Metallo Β-lactamase Research Articles

Rationale for the 2010 Revised Susceptibility Breakpoints for Cephalosporins, Aztreonam, and Carbapenems for Enterobacteriaceae.

β-Lactam antimicrobial agents are mainstays for treatment of infections due to many members of the Enterobacteriaceae. Since the introduction of these agents into clinical use in the 1970s and 1980s, there have been considerable changes in the potency of these drugs against clinical isolates of Enterobacteriaceae, largely due to the evolution and dissemination of extended spectrum β-lactamases (ESBLs) that hydrolyze penicillins, cephalosporins, and monobactams. The worldwide dissemination of these ESBLs played a major role in the first revision of susceptibility breakpoints (minimum inhibitory concentrations [MICs]) for these drugs by the Clinical Laboratory Standards Institute in June 2010. The increasing problem of β-lactamase-mediated resistance has recently extended to the carbapenem class of antimicrobial agents. The serine carbapenemase KPC (for Klebsiella pneumoniae carbapenemase) has been detected in Enterobacteriaceae from several major population regions in the United States, and is problematic in many institutions in Europe. More recently, a metallo β-lactamase (NDM-1) that hydrolyzes carbapenems and most β-lactams has spread rapidly from Asia to the United Kingdom and other regions in the world. The Centers for Disease Control and Prevention has issued alerts for both of these carbapenemases and recommendations for limiting its spread in hospitals [1, 2]. Carbapenemases of the KPC type produced a situation similar to that for ESBLs; strains producing KPC or ESBLs were often classified as susceptible using breakpoints established in the former era. Thus, new lower breakpoints for cephalosporins and carbapenems against Enterobacteriaceae were established to ensure optimal exposures with US Food and Drug Administration (FDA)-approved dosage regimens, and would also classify these isolates as nonsusceptible. Clinical, pharmacokinetic-pharmacodynamic (PKPD), and MIC distribution data were considered in revising the breakpoints [3, 4]. While highly desirable, clinical data were difficult to acquire. Critical information concerning MIC, concomitant drug treatment, site of infection, and cephalosporin dosage regimen was generally not available in studies of ESBL-producing isolates. Thus, other analyses were required. PK-PD analyses were conducted to determine whether usual FDA-approved dosage regimens of the Pediatric ID Consultant

Unusual Diversity of Acquired β-lactamases in Multidrug-ResistantPseudomonas aeruginosaIsolates in a Mexican Hospital

To investigate the presence of extended spectrum and metallo β-lactamases (MBLs) in Pseudomonas aeruginosa isolates which are resistant to imipenem and ceftazidime that were isolated in a hospital in Mexico. Pulsed-field gel electrophoresis (PFGE) revealed the presence of four clonal types among the 14 isolates. All these genes were found either alone or simultaneously in the P. aeruginosa strains in the following five different arrangements: <bla(GES-5)>; <bla(GES-5), bla(VIM-11)>; <bla(GES-5), bla(VIM-2), bla(VIM-11)>; <bla(GES-5), bla(OXA-2)>; and <bla(GES-5), bla(VIM-2), bla(VIM-11), and bla(OXA-2)>. Class 1 integrons were detected and contained the cassettes bla(GES-5) and bla(OXA-2), but not that of bla(VIM). bla(VIM) genes occurred only in the chromosome, while bla(GES-5) was located in the chromosome and in the plasmids. To our knowledge, this is the first description of P. aeruginosa strains simultaneously producing the VIM-2 and VIM-11 variants, and the combination of GES-5 and MBL carbapenemases, which determines a major challenge for the clinical microbiology laboratory and a remarkable epidemiological risk for the nosocomial spread of multidrug-resistant determinants.

Prevalence of β-Lactamase Production and Antimicrobial Susceptibility of Multidrug Resistant Clinical Isolates of Non-Fermenting Gram Negative Bacteria From Hospitalized Patients in Kerman/Iran

: Background: Non-fermenting Gram–negative bacteria are unable to ferment sugars in order to generate energy. They are ubiquitous in nature, and have a wide geographic distribution. They are also common in hospital settings, and may be isolated from humidifiers, ventilator machines, dialysis machines and other equipment, as well as from the skin of hospital personnel.Objectives: This study focused on the isolation of multidrug resistant (MDR) non-fermenting Gram negative bacteria from clinical samples. Antimicrobial susceptibility, detection of extended spectrum β-lactamases (ESBL), and the presence of CTX-M and Metallo β-lactamase (MBL) in the isolated bacteria were evaluated.Materials and Methods: Agar dilution method was used to test the susceptibility of the isolates to 10 antibacterial agents. All the isolates that were resistant to ≥ 3 antibacterial agents from different classes were regarded as MDR (111 isolates) and were selected for further studies.β-lactamase and ESBL production were detected by nitrocefin discs, combined discs (CD) and double discs plus CD (DCDT). blaCTX-M and MBL were detected by PCR and EDTA synergy methods respectively.Result: Among the MDR isolates the isolation frequency of Pseudomonas aeruoginosa, Stenotrophomonas maltophilia and Acinetoacter baumannii were 83.7%, 9.9% and 6.3% respectively. Resistance to imipenem (0.9%) and Ceftazidim (13.6%) was low, but resistance to other β-lactams was high, and 29.7% were resistant to ≥ 6 antibacterial agents from different classes simultaneously. β-lactamase was produced by 41.4% of the MDR isolates. Detection of ESBLs by a CD (59.4%) or DCDT test (46.8%) was not significantly different, but with a combination of CD and DCDT a higher percentage of ESBLs in the isolates (P ≤ 0.00) could be detected. The blaCTX-M and MBL phenotype were detected in two different strains of P. aeruoginosa.Conclusions: The presence of a high percentage of isolates producing ESBLs which are resistant to different antibacterial agents may result in treatment failure in infected patients. Careful detection of antimicrobial resistant strains is needed in order to avoid underestimation or misidentification of ESBLs. An effective hospital infection control policy is also necessary in order to prevent further resistance to antimicrobials in the region. Implication for health policy/practice/research/medical education:Detection of the frequency and type of extended spectrum β-lactamases (ESBL) involved in resistance to antimicrobial agents is important from a clinical and epidemiological point of view. Reporting the presence of highly resistant isolates of Pseudomonas aeruginosa producing ESBLs in this area will help physicians to select appropriate antimicrobials for empirical therapy. Please cite this paper as:Mansouri S, Razavi M, Norouzi F, Gholamhoseinian Najar S. Prevalence of β-Lactamase Production and Antimicrobial Susceptibility of Multidrug Resistant Clinical Isolates of Non-Fermenting Gram Negative Bacteria From Hospitalized Patients in Kerman/Iran. Jundishapur J Microbiol.2012;5(2):405-10. DOI: 10.5812/jjm.3399 Copyright © 2012 Kowsar Corp. All rights reserved.

VIM-2 metallo- -lactamase-producing Pseudomonas aeruginosa causing an outbreak in South Africa

Sir, Pseudomonas aeruginosa is an opportunistic pathogen that has been implicated in nosocomial outbreaks in immunocompromised patients worldwide. We present an outbreak caused by a multidrug-resistant P. aeruginosa (MRPA) clone in a haematology unit of a tertiary academic hospital in Cape Town, South Africa. Fifteen MRPA isolates were recovered from separate patients between January 2010 and April 2011, including 10 from blood, 2 from stool, 1 from bile, 1 from urine and 1 from a catheter tip. The majority of the patients were severely neutropenic following stem cell transplants and eight of them died. The genetic relatedness of the isolates was investigated using PFGE according to a previously published protocol with minor amendments. Whole genomic DNA was digested in situ with SpeI (New England Biolabs, Inc., UK) and the resulting restriction fragments were separated on a 1% agarose gel using a CHEF-DRII GeneNavigator apparatus (GE Healthcare, Piscataway, NJ, USA). Restriction profiles were analysed using GelCompar II version 5.1 (Applied Maths, St-Martens-Latem, Belgium). A dendrogram indicating the levels of similarity between the isolates was created using the Dice similarity coefficient. The band tolerance and optimizations were set at 1%, and a similarity threshold of 80% was used to define clusters. The profiles of 10 of the 15 isolates were indistinguishable and were assigned to cluster A. One isolate, with 77% similarity to cluster A, was assigned subtype A1. The remaining four isolates were all unique. To further characterize the genetic background of this MRPA clone, multilocus sequence typing (MLST) of three representative isolates from cluster A, spanning the defined outbreak period, were selected. MLST indicated that these three isolates belong to sequence type (ST) 233. Identification and susceptibility testing was performed on Vitek 2 (bioMerieux, Marcy l’Etoile, France). Imipenem and meropenem MICs were determined by Etests, according to the manufacturer’s specification (bioMerieux). Fourteen of the isolates expressed high levels of resistance to imipenem (MIC ≥128 mg/L) and meropenem (MIC ≥128 mg/L). The 15th strain was susceptible to both imipenem (MIC ≤1 mg/L) and meropenem (MIC ≤2 mg/L). As metallo-b-lactamases (MBLs) are considered a major mechanism of carbapenem resistance in P. aeruginosa, PCR assays were carried out using primers for the detection of the MBL-encoding genes blaIMP (IMPF 5′-ATTGACACTCCATTTAC-3′/ IMPR 5′-AACAACCAGTTTTGC-3′) and blaVIM (VIMF 5′-GTGAGTATCC GACAGTC-3′/VIMR 5′-GAGCAAGTCTAGACCG-3′). Although a PCR product of the expected size was obtained from a control P. aeruginosa strain carrying blaIMP-1 (gift from Y. Hirakata, Tohuku University Graduate School of Medicine, Japan), no products were obtained from the 15 Cape Town P. aeruginosa isolates. However, amplicons of the expected size for the blaVIM gene were obtained for the 10 strains in PFGE cluster A and the closely related strain, A1, as well as the blaVIM-2 positive control (gift from P. Nordmann, Hopital de Bicetre, France). No PCR products were obtained from the four unrelated P. aeruginosa isolates. PCR products from the 11 strains were purified (QIAquick PCR purification kit, QIAGEN, Germany) and sequencing analysis revealed the blaVIM-2 gene in all 11 strains. Additional PCR screening for the NDM, SPM, KPC and GES b-lactamase genes for all 15 isolates revealed that the single isolate, sensitive to both imipenem and meropenem and cultured from a catheter tip, carried a blaGES-2 gene, which has previously been implicated in a South African outbreak. MLST typing indicated that this carbapenem-susceptible strain belonged to ST625, a new genotype not previously described. Relatively little is known regarding the prevalence of carbapenem-hydrolysing enzymes in African countries. This outbreak of MBL blaVIM-2-carrying P. aeruginosa highlights the urgent need for the development of more active surveillance systems in South Africa and the importance of molecular epidemiology in a hospital outbreak situation.

Biomonitoring marine habitats in reference to antibiotic resistant bacteria and ampicillin resistance determinants from oviductal fluid of the nesting green sea turtle, Chelonia mydas

During the egg-laying process, oviductal fluid was collected using a non-invasive procedure from the cloacal vent of the green turtles. Forty-two independent isolates of antibiotic-resistant bacteria from 11 genera were obtained from 20 turtles during nesting. The dominant isolate was Citrobacter (52.4%), followed by Pseudomonas, Proteus, Enterobacter, Salmonella, Escherichia coli, Shigella, Edwardsiella, Morganella, Providencia and Arcomobacter. Most of the isolates were resistant to ampicillin. Ampicillin-resistant isolates showed variations in their resistance for the following classes of β-lactamases: extended-spectrum β-lactamases (EBSLs), AmpC type β-lactamases C (AmpC), and screen-positive β-lactamase. None of the isolates produced metallo β-lactamase. Some ampicillin-resistant genes were detected by multiplex polymerase chain reaction (PCR) only. Inhibitor based test (IBT) categorized some isolates as AmpC β-lactamase producers. β-Lactamase genes were detected from a few strains. The sequencing of those genes revealed the presence of cephamycinase (CMY) and AmpC β-lactamases. The oviductal fluid was used in this study as a source of bacterial antibiotic-resistant determinants for biomonitoring marine turtles exposed to contaminated effluents. This data can be of value in understanding the decline of this endangered species as a result of exposure to marine pollution which is threatening their survival.

Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 types

BackgroundGenes conferring antibiotic resistance to groups of bacterial pathogens are cause for considerable concern, as many once-reliable antibiotics continue to see a reduction in efficacy. The recent discovery of the metallo β-lactamase blaNDM-1 gene, which appears to grant antibiotic resistance to a variety of Enterobacteriaceae via a mobile plasmid, is one example of this distressing trend. The following work describes a computational analysis of pathogen-borne MBLs that focuses on the structural aspects of characterized proteins.ResultsUsing both sequence and structural analyses, we examine residues and structural features specific to various pathogen-borne MBL types. This analysis identifies a linker region within MBL-like folds that may act as a discriminating structural feature between these proteins, and specifically resistance-associated acquirable MBLs. Recently released crystal structures of the newly emerged NDM-1 protein were aligned against related MBL structures using a variety of global and local structural alignment methods, and the overall fold conformation is examined for structural conservation. Conservation appears to be present in most areas of the protein, yet is strikingly absent within a linker region, making NDM-1 unique with respect to a linker-based classification scheme. Variability analysis of the NDM-1 crystal structure highlights unique residues in key regions as well as identifying several characteristics shared with other transferable MBLs.ConclusionsA discriminating linker region identified in MBL proteins is highlighted and examined in the context of NDM-1 and primarily three other MBL types: IMP-1, VIM-2 and ccrA. The presence of an unusual linker region variant and uncommon amino acid composition at specific structurally important sites may help to explain the unusually broad kinetic profile of NDM-1 and may aid in directing research attention to areas of this protein, and possibly other MBLs, that may be targeted for inactivation or attenuation of enzymatic activity.

Biapenem Inactivation by B2 Metallo β-Lactamases: Energy Landscape of the Post-Hydrolysis Reactions

BackgroundThe first line of defense by bacteria against β-lactam antibiotics is the expression of β-lactamases, which cleave the amide bond of the β-lactam ring. In the reaction of biapenem inactivation by B2 metallo β-lactamases (MβLs), after the β-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5–C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo β-lactamase CphA in complex with product.Methodology/Principal FindingsQM/MM and metadynamics simulations of the post-hydrolysis steps in solution and in the enzyme reveal that while the rotation of the hydroxyethyl group can occur in solution or in the enzyme active site, formation of the bicyclic compound occurs primarily in solution, after which the final product binds back to the enzyme. The calculations also suggest that the rotation and cyclization steps can occur at a rate comparable to that observed experimentally for the enzymatic inactivation of biapenem only if the hydrolysis reaction leaves the N4 nitrogen of the β-lactam ring unprotonated.Conclusions/SignificanceThe calculations support the existence of a common mechanism (in which ionized N4 is the leaving group) for carbapenems hydrolysis in all MβLs, and suggest a possible revision of mechanisms for B2 MβLs in which the cleavage of the β-lactam ring is associated with or immediately followed by protonation of N4. The study also indicates that the bicyclic derivative of biapenem has significant affinity for B2 MβLs, and that it may be possible to obtain clinically effective inhibitors of these enzymes by modification of this lead compound.

Prevalence of imp type metallo-β-lactamase in carbapenem resistant pseudomonas aeruginosa and correlation of genotype to phenotype

Background Pseudomonas aeruginosa is a ubiquitous organism that is easily recovered from moist environments. It is encountered as a nosocomial pathogen and has become one of the most serious causes of nosocomial bacterial infections particularly in the immunocompromised and burned hosts as well as in patients with cystic fibrosis. Increasingly, Pseudomonas aeruginosa that are multidrug or pan-resistant are being isolated from clinical specimens. Metallo-b-lactamases (MBLs) which belong to Ambler class B b-lactamase have emerged as a therapeutic threat, as they often confer high-level resistance to all b-lactams except aztreo-nam. Aim The aim of this study is to identify the number of isolates that are carbapenem resistant due to the presence of an IMP type MBL from a storage collection in Queensland and to review the characteristics and epidemiology of these isolates. The secondary aim is to analyse whether the MBL genotypes correlate well with phenotypic detection. Method Previously stored carbapenem resistant Pseudomonas aeruginosa clinical isolates were retrieved. The minimum inhibitory concentrations (MICs) to two carbapenems (meropenem and imipenem) were defined using E-test (bioMerieux, France). Template DNA was prepared by heat lysis and polymerase chain reaction (PCR) was performed using IMP-1A and IMP-1B primers. Phenotypic testing was done using the same subculture by the modified Hodge test (MHT) and double disc synergy testing (DDST) methods. Results A total of 69 isolates was included in this study of which 14.5% (10 isolates) were positive for IMP type MBL. Of the 10 isolates, five were collected from intensive care units, three from medical wards and one each from the surgical ward and outpatients department. Phenotypic testing of the 10 isolates with the IMP type MBL showed negative results on MHT but was positive for the DDST. Conclusion This is the first study from Australia looking at the prevalence of IMP type MBL in carbapenem resistant Pseudomonas aeruginosa . The prevalence of IMP type MBL is 14.5% in carbapenem resistant Pseudomonas aeruginosa isolates in Queensland from as early back as 2002. Laboratory screening is important in identifying and preventing the further spread of MBL-producing organisms. The DDST is a relatively simple and economic test for this purpose. A positive screening test will need molecular confirmation to identify the MBL involved.

Phenotypic methods for detection of various β-lactamases in Gram-negative clinical isolates: Need of the hour

Background: Many clinical laboratories have problems detecting various β-lactamases. Confusion exists about the importance of these resistance mechanisms, optimal test methods, and appropriate reporting conventions. It is more imperative to use various phenotypic methods for detection of various β-lactamases in routine microbiology laboratory on day-to-day basis to prevent antimicrobial resistance by evidence-based judicious use of antimicrobials. Aims: In view of the multidrug-resistant organisms being reported world over, we planned a cross-sectional prospective analytical study to determine resistance mechanism by various β-lactamases in Gram-negative clinical isolates using various phenotypic methods. Materials and Methods: All nonrepeat, nonenteric clinical isolates of Gram-negative bacilli, resistant to at least two third-generation cephalosporins, were first screened by Novel disc placement method, and isolates showing multiple mechanisms of resistance and reduced zone of inhibition for imipenem were further confirmed for AmpC and metallo β-lactamases. Statistical Analysis: All the data was managed and analyzed in Microsoft Excel. Results: Out of 807 isolates tested, as many as 795 (98.51%) revealed the presence of extended-spectrum β-lactamases (ESBLs). Only 10 isolates of Escherichia coli and 2 of Klebsiella pneumoniae did not show production of ESBL. A total of 450 (55.76%) isolates produced single enzyme,while 345 (42.75%) strains revealed multiple enzyme production simultaneously. Only ESBL production was seen in 315 (39.03%) strains, only AmpC in 75 (9.29%) and only MBL in 60 (7.44%) strains, while ESBL and AmpC together were seen in 219 (27.14%) and AmpC plus MBL in 92 (11.40%) strains. However, ESBL plus MBL were never observed together. All three enzymes were simultaneously detected in 34 (4.21%) strains. Conclusion: This innovative method of disc placement makes it easy, affordable, and reliable method for routine use by basic microbiology laboratories for detection of various β-lactamases, pending confirmation for AmpC and metallo β-lactamase by three-dimensional test and double disc potentiation test, respectively.

Burden of different beta-lactamase classes among clinical isolates of AmpC-producing Pseudomonas aeruginosa in burn patients: A prospective study

Background:Pseudomonas aeruginosa is one of the most common pathogens causing infections in burns, and shows increasing resistance to β-lactam antibiotics by producing different classes of beta-lactamases. It is also not unusual to find a single isolate that expresses multiple β-lactamase enzymes, further complicating the treatment options. Thus, in this study, we aimed to determine the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa in the burn ward.Materials and Methods:A total of 101 clinical isolates of P. aeruginosa from the burn ward were identified and tested for the presence of different beta-lactamase enzymes (extended spectrum beta lactamase (ESBL), Amp C and metallo β-lactamases (MBL) from October 2006 to May 2009. In vitro susceptibility pattern of antipseudomonal antibiotics was done by the Kirby Bauer disc diffusion method.Results:A total of 33 (32.7%) isolates were confirmed to be positive for AmpC beta-lactamase. Co-production of AmpC along with ESBL and MBL was reported in 24.5% and 45.5% isolates, respectively. A total of 12 (11.9%) isolates were resistant to three or more antibiotic classes (multidrug resistance). Imipenem and piperacillin/tazobactum showed high sensitivity, with 86.1% and 82.2%, respectively.Conclusion:This study reveals the high prevalence of multidrug- resistant P. aeruginosa producing beta-lactamase enzymes of different mechanisms in this region from burn patients. The emerging antimicrobial resistance in burn wound pathogens poses serious therapeutic challenge. Thus proper antibiotic policy and measures to restrict the indiscriminate use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta -lactamase producing pathogen.

Molecular Characterization and Antimicrobial Susceptibility Study of Acinetobacter baumannii Clinical Isolates from Middle East, African and Indian Patients

The aim of the present investigation was to characterize the prevalence of extended-spectrum β-lactamases (ESBLs) and metallo β-lactamases (MBLs), and to study the antibiotic susceptibility profile among 250 clinical isolates of Acinetobacter baumannii. Phenotypic characterization was carried out by double disc synergy method and the prevalence of ESBLs and MBLs antibiotic resistant determinants were analyzed with Polymerase Chain Reaction (PCR). Susceptibility studies were performed by disc diffusion method according to Clinical and Laboratory Standards Institute guidelines 2009. Among the two hundred fifty isolates, two hundred nine isolates (83.6%) were positive for ESBLs whereas one hundred sixty seven isolates (79.9%) were positive for both ESBLs and MBLs. Moreover, five isolates (2.3%) which were positive for MBL on disc diffusion test, but negative in PCR showed MBL activity by spectrophotometric assay. Susceptibility study showed that all of the isolates were found to be more susceptible to ceftriaxone plus ethylenediaminetetraacetate plus sulbactam (90-93%), followed by meropenem (50-53%), imipenem (42-45%), cefoperazone plus sulbactam (40-42%), piperacillin plus tazobactam (38-42%) and amoxicillin plus clavulanic acid (28-31%). Among the ESBLs, TEM-types were varied from 82 to 87% followed by SHV-types (67-78%), CTX-M types (60 to 67) and OXA types (51 to 56%) in all of the isolates. Among the MBLs, NDM-1 varied from 40 to 49% followed by IMP-1 (51 to 55%), VIM-1 (55 to 59%) and KPC (47 to 55%) in all of the isolates. Moreover, results of the present study revealed that all of the clinical isolates were susceptible to ceftriaxone plus EDTA plus sulbactam and can be a potent antibacterial agent for the treatment of severe bacterial infections caused by A. baumannii.

Prevalence and Risk Factors of Metallo β-lactamase Producing Pseudomonas aeruginosa and Acinetobacter species in Burns and Surgical Wards in a Tertiary Care Hospital

Introduction:The production of Metallo-β-lactamases (MBLs) is one of the resistance mechanisms of Pseudomonas aeruginosa and Acinetobacter species. There is not much Indian data on the prevalence of MBLs in burns and surgical wards.Materials and Methods:A total of 145 non-duplicate isolates of carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter species, isolated from pus/wound swabs and endotracheal secretions from burns and surgical wards, were tested for MBL production by modified ethylene diamine tetra acetic acid (EDTA) disc synergy and double disc synergy tests.Results:Prevalence of MBLs was 26.9% by both the above tests. All MBL-positive isolates were multidrug resistant. Only 6.06% (2/33) P.aeruginosa and 16.67% (1/06) Acinetobacter species were susceptible to piperacillin-tazobactam and netilmycin, respectively. These patients had multiple risk factors like >8 days hospital stay, catheterization, IV lines, previous antibiotic use, mechanical ventilation, etc. Graft application and surgical intervention were significant risk factors in MBL-positive patients. Overall mortality in MBL-positive patients was 34.21%.Conclusion:Emergence of MBL-producing Pseudomonas aeruginosa and Acinetobacter species in this hospital is alarming, which reflect excessive use of carbapenems and at the same time, pose a therapeutic challenge to clinicians as well as to microbiologists. Therefore, a strict antibiotic policy and implementation of proper infection control practices will go a long way to prevent further spread of MBLs. Detection of MBLs should also become mandatory in all hospitals.

Emergence of Klebsiella pneumoniae isolate co-producing NDM-1 with KPC-2 from India

Sir, The emergence of NDM-1-producing isolates and their sources have been clearly identified in several countries worldwide. In particular, the blaNDM-1 gene was identified in various genera of Enterobacteriaceae and in non-fermenting Gram-negative bacilli from environmental samples in India. Furthermore, the increasing co-production of NDM-1 with other carbapenemases has been detected amongst isolates of Enterobacteriaceae and Acinetobacter sp. in many parts of India. – 7 We report here the isolation of a strain of Klebsiella pneumoniae (designated IR98) from a urine sample from a middle-aged patient admitted to the intensive care unit of a tertiary care hospital in Chennai, India in July 2010. Species identification and antibiotic susceptibility, determined using an automated system (VITEK-2, bioMerieux Inc.), showed wide-spectrum resistance to b-lactams, aminoglycoside, fluoroquinolones, co-trimoxazole, nitrofurantoin and tigecycline, but susceptibility to colistin and fosfomycin, according to CLSI guidelines. MICs of various antibiotics were determined using the agar dilution method, while the tigecycline MIC was determined using the broth microdilution method. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied to interpret the susceptibilities. The isolate was highly resistant to imipenem (256 mg/L), meropenem (128 mg/L), ceftazidime (.256 mg/L), cefotaxime (.256 mg/L), amikacin (.512 mg/L), gentamicin (.512 mg/L), tobramycin (.512 mg/L), netilmicin (.512 mg/L), co-trimoxazole (.32 mg/L), ciprofloxacin (.32 mg/L) and tigecycline (4 mg/L), but remained susceptible to colistin (0.5 mg/L). The double-disc synergy test (DDST), modified Hodge test (MHT) and combined-disc synergy test (CDST) were used for the detection of metallo-b-lactamases (MBLs), other carbapenemases and KPC, or KPC with MBLs. The simultaneous production of MBL and KPC-like carbapenemases was confirmed by positive DDST, MHT and CDST with meropenem discs containing both EDTA and phenylboronic acid (PBA) or EDTA, while meropenem discs supplemented with PBA were negative. PCR assays for genes encoding b-lactamases and 16S rRNA methylases revealed the presence of blaNDM-1, blaKPC-2, blaCTX-M-15, blaSHV-12, blaTEM-1, blaOXA-1 and rmtB genes. Plasmid analysis using the Kieser technique revealed that K. pneumoniae IR98 harboured four plasmids, with sizes of 160, 120, 70 and 40 kb, using Escherichia coli NCTC 50192 as a reference. To study the transferability of these plasmids (encoding the resistance determinants), transconjugation and transformation experiments were performed using E. coli J53 (Azide-R) and E. coli TOP10 as recipient strains. Transconjugants were selected on MacConkey agar plates using sodium azide (200 mg/L) with ceftazidime (2 mg/L), meropenem (0.5 mg/L) or amikacin (20 mg/L). The plasmid extract of K. pneumoniae IR98 was transformed into E. coli TOP10, and transformants were selected on MacConkey agar plates containing 2 mg/L ceftazidime. Selected colonies were replica-plated onto MacConkey agar plates with or without meropenem (0.5 mg/L) or amikacin (20 mg/L). The genes encoding NDM-1, CTX-M15 and 16S rRNA methylase were transferred in conjugation experiments, whereas transfer of KPC-2 was successful only by transformation. The plasmids purified from the clinical isolate, transconjugants and transformants were typed by PCR-based replicon typing (PBRT). The E. coli J53-p98A transconjugant obtained from the meropenem plate showed an MBL phenotype and elevated MICs of all the b-lactams (except aztreonam) and susceptibility to non-b-lactam antibiotics. Plasmid analysis revealed that E. coli J53-p98A harboured a 160 kb plasmid that belonged to the Inc A/C type. Subsequently, this plasmid was found by PCR to carry the blaNDM-1 gene. In addition, the E. coli J53-p98B transconjugant grown on both ceftazidime and amikacin plates showed decreased susceptibility to aminoglycosides and cephalosporins except cefoxitin, and was positive for extendedspectrum b-lactamase (ESBL) production on DDST. Both blaCTX-M15 and rmtB genes were carried on a 120 kb IncF plasmid that was identified in E. coli J53-p98B. In contrast, the ESBL-negative transformant (E. coli TOP10-p98C) from both ceftazidime and meropenem plates was resistant to all the b-lactams except carbapenems (MICs 2 mg/L). The blaKPC-2 gene together with blaTEM-1 was carried on a 70 kb non-typeable plasmid that was identified in E. coli TOP10-p98C. Although the co-existence of blaNDM-1 with different carbapenemase genes has been reported in India, we believe this to be the first report of the co-occurrence of blaNDM-1 with blaKPC-2 and rmtB in a clinical isolate of K. pneumoniae from India. This co-production of NDM-1 with an unrelated carbapenemase and 16S RNA methylase results in very broad-spectrum antibiotic resistance profiles. The growing emergence of these powerful resistance mechanisms in India is cause for great concern as treatment options are virtually exhausted.

Current Trends in &amp;#946;-Lactam Based &amp;#946;-Lactamases Inhibitors

The introduction of antibiotics to treat bacterial infections either by killing or blocking their growth has been accompanied by the development of resistance mechanism that allows the bacteria to survive and proliferate. In particular the successive series of β-lactams have selected several generations of β-lactamases including ESBLs, AmpC β-lactamases, KPC carbapenamases in Enterobacteriaceae, the metallo β-lactamases VIMs and IMPs, and very recently the threatening NDM-1 that confers resistance to virtually any clinically used antibiotic. The increasing use of carbapenems due to the spread of resistance to other existing antibacterial agents has facilitated the spread of resistance, especially in Acinetobacter spp. due to OXA- and metallo-carbapenemases. The pharmaceutical industry, that abandoned this field at the end of the nineties, is now trying to recover by developing some novel β-lactam antibiotics and novel β-lactamase-inhibitors, the latter to be used in combination with new as well as seasoned β-lactam antibiotics. This article provides a survey of patent and scientific literature for β-lactamase inhibitors discovered in the period 2006-2010.

Silent spread of IMP-13-producing Pseudomonas aeruginosa belonging to sequence type 621 in Belgium

Sir, Organisms with acquired metallo-b-lactamases (MBLs) have been identified worldwide in numerous species, including Pseudomonas aeruginosa. VIM and IMP types are the most prevalent MBLs, being encoded by gene cassettes inside class 1 integrons. IMP-13, initially detected in clinical isolates of P. aeruginosa from Italy, has become a widespread carbapenem resistance determinant, being involved in relatively large outbreaks in Italy. IMP-13 has also been occasionally detected in P. aeruginosa isolates from Austria and Romania. Recently, IMP-13-producing isolates were found in Argentina that belonged to the same clonal sequence type (ST), i.e. ST621, as the Italian isolates. In these isolates, the blaIMP-13 gene was found to be carried in different class 1 integron structures (InPSG, In88 and In89), usually located on the chromosome or, less frequently, on plasmids. From November 2006 to September 2008, four multidrug-resistant (MDR) P. aeruginosa isolates with reduced susceptibility to carbapenems (being susceptible only to colistin) were isolated from clinical samples of four patients hospitalized in three Belgian hospitals (two in the Brussels area and one located 70 km south of Brussels) (Table 1). Despite multiple investigations, no other carbapenem-resistant P. aeruginosa isolates were recovered from other patients hospitalized in the same wards before or concomitantly to the infected patients, and no overlap in hospital stay could explain cross-transmission between patients. Furthermore, none of these patients had recently travelled or had been hospitalized abroad, suggesting that these isolates were acquired in Belgium. Two of these isolates (P1 and P2) displayed only slightly decreased susceptibility to imipenem and meropenem (MICs of 4 mg/L) and were categorized as susceptible according to CLSI breakpoints [according to current breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST), P1 and P2 isolates would be classified as susceptible to imipenem (susceptible breakpoint is ≤4 mg/L) and intermediately resistant to meropenem (susceptible breakpoint is ≤2 mg/L)], while isolates P3 and P4 displayed high-level resistance to carbapenems (Table 1). The Etest MBL, performed according to the manufacturer’s recommendations, showed a reduction in the imipenem MICs in the presence of EDTA for all of the MDR isolates (Table 1). However, the extent of MIC reduction (2to 4-fold) was insufficient to indicate the production of an MBL, for isolates P1 and P2, according to the manufacturer’s recommendations. Enzyme assays revealed the presence of EDTA-inhibitable carbapenemase activity in crude extracts of the four P. aeruginosa isolates. Analytical isoelectric focusing revealed the presence of two b-lactamase bands with pIs of 8.0 and of 8.2 (data not shown). PCR experiments followed by sequencing identified b-lactamase genes encoding the carbapenemase IMP-13 and the chromosome-encoded AmpC and OXA-50. Characterization of the variable regions of the class 1 integrons carrying the blaIMP-13 cassette from the Belgian P. aeruginosa isolates revealed structures identical to those previously found in the Italian epidemic clone, harbouring integron InPSG, which was also the most common integron platform among Italian isolates, and in isolates from Romania and Austria. These isolates carried the blaIMP-13 and aacA4 cassettes. Moreover, PCR mapping showed that InPSG was inserted into a Tn5051-like transposon as described for the InPSG-harbouring Italian isolates, suggesting a common origin for these genetic structures. In order to investigate the genomic relatedness of the P. aeruginosa isolates, a novel semi-automated rep-PCR technique (DiversiLabTM, bioMerieux) was carried out. This technique revealed that all four Belgian isolates exhibited banding patterns that were identical to the Italian reference clone AV65 (data not shown), suggesting a clonal relatedness between the Belgian isolates and the Italian epidemic clone. By multilocus sequence typing analysis the AV65 isolate and all the Belgian isolates were identified as belonging to ST621 and by serotyping all were identified as serotype O4. Interestingly, the same ST was also reported for the IMP-13-producing isolates from Austria and Argentina. Altogether, these results revealed a common genetic lineage for the IMP-13-producing P. aeruginosa isolates circulating worldwide. Efforts aimed at purifying plasmids carrying blaIMP-13 or transferring the resistance determinant by conjugation or transformation were unsuccessful. Southern blot hybridization, using a blaIMP-13 probe and a 16S rRNA probe after I-Ceu I digestion and PFGE separation, showed that the blaIMP-13 gene was chromosomally located, since the bands that hybridized with the blaIMP-13 probe also hybridized with the 16S rRNA probe (data not shown). The overall genomic relatedness of the IMP-13-producing isolates and the fact that the blaIMP-13 gene was inserted in a conserved genetic context point to a common ancestry for the IMP-13-producing strains disseminated in South America, Italy, Romania, Austria and now in Belgium, thus underscoring the role of the ST621 clonal lineage as a highly successful P. aeruginosa epidemic clone.

The telomeric protein SNM1B/Apollo is required for normal cell proliferation and embryonic development

Conserved metallo β-Lactamase and β-CASP (CPSF-Artemis-Snm1-Pso2) domain nuclease family member SNM1B/Apollo is a shelterin-associated protein that localizes to telomeres through its interaction with TRF2. To study its in vivo role, we generated a knockout of SNM1B/Apollo in a mouse model. Snm1B/Apollo homozygous null mice die at birth with developmental delay and defects in multiple organ systems. Cell proliferation defects were observed in Snm1B/Apollo mutant mouse embryonic fibroblasts (MEFs) owing to high levels of telomeric end-to-end fusions. Deficiency of the nonhomologous end-joining (NHEJ) factor Ku70, but not p53, rescued the developmental defects and lethality observed in Snm1B/Apollo mutant mice as well as the impaired proliferation of Snm1B/Apollo-deficient MEFs. These findings demonstrate that SNM1B/Apollo is required to protect telomeres against NHEJ-mediated repair, which results in genomic instability and the consequent multi-organ developmental failure. Although Snm1B/Apollo-deficient MEFs exhibited high levels of apoptosis, abrogation of p53-dependent programmed cell death did not rescue the multi-organ developmental failure in the mice.

Characterization of ertapenem resistance in Klebsiella pneumoniae from Croatia

ABSTRACT Klebsiella pneumoniae isolates with reduced susceptibility to carbapenems were recently reported in USA, UK, Turkey and some other countries of the world. Recently eight Klebsiella pneumoniae strains with reduced susceptibility to carbapenems were isolates in four different hospitals in Croatia. The aim of the study was to determine the mechanisms of ertapenem resistance in these strains. Antibiotic susceptibilities were determined by broth microdilution method. Transferability of ertapenem resistance was determined by conjugation (broth mating method) using E. coli A15 R- as recipient. Production of metallo β-lactamases was detected by double-disk synergy test and E test. Β-lactamases were characterized by PCR with primers specific for extended-spectrum β-lactamases, plasmid-mediated ampC β-lactamases, metallo β-lactamases of VIM and IMP series, KPC and OXA-48 β-lactamases. Genotyping of the strains was performed by pulsed-field gel electrophoresis. All strains were resistant to ceftazidime, cefotaxime, ceftriaxone, piperacillin alone and combined with tazobactam, amoxycillin/clavulanate, gentamicin and ciprofloxacin. All except one strains showed resistance to ertapenem, intermediate susceptibility or resistance to meropenem and intermediate susceptibility or full susceptiblity to imipenem. One strain was resistant to all three carbapenems. Ertapenem resistance was not transferable by conjugation to E. coli recipient in neither of our strains. PCR revealed the presence of blaSHV and blaCTX-M genes. Multiplex PCR was positive for group 1 CTX-M β-lactamases. The strain resistant to all three carbapenem was positive by E test for MBLs. However, PCR was negative for VIM and IMP β-lactamases. No KPC or plasmid-mediated ampC β-lactamases were found. The strains were not clonally related as shown by PFGE and displayed distinct PFGE fingerprints. This is the first report of carbapenem resistant Klebsiellae in Croatia. Ertapenem resistance in Klebsiella was previously reported in UK, Turkey and Israel mainly due to the production of CTX-M β-lactamases of group 1 combined with porin loss (OmpK36 or OmpK35). The characterization of outer membrane porins needs to be done to clarify the mechanisms of ertapenem resistance in our strains. Further testing is necessary to determine the mechanism of carbapenem resistance in the strain with positive E test–MBL.

Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España

Pseudomonas aeruginosa strains producing metallo-beta-lactamases (MbetaL) are uncommon in Spain. This study describes the characterization of 9 new clonally related multiresistant P. aeruginosa isolates possessing the bla(VIM-2) gene in Cantabria (Northern Spain). P. aeruginosa clinical strains (1 per patient) were isolated in the Microbiology Service of Marqués de Valdecilla University Hospital between January 2004 and December 2006. Identification and preliminary susceptibility studies were performed with the MicroScan WalkAway system (Dade Behring, Sacramento, CA) and results were verified by a microdilution reference method. MICs of imipenem and meropenem for the 9 isolates ranged from 32 to 128 and 16 to 64 microg/mL, respectively. Nine isolates had a single Rep-PCR pattern and were intermediate or resistant to ceftazidime, cefepime, gentamicin, tobramycin, amikacin and ciprofloxacin. Eight of the 9 isolates were susceptible to aztreonam. Hydrolysis activity of imipenem in MbetaL-positive isolates ranged from 162+/-18 to 235+/-28 pmol/min/microg protein and was abolished in the presence of 5 mM EDTA. All isolates possessed an integron with genes aac(6')32, bla(VIM-2) and a putative transposase-encoding gene, flanked by the conserved 5'CS and 3'CS regions. In the clinical isolates studied, the presence of MbetaL VIM-2 sufficed to explain their resistance to carbapenems.

Carbapenem-Resistant &lt;i&gt;Acinetobacter baumannii&lt;/i&gt; Producing OXA-23 in Thailand

We investigated the resistance determinant of 13 clinical isolates of carbapenem-resistant Acinetobacter baumannii collected from a regional hospital in the north of Thailand. All isolates were multidrug resistant and produced the OXA-23 carbapenemase. The blaOXA-23 gene was found adjacent to ISAba1. Furthermore, two isolates carried the metallo β-lactamase gene, blaIMP. The blaOXA-23 and blaIMP genes were plasmid-mediated according to the transformation assays. This is the first description of OXA-23-producing A. baumannii from Thailand.

Metallo-β-lactamase-producing Pseudomonas aeruginosa isolated from a large tertiary centre in Kenya

This study was designed to characterize the beta-lactamase content of carbapenem-resistant Pseudomonas aeruginosa isolates recovered during 2006 and 2007 in a large tertiary-care centre in Nairobi, Kenya. Molecular characterization was done using PCR and sequencing, and typing was performed using pulsed-field gel electrophoresis (PFGE). In total, 416 P. aeruginosa isolates were obtained during that period, of which 57 (13.7%) were resistant to carbapenems. All carbapenem-resistant isolates tested positive for metallo-beta-lactamase (MBL) production. All MBL isolates produced VIM-2 with two types of integron structures. PFGE identified three clonally related groups of VIM-2-producing P. aeruginosa, including a pan-resistant clone that was responsible for nosocomial outbreaks during 2006 and 2007 in the intensive-care unit. These findings suggest that continuous molecular surveillance needs to be performed to monitor the spread within the hospital of this pan-resistant strain. This study is the first report of VIM-2-producing P. aeruginosa from the African continent.