Acinetobacter pittii has emerged as an opportunistic nosocomial pathogen associated with hospital-acquired infections. The purpose of this study was to investigate the genetic structures of plasmids carrying carbapenemase genes blaOXA-58 and blaOXA-72 in A. pittii strains AR3676 and AR3651 isolated from patients. METHODS: Antimicrobial susceptibility testing was performed using broth microdilution. Whole-genome sequencing and bioinformatics analysis were performed to characterize the genome of A. pittii AR3676 and AR3651. Conjugation experiments were conducted to evaluate plasmids transferability. Phylogenetic and comparative genomic analysis were performed to explore the characteristics of carbapenem-resistant A. pittii isolates worldwide. RESULTS: The AR3676 strain exhibited resistance to imipenem. The 19,700-bp plasmid pAR3676-OXA-58 harbored blaOXA-58 with genetic contexts consisting of a truncated ISAba3-like-blaOXA58-ISAba3. Additionally, the AR3651 strain exhibited resistance to imipenem and meropenem. The genome of AR3651 comprised one 9,837-bp RepA_AB plasmid pAR3651-OXA-72 harboring blaOXA-72. The blaOXA-72 was flanked by XerC/XerD recombination sites. The conjugation of plasmids pAR3676-OXA-58 and pAR3651-OXA-72 from A. pittii to A. baumannii ATCC 17978RIFR failed three independent times. Phylogenetic analysis of A. pittii strains AR3676, AR3651 and other 504 A. pittii strains collected between 1966 and 2022 from various geographic localities, revealed genetic diversity with a heterogeneous distribution of carbapenemase genes. CONCLUSION: A. pittii strains with plasmid carrying blaOXA-58 or blaOXA-72 may serve as an important reservoir of carbepenemase genes. The carbepenemase genes on a single plasmid may facilitate their dissemination and persistence. Additionally, the pdif sites and mobile elements play an important role in the mobilization of resistance genes and plasmid evolution.

Detection of rare carbapenemases in Enterobacterales-comparison of two colorimetric and three CIM-based carbapenemase assays.

Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) is crucial for prompt treatment and infection control. Most assays target the primary four enzymes (KPC, OXA-48-like, VIM, and NDM), often missing less common variants (e.g., GES, IMI, OXA-23, and OXA-58). Therefore, assays based on the hydrolysis of carbapenems are recommended in addition to differentiation tests such as PCR or immunochromatographic assays. The aim of this study was to compare the currently Clinical and Laboratory Standards Institute (CLSI)-recommended tests mCIM (modified carbapenem inactivation method) and Carba NP with new colorimetric tests (NitroSpeed-Carba NP) and novel variations of the carbapenem inactivation method (CIM) such as simplified CIM (sCIM) or modified zinc-supplemented CIM (mzCIM). The challenge collection included 205 clinical isolates, 139 CPE vs 66 non-CPE. Among all 205 isolates, the sensitivity/specificity of mCIM was 81.3%/98.5%, Carba NP 76.3%/100%, NitroSpeed-Carba NP 86.3%/78.8%, sCIM 100%/94%, and mzCIM 97.8%/98.5%. For rare carbapenemases (n = 48), the sensitivity of mzCIM (98.3%) and sCIM (100%) was higher than that of mCIM (60.4%), Carba NP (50%), or NitroSpeed-Carba NP (70.2%). Most indeterminate results occurred for mCIM (14.4%), Carba NP (8.2%), and sCIM (6.3%). The detection of rare carbapenemases remains challenging with the currently recommended assays. The CIM-based tests demonstrated superior sensitivity, with sCIM and mzCIM outperforming the currently recommended mCIM and Carba NP, especially among isolates with weakly hydrolyzing carbapenemases (e.g., OXA-23 and OXA-58). Although colorimetric assays provide more rapid results, laboratories have to be aware of the low sensitivity for rare carbapenemases. Both sCIM and the new mzCIM performed well, are cost-effective, and can easily be implemented in any laboratory.IMPORTANCEDetection of so-called rare carbapenemases (e.g., GES, IMI, OXA-23, and OXA-58) in Enterobacterales is challenging, and data on the performance of currently available assays are scarce. This study systematically assessed the performance of currently recommended and novel hydrolysis-based assays on a set of molecularly characterized isolates. It demonstrates that the currently recommended assays mCIM and Carba NP perform well on isolates producing common carbapenemases such as KPC, VIM, NDM, and OXA-48, but have only a moderate sensitivity in the detection of rare carbapenemases. In contrast, the newer CIM-based variants, sCIM and mzCIM, are equally capable of detecting frequent and uncommon carbapenemases. These assays could potentially help to improve our knowledge on the epidemiology of these "rare" enzymes.

Complete genetic characterization of carbapenem-resistant Acinetobacter johnsonii, co-producing NDM-1, OXA-58, and PER-1 in a patient source.

The emergence of carbapenemase-producing Acinetobacter spp. has been widely reported and become a global threat. However, carbapenem-resistant A. johnsonii strains are relatively rare and without comprehensive genetic structure analysis, especially for isolates collected from human specimen. Here, one A. johnsonii AYTCM strain, co-producing NDM-1, OXA-58, and PER-1 enzymes, was isolated from sputum in China in 2018. Antimicrobial susceptibility testing showed that it was resistant to meropenem, imipenem, ceftazidime, ciprofloxacin, and cefoperazone/sulbactam. Whole-genome sequencing and bioinformatic analysis revealed that it possessed 11 plasmids. bla OXA-58 and bla PER-1 genes were located in the pAYTCM-1 plasmid. Especially, a complex class 1 integron consisted of a 5' conserved segment (5' CS) and 3' CS, which was found to carry sul1, arr-3, qnrVC6, and bla PER-1 cassettes. Moreover, the bla NDM-1 gene was located in 41,087 conjugative plasmids and was quite stable even after 70 passages under antibiotics-free conditions. In addition, six prophage regions were identified. Tracking of closely related plasmids in the public database showed that pAYTCM-1 was similar to pXBB1-9, pOXA23_010062, pOXA58_010030, and pAcsw19-2 plasmids, which were collected from the strains of sewage in China. Concerning the pAYTCM-3 plasmids, results showed that strains were collected from different sources and their hosts were isolated from various countries, such as China, USA, Japan, Brazil, and Mexico, suggesting that a wide spread occurred all over the world. In conclusion, early surveillance is warranted to avoid the extensive spread of this high-risk clone in the healthcare setting.

Proteus mirabilis - analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection.

OBJECTIVES: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays. METHODS: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing. RESULTS: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-ß-lactamase (NDM) [n = 2]; Verona integron-encoded metallo-ß-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17-46%)/89% (CI: 75-97%) for CARBA NP, 74% (CI: 60-85%)/82% (CI: 67-91%) for faropenem, 91% (CI: 78-97%)/82% (CI: 66-92%) for simplified CIM, and 93% (CI: 81-99%)/100% (CI: 91-100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92-100%)/100% (CI: 91-100%) on the 81 isolates, and 100% (CI: 29-100%)/100% (CI: 96-100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France. DISCUSSION: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.

Dynamic state of plasmid genomic architectures resulting from XerC/D-mediated site-specific recombination in Acinetobacter baumannii Rep_3 superfamily resistance plasmids carrying blaOXA-58 - and TnaphA6-resistance modules.

The acquisition of bla OXA genes encoding different carbapenem-hydrolyzing class-D ß-lactamases (CHDL) represents a main determinant of carbapenem resistance in the nosocomial pathogen Acinetobacter baumannii. The blaOXA-58 gene, in particular, is generally embedded in similar resistance modules (RM) carried by plasmids unique to the Acinetobacter genus lacking self-transferability. The ample variations in the immediate genomic contexts in which blaOXA-58 -containing RMs are inserted among these plasmids, and the almost invariable presence at their borders of non-identical 28-bp sequences potentially recognized by the host XerC and XerD tyrosine recombinases (pXerC/D-like sites), suggested an involvement of these sites in the lateral mobilization of the gene structures they encircle. However, whether and how these pXerC/D sites participate in this process is only beginning to be understood. Here, we used a series of experimental approaches to analyze the contribution of pXerC/D-mediated site-specific recombination to the generation of structural diversity between resistance plasmids carrying pXerC/D-bounded bla OXA-58- and TnaphA6-containing RM harbored by two phylogenetically- and epidemiologically-closely related A. baumannii strains of our collection, Ab242 and Ab825, during adaptation to the hospital environment. Our analysis disclosed the existence of different bona fide pairs of recombinationally-active pXerC/D sites in these plasmids, some mediating reversible intramolecular inversions and others reversible plasmid fusions/resolutions. All of the identified recombinationally-active pairs shared identical GGTGTA sequences at the cr spacer separating the XerC- and XerD-binding regions. The fusion of two Ab825 plasmids mediated by a pair of recombinationally-active pXerC/D sites displaying sequence differences at the cr spacer could be inferred on the basis of sequence comparison analysis, but no evidence of reversibility could be obtained in this case. The reversible plasmid genome rearrangements mediated by recombinationally-active pairs of pXerC/D sites reported here probably represents an ancient mechanism of generating structural diversity in the Acinetobacter plasmid pool. This recursive process could facilitate a rapid adaptation of an eventual bacterial host to changing environments, and has certainly contributed to the evolution of Acinetobacter plasmids and the capture and dissemination of bla OXA-58 genes among Acinetobacter and non-Acinetobacter populations co-residing in the hospital niche.

The molecular characterization of colistin-resistant isolates of Acinetobacter baumannii from patients at intensive care units.

Background and Objectives: The objective of this study was to determine molecular characterization and genetic diversity of colistin-resistant A. baumannii clinical isolates in Intensive Care Unit hospitalized patients. Materials and Methods: A total of 127 A. baumannii clinical isolates were evaluated for antimicrobial susceptibility. PCR reaction and sequencing were performed for the detection of mutations in pmrAB and lpx ACD genes. Results: Based on antimicrobial susceptibility testing, 40.94% and 33.85% of the isolates were MDR and XDR respectively whereas 3.93% of them were found to be PDR. Results of agar dilution MIC and E-test indicated that 76% of the isolates were sensitive to colistin. All of the isolates were positive for bla OXA-51 and 50% of them were positive for both bla OXA-23 -like and bla OXA-143 -like genes while only 25% of the isolates were positive for bla OXA-72 . None of them were positive for the bla OXA-58 -like gene. There is no mutation in pmrA. The V162A substitution for pmrB gene was repeated in two isolates, and E394D and Y292H substitutions in lpxA were observed in two isolates; also, C120R and F165L substitutions in lpxC gene was repeated in two isolates. Analysis of phylogenetic tree based on alterations in lpxACD and pmrB genes indicated the appearance of new isolates compared to the reference strain ATCC17978 A. baumannii isolates. Conclusion: The present study indicated the prevalence of MDR and XDR A. baumannii isolates and the emergence of PDR isolates in the northwest portion of Iran. The appearance of colistin-resistant isolates with new mutations in pmrB, lpxACD genes indicates new resistance mechanisms.

Co-infection of ST2IP carbapenem-resistant Acinetobacter baumannii with SARS-CoV-2 in the patients admitted to a Tehran tertiary referral hospital.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is among the most concerning cause of healthcare-associated infections (HAI) due to its high level of antibiotic resistance and high mortality. In the era of the COVID-19 pandemic, the key priority of infection control committees is to contain the dissemination of antibiotic resistant Gram-negative bacteria. Here, we aimed to timely recognize the emergence of CRAB in COVID-19 cases admitted to the wards of a tertiary referral hospital and to identify the genetic relatedness of the isolates. METHODS: From 30 March to 30 May 2020, a total of 242 clinical samples from COVID-19 cases were screened for CRAB isolates using standard microbiologic and antibiotic susceptibility tests. The PCRs targeting oxa23, oxa24, oxa58, blaTEM and blaNDM-1 genes were performed. Two multiplex PCRs for identifying the global clones (GC) of A. baumannii were also performed. The sequence type of CRABs was determined using Institut Pasteur (IP) multilocus sequence typing (MLST) scheme. RESULTS: Eighteen CRAB isolates were recovered from COVID-19 patients with the mean age of 63.94 ± 13.8 years. All but 4 COVID-19 patients co-infected with CRAB were suffering from an underlying disease. Death was recorded as the outcome in ICUs for 9 (50%) COVID-19 patients co-infected with CRAB. The CRAB isolates belong to GC2 and ST2IP and carried the oxa23 carbapenem resistance gene. CONCLUSION: This study demonstrated the co-infection of CRAB isolates and SARS-CoV-2 in the patients admitted to different ICUs at a referral hospital in Tehran. The CRAB isolates were found to belong to ST2IP, share the oxa23 gene and to have caused several outbreaks in the wards admitting COVID-19 patients.

Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases.

Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-ß-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.

Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen that has emerged as a global threat because of high levels of resistance to many antibiotics, particularly those considered to be last-resort antibiotics, such as carbapenems. Although alterations in the efflux pump and outer membrane proteins can cause carbapenem resistance, the main mechanism is the acquisition of carbapenem-hydrolyzing oxacillinase-encoding genes. Of these, oxa23 is by far the most widespread in most countries, while oxa24 and oxa58 appear to be dominant in specific regions. Historically, much of the global spread of carbapenem resistance has been due to the dissemination of two major clones, known as global clones 1 and 2, although new lineages are now common in some parts of the world. The analysis of all publicly available genome sequences performed here indicates that ST2, ST1, ST79 and ST25 account for over 71 % of all genomes sequenced to date, with ST2 by far the most dominant type and oxa23 the most widespread carbapenem resistance determinant globally, regardless of clonal type. Whilst this highlights the global spread of ST1 and ST2, and the dominance of oxa23 in both clones, it could also be a result of preferential selection of carbapenem-resistant strains, which mainly belong to the two major clones. Furthermore, ~70 % of the sequenced strains have been isolated from five countries, namely the USA, PR China, Australia, Thailand and Pakistan, with only a limited number from other countries. These genomes are a vital resource, but it is currently difficult to draw an accurate global picture of this important superbug, highlighting the need for more comprehensive genome sequence data and genomic analysis.

Clonal relatedness and resistance characteristics of OXA-24 and -58 producing carbapenem-resistant Acinetobacter baumannii isolates in Tehran, Iran.

AIMS: The present study was conducted to investigate the mechanism of carbapenem resistance and the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates collected from two nearby hospitals in Tehran, Iran. METHODS AND RESULTS: A total of 180 CRAB isolates were studied. Antimicrobial susceptibility testing was performed using disk diffusion and Epsilometer tests. The detection of OXA-23, -24 and -58 was implemented for all isolates using polymerase chain reaction. Subsequently, isolates harbouring OXA-24 and -58 were investigated for the presence of resistance determinants of Ambler class A, metallo-ß-lactamases (MBLs), and carbapenem-hydrolysing class D ß-lactamases, ISAba1, and the genetic relatedness between them was analysed using pulsed-field gel electrophoresis (PFGE). All isolates were found to be resistant to imipenem with a MIC of ≥8 µg ml-1 and were susceptible to colistin with a MIC of ≤1·5 µg ml-1 . Sixty percent of the isolates had OXA-23. OXA-24 and -58 were detected in 31 of 180 CRAB isolates. These chosen isolates were devoid of MBLs and blaSHV , blaC TX-M , blaVEB ESBL genes. The PER determinant was detected in 38% of isolates as the most common extended spectrum ß-lactamases (ESBLs). Of these isolates, 51·6% had OXA-23, and ISAba1 was found to be upstream of OXA-23 and OXA-51 in 16 and 8 isolates, respectively. The band patterns produced by PFGE showed nine clonal pulsotypes distributed between the two hospitals. CONCLUSION: The findings showed that the refractory CRAB isolates were transmitted intra- and inter-hospital, particularly in the ICU due to shortcomings in infection control surveillance. SIGNIFICANCE AND IMPACT OF THE STUDY: Carbapenem resistance is a substantial threat in the treatment of infections caused by A. baumannii due to limitations in the therapeutic options.

Detection of co-harboring OXA-58 and NDM-1 carbapenemase producing genes resided on a same plasmid from an Acinetobacter pittii clinical isolate in China.

OBJECTIVES: Acinetobacter pittii has become an emerging opportunistic noscomial pathogen worldwide with multi-drug resistance. In the present study, an A. pittii strain was isolated from bronchoalveolar lavage fluid sample harboring both OXA-58 and NDM-1carbapenemase producing genes. The mechanisms of carbapenem resistance of the A. pittii strain was investigated. MATERIALS AND METHODS: Carbapenemase producing genes were examined by PCR and DNA sequencing. S1-PFGE was used to localize carbapenemase encoding genes. Filter mating and electrotransformation were used to investigate the transferability of such carbapenemase encoding genes between different strains. Genetic surroundings of bla OXA-58 and bla NDM-1 genes were detected as well. RESULTS: The A. pittii strain, carrying both OXA-58 and NDM-1 carbapenemase encoding genes, was resistant to all ß-lactam antibiotics, while suscepitible to ciprofloxacin, levofloxacin, tobramycin, cotrimoxazole and tigecycline. Southern blot hybridization for the bla OXA-58 and bla NDM-1 gene indicated that the two genes locate in the same plasmid with molecular weight of 310.1-336.5kb. Bla OXA-58 was located in an ISAba3-bla OXA-58-ISAba3-like structure, and the blaNDM-1 gene cluster was embedded in an ISAba125-aphA6- bla NDM-1 -ble MBL -ΔtrpF-dsbC-cutA structure sequentially. CONCLUSION: In the present study, it is first reported an A. pittii clincal strain in China, co-harboring OXA-58 and NDM-1 carbapenemase producing genes residing on a same plasmid. In hospital and community settings, it is of great significance and urgence to increase the surveillance of these kinds of organisms.

The pollution level of the blaOXA-58 carbapenemase gene in coastal water and its host bacteria characteristics.

This paper investigated 10 carbapenemase genes and selected the hosts of these genes in the estuary of Bohai Bay. The results showed that the OXA-58 producer accounted for a large percentage of carbapenem resistant bacteria in the sampling points, whereas the VIM, KPC, NDM, IMP, GES, OXA-23, OXA-24, OXA-48 and OXA-51 producers were not detected in the study. In addition, 9 bacterial genera with 100% identical blaOXA-58 sequences, including Pseudomonas, Rheinheimera, Stenotrophomonas, Shewanella, Raoultella, Vibrio, Pseudoalteromonas, Algoriphagus, Bowmanella and Thalassospira, were isolated from seawater. It is suggested that the host of blaOXA-58 gene were varied and many kinds of them could survive in the seawater. Moreover, we preformed the quantitative RT-PCR and the result shown the abundance of blaOXA-58 fluctuated between 2.8×10-6 copies/16S and 2.46×10-4 copies/16S, which was of the same order of magnitude as some common antibiotic resistance genes in environment. Furthermore, the variation trend of blaOXA-58 gene suggested that pollution discharge and horizontal gene transfer could contribute to the increase of the gene in coastal area.

Whole-genome sequencing of an NDM-1- and OXA-58-producing Acinetobacter towneri isolate from hospital sewage in Sichuan Province, China.

OBJECTIVES: Acinetobacter spp. isolates carrying the blaNDM-1 gene are frequently reported. However, most reported blaNDM-1 genes are carried by clinical strains. Here we report a carbapenem-resistant Acinetobacter towneri isolate from hospital sewage in China co-harbouring blaNDM-1 and blaOXA-58 in the genome. METHODS: Whole-genome sequencing was performed using a single molecule, real-time (SMRT) sequencing platform with a Pacific Biosciences RS II Sequencer and MiSeq system. Reads were de novo assembled using Celera Assembler v.8.0. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and the genome sequence was analysed by bioinformatics methods. RESULTS: The 2963729-bp genome with a G+C content of 41.30% displayed 11 antimicrobial resistance genes, including blaNDM-1 and blaOXA-58. Meanwhile, 2 plasmids and 19 genomic islands were predicted within the genome. CONCLUSION: The whole-genome sequence reported here can be compared with other genomes of NDM-1-producing Acinetobacter spp. These data could facilitate further understanding of the specific genomic features of carbapenem-resistant Acinetobacter spp. in China.

Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate.

Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired ß-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of blaOXA-58 class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a blaAmpC-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.

CHROMagar Acinetobacter medium for detection of carbapenemase-producing Acinetobacter spp. strains from spiked stools.

The recently modified CHROMagar Acinetobacter medium was evaluated for detection of carbapenemase-producing Acinetobacter baumannii from spiked stools. A total of 45 Acinetobacter spp. isolates were tested. The CHROMagar Acinetobacter medium had a high sensitivity of 86.5% and a specificity of 75%. This medium is likely to be most useful for controlling outbreaks and in endemic situations.

Genetic characterisation of drug resistance and clonal dynamics of Acinetobacter baumannii in a hospital setting in Mexico.

The aim of this study was to determine the clinical characteristics, molecular epidemiology and biofilm production of Acinetobacter baumannii clinical isolates obtained from a tertiary-care hospital in Mexico. Clinical isolates of A. baumannii (n=152) isolated from 2007 to 2012 were included. Clonal diversity was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined using the broth microdilution method. IMP, VIM, NDM and OXA-type genes were screened by PCR. Biofilm production was analysed using the crystal violet method. Mortality attributable to A. baumannii infection was 14.5%. Fifty-four clones were detected, of which five predominated. MLST results showed three new sequence types and two reported sequence types. More than 86% of the isolates were resistant to ciprofloxacin, ceftazidime and cefotaxime. Furthermore, 50.7% and 35.5% of the isolates were resistant to imipenem and meropenem, respectively. Of the isolates evaluated, 28.3% and 25.7% were positive for the blaOXA-58 and blaOXA-72 genes, respectively. Biofilm production was associated with resistance to imipenem (P=0.002).

Variations in IS6 promoters alter the expression of carbapenem resistance in related strains of Acinetobacter baumannii.

The aim of this work was to investigate the role of the IS6 family of insertion sequences present upstream of blaOXA-58 in two clonally related carbapenem-resistant Acinetobacter baumannii isolates obtained from paediatric cancer patients in Egypt. To determine their relatedness, the isolates were typed by pulsed-field gel electrophoresis (PFGE), and the intrinsic blaOXA-51-like gene was amplified and sequenced. Minimum inhibitory concentrations (MICs) to imipenem and meropenem was determined according to British Society of Antimicrobial Chemotherapy (BSAC) guidelines. PCR and sequencing of blaOXA-58 and the upstream and downstream regions was performed to determine the genetic environment. The two isolates were positive for the intrinsic blaOXA-64 gene, and the MICs for isolates AB-14298 and AB-P67 were 8mg/L and 64mg/L for imipenem and 2mg/L and 16mg/L for meropenem, respectively. The blaOXA-58 gene in AB-14298 was flanked by ISAba3 interrupted with IS1006, whereas AB-P67 had ISAba3 interrupted by IS1008, both belonging to the IS6 family of insertion sequences. In conclusion, both IS1006 and IS1008 provided suitable promoter sequences for expression of the downstream blaOXA-58 gene.