Carbapenem Resistance Determinants Research Articles

Dissemination of multiple carbapenem-resistant clones of Acinetobacter baumannii in the Eastern District of Saudi Arabia.

It has previously been shown that carbapenem-resistant Acinetobacter baumannii are frequently detected in Saudi Arabia. The present study aimed to identify the epidemiology and distribution of antibiotic resistance determinants in these bacteria. A total of 83 A. baumannii isolates were typed by pulsed-field gel electrophoresis (PFGE), and screened by PCR for carbapenemase genes and insertion sequences. Antibiotic sensitivity to imipenem, meropenem, tigecycline, and colistin were determined. Eight different PFGE groups were identified, and were spread across multiple hospitals. Many of the PFGE groups contained isolates belonging to World-wide clone 2. Carbapenem resistance or intermediate resistance was detected in 69% of isolates. The blaVIM gene was detected in 94% of isolates, while blaOXA–23–like genes were detected in 58%. The data demonstrate the co-existence and wide distribution of a number of clones of carbapenem-resistant A. baumannii carrying multiple carbapenem-resistance determinants within hospitals in the Eastern Region of Saudi Arabia.

Increased prevalence of carbapenem resistant Enterobacteriaceae in hospital setting due to cross-species transmission of the blaNDM-1 element and clonal spread of progenitor resistant strains

This study investigated the transmission characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains collected from a hospital setting in China, in which consistent emergence of CRE strains were observable during the period of May 2013 to February 2014. Among the 45 CRE isolates tested, 21 (47%) strains were found to harbor the blaNDM-1 element, and the rest of 24 CRE strains were all positive for blaKPC-2. The 21 blaNDM-1—borne strains were found to comprise multiple Enterobacteriaceae species including nine Enterobacter cloacae, three Escherichia coli, three Citrobacter freundii, two Klebsiella pneumoniae, two Klebsiella oxytoca, and two Morganella morganii strains, indicating that cross-species transmission of blaNDM-1 is a common event. Genetic analyses by PFGE and MLST showed that, with the exception of E. coli and E. cloacae, strains belonging to the same species were often genetically unrelated. In addition to blaNDM-1, several CRE strains were also found to harbor the blaKPC-2, blaVIM-1, and blaIMP-4 elements. Conjugations experiments confirmed that the majority of carbapenem resistance determinants were transferable. Taken together, our findings suggest that transmission of mobile resistance elements among members of Enterobacteriaceae and clonal spread of CRE strains may contribute synergistically to a rapid increase in the population of CRE in clinical settings, prompting a need to implement more rigorous infection control measures to arrest such vicious transmission cycle in CRE-prevalent areas.

Characterization of Acinetobacter baumannii clinical isolates carrying bla(OXA-23) carbapenemase and 16S rRNA methylase armA genes in Yemen.

The aim of this study was to investigate the molecular support of resistance to carbapenems, aminoglycosides, and fluoroquinolones in Acinetobacter baumannii clinical isolates collected from Yemen hospital. Three A. baumannii were isolated in February 2013 from three patients hospitalized at Al-Thawra University Hospital in Sana'a, Yemen. Antibiotic susceptibility testing was performed using the disk diffusion and E-test methods. Carbapenemase production was carried out by the modified Hodge test (MHT) and imipenem-ethylenediaminetetraacetic acid (EDTA) methods. Carbapenem, aminoglycoside, and fluoroquinolone resistance determinants were studied by polymerase chain reaction and sequencing. The epidemiological relatedness of the three strains was studied using multilocus sequence typing (MLST). The isolates were resistant to almost all antibiotics tested with very high imipenem, amikacin, and ciprofloxacin minimum inhibitory concentrations (>32, >256, and >32 mg/L, respectively). The microbiological tests showed that the three A. baumannii were MHT positive, besides, the activity of β-lactamases was not inhibited by EDTA. All the three isolates contained the naturally occurring bla(OXA-51)-like gene and the bla(OXA-23)-like carbapenemase-encoding gene. The 16S rRNA methylase armA gene was detected in the three isolates. In addition, screening for genes encoding the aminoglycoside-modifying enzymes (AMEs) demonstrated that one isolate contained the acetyltransferase gene aac(6')-Ib. Fluoroquinolone resistance was associated with a single mutation Ser83Leu in the quinolone resistance determining region of the gyrA gene in all isolates. The MLST showed that the sequence type (ST) obtained corresponds to ST2 for the three strains. Here we report the first identification of multidrug-resistant A. baumannii isolates harboring the bla(OXA-23)-like gene, AMEs [aac(6')-Ib], and the 16S rRNA methylase (armA) in the Yemen hospital.

First report of 16S rRNA methylase ArmA-producing Acinetobacter baumannii and rapid spread of metallo-β-lactamase NDM-1 in Algerian hospitals.

The aim of the present study was to characterize the molecular support of resistance to carbapenems, aminoglycosides and fluoroquinolones in carbapenem-resistant Acinetobacter baumannii clinical isolates recovered between January 2011 and April 2013 from Algerian hospitals. Antibiotic susceptibility testing was performed using disk diffusion and Etest methods. Carbapenemase activity was detected using both MALDI-TOF mass spectrometry assay and via microbiological tests. Carbapenem, aminoglycoside and fluoroquinolone resistance determinants were studied by PCR and sequencing. Clonal relationships between strains were determined using Multi Locus Sequence Typing (MLST). A total of 47 imipenem-resistant A. baumannii were isolated and identified by MALDI-TOF mass spectrometry. All imipenem-resistant strains were positive in the modified Hodge test, and EDTA inhibited the activity of metallo-β-lactamases enzymes in 11 strains. The blaOXA-23 gene was detected in 33 strains and the blaOXA-24 gene in 10 strains. The metallo-β-lactamase blaNDM-1 gene was detected in 11 isolates (23.4%) from Algiers and Sétif, including 7 that co-expressed a blaOXA-23 gene. Resistance to aminoglycosides was due to the production of aminoglycoside-modifying enzymes, AAC(3)-Ia, AADA, ANT(2″)-I, APH(3')-VI, and 16S rRNA methylases, ArmA. The fluoroquinolone resistance was mainly associated with mutations at Ser83Leu and Ser80Leu of the gyrA and parC genes, respectively. MLST revealed five sequence types (STs), 1, 2, 19, 25, and 85. The imipenem-resistant A. baumannii ST2 was the predominant clone (35/47). Here we report for the first time clinical multidrug-resistant A. baumannii isolates harboring 16S rRNA methylase gene, armA, and rapid spread of metallo-β-lactamase NDM-1 isolated from patients in Algeria.

Molecular mechanisms of β-lactam resistance in carbapenemase-producing Klebsiella pneumoniae from Sri Lanka.

Carbapenemases are increasingly important antimicrobial resistance determinants. Little is known about the carbapenem resistance mechanisms in Sri Lanka. We examined 22 carbapenem-resistant Klebsiella pneumoniae from Sri Lanka to determine their β-lactam resistance mechanisms. The predominant resistance mechanisms we detected in this study were OXA-181, NDM-1 carbapenemases and extended-spectrum β-lactamase CTX-M-15. All isolates were then genotyped by pulsed-field gel electrophoresis, variable-number tandem repeat sequence analysis and multilocus sequence typing, and seven distinct genotypes were observed. Five OXA-181-positive Klebsiella pneumoniae isolates were genotypically related to an isolate of Indian origin. Multilocus sequence typing found that these related isolates belong to ST-14, which has been associated with dissemination of OXA-181 from the Indian subcontinent. Other genotypes we discovered were ST-147 and ST-340, also associated with intercontinental spread of carbapenemases of suspected subcontinental origin. The major porin genes ompK35 and ompK36 from these isolates had insertions, deletions and substitutions. Some of these were exclusive to strains within single pulsotypes. We detected one ompK36 variant, ins AA134-135GD, in six ST-14- and six ST-147, blaOXA-181-positive isolates. This porin mutation was an independent predictor of high-level meropenem resistance in our entire Sri Lankan isolate collection (P=0.0030). Analysis of the Sri Lankan ST-14 and ST-147 ins AA134-135GD-positive isolates found ST-14 was more resistant to meropenem than other isolates (mean MIC: 32±0 µg ml(-1) and 20±9.47 µg ml(-1), respectively, P=0.0277). The likely international transmission of these carbapenem resistance determinants highlights the need for regional collaboration and prospective surveillance of carbapenem-resistant Enterobacteriaceae.

Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae.

BackgroundMultidrug resistance and, in particular, carbapenem resistance is spreading worldwide at an alarming rate, comprehending a variety of bacterial species and causing both nosocomial and community acquired outbursts. Early and efficient detection of infected patients or colonized carriers are mandatory steps in infection control and prevention of multidrug resistance diffusion. The latest EUCAST guidelines for detection of carbapenemase-producing Enterobacteriaceae have set low clinical breakpoints to ensure the maximum detection sensitivity of positive samples. Current workflows involve an initial screening step for species and resistance pattern detection, followed by phenotypic and/or genotypic confirmation. The aim of the present study was to assess the efficiency of six widely used and validated phenotypic assays for the detection of carbapenemases/AmpC in Enterobacteriaceae, to estimate the best workflow in the routine characterization of Enterobacteriaceae isolates.MethodsA panel of 108 non-repetitive Enterobacteriaceae isolates with reduced susceptibility to carbapenems was analyzed by means of 1) Modified Hodge Test, 2) Metallo Beta Lactamase Etest, 3) Double disk test with EDTA, 4) Rosco Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test. Confirmation and validation of results was achieved by genotypic analysis.ResultsThe most accurate identification of resistance determinants was obtained with the combined disc test (Rosco Diagnostica KPC and MBL confirm kit) which had to be coupled with the cloxacillin inhibition test for correct detection of AmpC enzymes. However, in general, phenotypic tests failed to characterize isolates harboring multiple carbapenem resistance determinants, which were successfully assessed only by PCR-based analysis.ConclusionsTo detect and control the spread of pathogens with complicated resistance patterns, both optimized phenotypic analysis (i.e. Rosco Diagnostica KPC and MBL confirm kit coupled with the cloxacillin inhibition test) and genotypic assays are recommended in the routine diagnostic of clinical laboratories.

Surveillance of carbapenem-resistant Klebsiella pneumoniae: tracking molecular epidemiology and outcomes through a regional network.

Carbapenem resistance in Gram-negative bacteria is on the rise in the United States. A regional network was established to study microbiological and genetic determinants of clinical outcomes in hospitalized patients with carbapenem-resistant (CR) Klebsiella pneumoniae in a prospective, multicenter, observational study. To this end, predefined clinical characteristics and outcomes were recorded and K. pneumoniae isolates were analyzed for strain typing and resistance mechanism determination. In a 14-month period, 251 patients were included. While most of the patients were admitted from long-term care settings, 28% of them were admitted from home. Hospitalizations were prolonged and complicated. Nonsusceptibility to colistin and tigecycline occurred in isolates from 7 and 45% of the patients, respectively. Most of the CR K. pneumoniae isolates belonged to repetitive extragenic palindromic PCR (rep-PCR) types A and B (both sequence type 258) and carried either blaKPC-2 (48%) or blaKPC-3 (51%). One isolate tested positive for blaNDM-1, a sentinel discovery in this region. Important differences between strain types were noted; rep-PCR type B strains were associated with blaKPC-3 (odds ratio [OR], 294; 95% confidence interval [CI], 58 to 2,552; P < 0.001), gentamicin nonsusceptibility (OR, 24; 95% CI, 8.39 to 79.38; P < 0.001), amikacin susceptibility (OR, 11.0; 95% CI, 3.21 to 42.42; P < 0.001), tigecycline nonsusceptibility (OR, 5.34; 95% CI, 1.30 to 36.41; P = 0.018), a shorter length of stay (OR, 0.98; 95% CI, 0.95 to 1.00; P = 0.043), and admission from a skilled-nursing facility (OR, 3.09; 95% CI, 1.26 to 8.08; P = 0.013). Our analysis shows that (i) CR K. pneumoniae is seen primarily in the elderly long-term care population and that (ii) regional monitoring of CR K. pneumoniae reveals insights into molecular characteristics. This work highlights the crucial role of ongoing surveillance of carbapenem resistance determinants.

Detection of OXA-370, an OXA-48-Related Class D β-Lactamase, in Enterobacter hormaechei from Brazil

The class D β-lactamase OXA-48 ([1][1]) and its variants have emerged as important determinants of carbapenem resistance in Enterobacteriaceae, representing a public health concern in some countries ([2][2][–][3][4][4]). The objective of this study was to report a new OXA-48 variant. In May 2013

Distribution of carbapenem resistance determinants among epidemic and non-epidemic types of Acinetobacter species in Japan

We performed a comparative molecular analysis on three types of clinically isolated Acinetobacter spp.: epidemic sequence types (STs) of Acinetobacter baumannii (epidemic ST-AB), non-epidemic sequence types of A. baumannii (non-epidemic ST-AB) and non-baumannii Acinetobacter spp. A total of 87 isolates - 46 A. baumannii, 25 A. pittii and 16 A. nosocomialis - from 43 hospitals were analysed. Of these, 31 A. baumannii isolates were ST1 or ST2 according to the Pasteur Institute multilocus sequence typing scheme and were defined as epidemic ST-AB. The other 15 A. baumannii isolates were defined as non-epidemic ST-AB. The epidemic ST-AB isolates harboured the blaOXA-23-like gene or had an ISAba1 element upstream of blaOXA-51-like, or both, whereas non-epidemic ST-AB and non-baumannii Acinetobacter spp. isolates harboured blaOXA-58-like or metallo-β-lactamase genes, or both. The proportion of multidrug-resistant isolates was significantly higher in the epidemic ST-AB isolates (48 %) than that in the other types of Acinetobacter isolates (5 %) (P<0.05). In addition, epidemic ST-AB isolates exhibited a relatively higher proportion of fluoroquinolone resistance. We demonstrated that, in terms of genotypes and phenotypes of antimicrobial resistance, non-epidemic ST-AB isolates shared more similarity with non-baumannii Acinetobacter spp. isolates than with epidemic ST-AB isolates, regardless of bacterial species. In addition, this study revealed that, even in Japan, where IMP-type metallo-β-lactamase producers are endemic, epidemic ST-AB harbouring blaIMP have not yet emerged.

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.

Draft Genome Sequence of Serratia fonticola UTAD54, a Carbapenem-Resistant Strain Isolated from Drinking Water

Serratia fonticola UTAD54 is an environmental isolate that is resistant to carbapenems due to the presence of a class A carbapenemase and a metallo-β-lactamase that are unique to this strain. Its draft genome sequence was obtained to clarify the molecular basis of its carbapenem resistance and identify the genomic context of its carbapenem resistance determinants.

Spread of Carbapenem-Resistant Acinetobacter baumannii Global Clone 2 in Asia and AbaR-Type Resistance Islands

In this surveillance study, we identified the genotypes, carbapenem resistance determinants, and structural variations of AbaR-type resistance islands among carbapenem-resistant Acinetobacter baumannii (CRAB) isolates from nine Asian locales. Clonal complex 92 (CC92), corresponding to global clone 2 (GC2), was the most prevalent in most Asian locales (83/108 isolates; 76.9%). CC108, or GC1, was a predominant clone in India. OXA-23 oxacillinase was detected in CRAB isolates from most Asian locales except Taiwan. blaOXA-24 was found in CRAB isolates from Taiwan. AbaR4-type resistance islands, which were divided into six subtypes, were identified in most CRAB isolates investigated. Five isolates from India, Malaysia, Singapore, and Hong Kong contained AbaR3-type resistance islands. Of these, three isolates harbored both AbaR3- and AbaR4-type resistance islands simultaneously. In this study, GC2 was revealed as a prevalent clone in most Asian locales, with the AbaR4-type resistance island predominant, with diverse variants. The significance of this study lies in identifying the spread of global clones of carbapenem-resistant A. baumannii in Asia.

NDM-1- and OXA-163-producing Klebsiella pneumoniae isolates in Cairo, Egypt, 2012.

Here we describe carbapenem resistance determinants in two Klebsiella pneumoniae isolates recovered from two hospitalised patients in the same intensive care unit of a cancer hospital in Cairo, Egypt. PCR and sequencing were used to detect and characterise β-lactamase genes. Clonal relationships between the isolates were analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The first K. pneumoniae isolate carried the blaNDM-1 gene and the second isolate carried the blaOXA-163 gene. Both isolates co-expressed the extended-spectrum β-lactamase CTX-M-15. The two isolates belonged to different sequence types (STs), ST11 and ST16, respectively. No history of travel was established for the two patients. The first identification of NDM-1-producing K. pneumoniae in Egypt adds further evidence to the spread of NDM-1-producing Gram-negative micro-organisms in North Africa. The additional detection of blaOXA-163 in a K. pneumoniae isolate confirms its endemic presence in a critical healthcare setting of this geographic area.

Klebsiella pneumoniae Carbapenemase-Producing Klebsiella Pneumoniae in the Intensive Care Unit

The prevalence of multidrug-resistant pathogens, especially in intensive care units, has increased and represents a great concern for medical and scientific community. Infections caused by these pathogens are associated with increased costs, length of hospitalization, and morbidity/mortality rates. The last decade was marked by the spread of carbapenem resistance determinants especially in Enterobacteriaceae isolates. In this review, the Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae is used as an example to discuss the difficulties in dealing with multidrug-resistant pathogens in the intensive care unit setting and how they represent a challenge to the medical-scientific community and, ultimately, the whole society.

OXA-235, a Novel Class D β-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-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.

OXA-162, a novel variant of OXA-48 displays extended hydrolytic activity towards imipenem, meropenem and doripenem

Context: Isolation and characterization of OXA-162, a novel variant of OXA-48.Objectives: Klebsiella pneumoniae isolate with decreased susceptibility to carbapenems was recovered from a Turkish patient. This study aimed at characterizing the carbapenem resistance determinants of this isolate.Materials and methods: Antibiotic susceptibility tests, analytic isoelectric focusing (IEF), cloning and sequencing were performed. Cloned β-lactamase was purified by means of preparative gel electrophoresis and the kinetic constants were determined under initial rate conditions.Results: The identified blaOXA-162 gene was located on a ca. 45-kb plasmid carrying a transposon consisted of two IS1999-2 elements. OXA-162 differed from OXA-48 by a single amino acid substitution (Thr213Ala) which increased the catalytic efficiency (kcat/KM) of OXA-162 towards imipenem and meropenem. Also this substitution caused a gain of hydrolysis ability towards doripenem. Analysis of OXA-162 model implied that the amino acid change might generate an extension in the opening of the substrate entry site and might cause extended hydrolytic activity towards imipenem, meropenem and doripenem.Discussion and conclusion: OXA-162, a derivative of OXA-48 has enhanced catalytic properties.

Emergence and spread of a multidrug-resistant Acinetobacter baumannii clone producing both the carbapenemase OXA-23 and the 16S rRNA methylase ArmA

Acinetobacter baumannii is a Gram-negative organism reported worldwide as a cause of health-care-associated infections, particularly in intensive care units (ICUs). The aim of this study is to describe the emergence and spread of carbapenem-resistant A. baumannii (CRAB) isolates in hospitalized patients. From March to November 2009, multidrug-resistant CRAB isolates were obtained from 21 patients hospitalized in different wards (mostly ICUs). Antimicrobial susceptibility was determined by using the Etest method. Carbapenem and aminoglycoside resistance determinants were studied by PCR and sequencing. Genetic relatedness was investigated by pulsed-field gel electrophoresis and multiplex PCR identification of sequence groups. Clinical records of patients were examined retrospectively. CRAB isolates were consistently resistant to multiple drugs including fluoroquinolones and aminoglycosides, whereas they retained a susceptibility to colistin. Molecular analysis revealed that 19 of the 21 CRAB isolates belonged to a single clone producing both the carbapenemase OXA-23 and the 16S rRNA methylase ArmA. Based on clinical data, the patients included in the study were classified as infected (n=13) or colonized (n=8). Colistin alone or in combination with ampicillin-sulbactam was administered to 11 of the 13 infected patients. A complete or partial response was obtained in eight cases, whereas a failure to respond was observed in one patient and a relapse was observed in two patients. An A. baumannii clone producing both OXA-23 and ArmA has been identified as an emerging and rapidly spreading pathogen. To our knowledge, this is the first report of the ArmA enzyme in A. baumannii in Italy and is the first report of hospital dissemination of A. baumannii carrying both bla(OXA-23) and armA genes.

Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D β-lactamase OXA-10 by rational protein design

Class D β-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae. Carbapenemase activity is an unusual feature among class D β-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands β5 and β6 (the β5-β6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D β-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the β5-β6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10-derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted β5-β6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the β5-β6 loop in the carbapenemase activity of class D β-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.

Sequential outbreaks of multidrug-resistant Acinetobacter baumannii in intensive care units of a tertiary referral hospital in Italy: combined molecular approach for epidemiological investigation

A laboratory-based surveillance study was conducted from January 2007 to May 2010 in San Martino Tertiary Referral Hospital in Genoa, Italy in which the molecular epidemiology of multidrug-resistant Acinetobacter baumannii was investigated in the five intensive care units (ICUs). A total of 53 A.baumannii strains were isolated from patients admitted to ICUs (69.8%) and to other epidemiologically linked hospital wards (30.2%) and were genotyped by repetitive extragenic palindromic polymerase chain reaction (REP-PCR), multilocus sequence typing (MLST) and adeB sequence typing. REP-PCR fingerprinting analysis, MLST and adeB typing results were well correlated and allowed us to classify strains causing epidemic events into three major epidemic clones: A (REP-I/ST4, adeB-STII genotype) isolated for the first time in May 2007, B (REP-IV/ST95, adeB-STI genotype) from November 2007 to May 2009 and C (REP-VII/ST118, adeB-STII genotype) from July 2008 to May 2010. MLST results demonstrated that epidemic clones A and C were related as they were members of the widespread clonal complex CC92. The genetic determinants of carbapenem resistance were investigated and resistance associated with the presence of the bla(OxA-58-like) gene with ISAba2 and ISAba3 elements flanking it in clone A, and with the bla(OxA-23-like) gene flanked by ISAba1 in clones B and C. Amolecular approach allowed the prompt introduction of infection control measures and the evaluation of data in a global epidemiological context.

Carbapenem Susceptibility Testing Errors Using Three Automated Systems, Disk Diffusion, Etest, and Broth Microdilution and Carbapenem Resistance Genes in Isolates of Acinetobacter baumannii-calcoaceticus Complex

The Acinetobacter baumannii-calcoaceticus complex (ABC) is associated with increasing carbapenem resistance, necessitating accurate resistance testing to maximize therapeutic options. We determined the accuracy of carbapenem antimicrobial susceptibility tests for ABC isolates and surveyed them for genetic determinants of carbapenem resistance. A total of 107 single-patient ABC isolates from blood and wound infections from 2006 to 2008 were evaluated. MICs of imipenem, meropenem, and doripenem determined by broth microdilution (BMD) were compared to results obtained by disk diffusion, Etest, and automated methods (the MicroScan, Phoenix, and Vitek 2 systems). Discordant results were categorized as very major errors (VME), major errors (ME), and minor errors (mE). DNA sequences encoding OXA beta-lactamase enzymes (bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), and bla(OXA-51-like)) and metallo-β-lactamases (MBLs) (IMP, VIM, and SIM1) were identified by PCR, as was the KPC2 carbapenemase gene. Imipenem was more active than meropenem and doripenem. The percentage of susceptibility was 37.4% for imipenem, 35.5% for meropenem, and 3.7% for doripenem. Manual methods were more accurate than automated methods. bla(OXA-23-like) and bla(OXA-24-like) were the primary resistance genes found. bla(OXA-58-like), MBLs, and KPC2 were not present. Both automated testing and manual testing for susceptibility to doripenem were very inaccurate, with VME rates ranging between 2.8 and 30.8%. International variability in carbapenem breakpoints and the absence of CLSI breakpoints for doripenem present a challenge in susceptibility testing.