Metallo Β-lactamase Research Articles

Serotyping and Antimicrobial Susceptibility Pattern of Escherichia coli Isolates from Urinary Tract Infections in Pediatric Population in a Tertiary Care Hospital.

Urinary tract infections (UTIs) in pediatric population are associated with high morbidity and long term complications. In recent years, there is increased prevalence of Escherichia coli (E. coli) strains producing extended spectrum β-lactamase, Amp C, and Metallo β-lactamase, making the clinical management even more difficult. This study was aimed to detect the serotypes and to determine antimicrobial susceptibility profile of E. coli isolates from urine samples of children <10 yrs old. A total of 75 pure E. coli strains isolated from patients with symptoms of UTI and colony count ≥105 organisms/mL were included in the study. Antibiotic sensitivity pattern showed maximum resistance to nalidixic acid (98.7%), followed by ampicillin (97.3%), amoxi-clavulanate (96%), and fluoroquinolones (92%) while most of the isolates were found sensitive to piperacillin-tazobactam (13.3%), nitrofurantoin (5.3%), and meropenem (1.3%). 48% of the strains were ESBL producer (extended spectrum beta lactamase). 44% strains were typable withantisera used in our study and the most common serogroup was O6 (33.3%) followed by O1 (15.1%) and O15 (15.1%). To conclude, judicious use of antibiotics according to hospital antibiotic policy and infection control measures should be implemented to prevent spread of multidrug resistant organisms.

Mechanisms of Carbapenem Resistance in K.pneumoniae and E. coli from Bloodstream Infections in India

Introduction: Emergence and global spread of carbapenemase producing Enterobacteriaceae (CPE) are of great concern in healthcare settings. Resistance to carbapenem is mostly conferred by metallo β-lactamase (IMP, VIM and NDM) and carbapenem hydrolyzing class D β-lactamase (OXA-48 like). The aim of this study was to characterise the molecular mechanism of resistance in the clinical isolates of Enterobacteriaceae causing bacteremia and showing resistance to β-lactams, including carbapenems. Materials and Methods: Isolates of E.coli (n=42) and K. pneumoniae (n=134) from blood culture collected during 2013-2015 were screened for carbapenemase production by using carba NP test and the presence of carbapenem resistant genes (KPC, IMP, VIM, NDM and OXA- 48 like). Sequencing was performed for the randomly selected isolates positive for NDM and OXA-48 like.Results: Of the 176 isolates, 97% of the isolates were found to be positive with carba NP test. Carba NP test has the sensitivity, specificity, PPV and NPV of 98%, 50%, 99% and 20% respectively. Each of blaNDM and blaOXA-48 like was seen in 32% of the tested isolates. Co-production of blaNDM and blaOXA48 like and blaVIM and blaOXA48 were seen in 13% and 8% of isolates respectively. Noticeably, 3% of isolates were identified as co-producers of blaNDM, blaVIM and blaOXA48 like. All of the sequenced NDM and OXA-48 like were identified as NDM-1 and OXA-181 variants. Conclusion: Increasing incidence of OXA-48 like is worrisome in developing countries. Because of its weak hydrolytic acivity against broad spectrum cephalosporin and carbapenems, these may go undetected in routine screening. In particular, blaOXA48 like gene is mostly identified on the plasmid and is implicated as the cause for silent spread and outbreaks in hospitalized patients.

A Pilot Study on Carbapenemase Detection: Do We See the Same Level of Agreement as with the CLSI Observations.

Rapid identification of carbapenemase producing organisms is of great importance for timely detection, treatment and implementation of control measures to prevent the spread. The Modified Hodge Test (MHT) and Carba NP test is recommended by CLSI for the detection of carbapenemases in Enterobacteriaceae. However, MHT may give false positive results or fail to detect metallo β-lactamases (MBLs). In the US, MHT is the most widely used test for detection of carbapenemases and has been found to have a sensitivity and specificity of >90% for bla KPC producers. However, in India, the prevalence of bla NDM is higher than bla KPC producers. To evaluate the usefulness of CarbaNP in an Indian setting. A total of 260 isolates of carbapenem resistant E.coli (n=57), Klebsiella spp. (n=85), Pseudomonas aeruginosa (n=60), and Acinetobacter baumannii (58) isolated from clinical specimens between 2012-2014 at the Christian Medical College, Vellore were included in the study. All the carbapenem resistant isolates were subjected to CarbaNP, MHT and multiplex PCR for detection of carbapenemase genes. CarbaNP was found to be positive in 88% (n=50/57), 81% (n=69/51), 38% (n=23/60) and 81% (n=47/58) for E.coli, Klebsiella spp., P. aeruginosa, and A. baumannii respectively. While in MHT it showed, 89% (n=51/57) and 81 % (n=69/85) for E.coli and Klebsiella spp. respectively. In P.aeruginosa, synergy testing of imipenem plus cloxacillin showed that, 65% of CarbaNP negatives were ampC producers. Overall, the sensitivity and specificity of CarbaNP was found to be 94% and 100 for bla NDM; 77% and 100 % for bla OXA-48 like producers and 81% and 100% for CarbAcinetoNP respectively. This observation was more than what was reported in CLSI guidelines. Therefore, it is advisable to evaluate an assay for better laboratory diagnosis at respective regions.

Syntheses and Biological Evaluations of Highly Functionalized Hydroxamate Containing and N-Methylthio Monobactams as Anti-Tuberculosis and β-Lactamase Inhibitory Agents.

Both the resurgence of tuberculosis (TB) and antibiotic resistance continue to threaten modern healthcare and new means of combating pathogenic bacterial infections are needed. The syntheses of monobactams possessing hydroxamate and N-methylthio functionality are described, as well as their anti-TB, in vitro β-lactamase inhibitory, and general antimicrobial evaluations. A number of compounds exhibited significant anti-TB and β-lactamase inhibitory activity, with MIC values in the range of 25 to < 0.19 μM against Mycobacteria tuberculosis (M.tb), and Ki values in the range of 25-0.03 μM against purified NDM-1 and VIM-1 lystate metallo β-lactamases. This work suggests that these scaffolds may serve as promising leads in developing new antibiotics and/or β-lactamase inhibitors.

Antimicrobial Effect of Imipenem-Functionalized Fe2O3 Nanoparticles on Pseudomonas aeruginosa Producing Metallo β-lactamases.

Resistant strains of Pseudomonas aeruginosa to imipenem was medical treatment problem, especially in burnt units of hospitals. This study was conducted to evaluate the antimicrobial effect of Fe2O3 nanoparticles alone and functionalized with imipenem on P. aeruginosa starins producing metallo β-lactamases (MBL). A disk diffusion method was used to isolate a clinical P. aeruginosa producing Metallo β-lactamases with imipenem resistance. The minimum inhibitory concentration (MIC) of Fe2O3 nanoparticles and imipenem were calculated against the bacteria. The antimicrobial effect of nanoparticles functionalized with the antibiotic was determined. Standard strain of P. aeruginosa ATCC: 27853 was used as control. The clinical sample was resistant to imipenem (up to 28 μg.mL-1). Similarly, MIC of the nanoparticles against the isolate was 160 μg.mL-1. Subsequently, the combination of 16 pg.mL-1 of antibiotic with 80 μg.mL-1 of Fe2O3 nanoparticles were able to inhibit the growth of the isolate. Fe2O3 nanoparticles functionalized with imipenem can impair antibiotic resistance mechanisms of bacteria as it can make the imipenem resistant the aforementioned bacterium more susceptible to weaker concentrations of antibiotic. It also has its own antibacterial effect in certain concentrations.

Community acquired multi-drug resistant clinical isolates of Escherichia coli in a tertiary care center of Nepal.

BackgroundMulti-drug resistance (MDR) in Gram-negative organisms is an alarming problem in the world. MDR and extensively-drug resistance (XDR) is in increasing trend due to the production of different types of beta (β)-lactamases. Thus the aim of this study was to document the incidence of MDR and XDR in clinical isolates of Escherichia coli and also to find out the enzymatic mechanisms of β-lactam antibiotics resistance.MethodsTwo hundred clinical isolates of Escherichia coli (E. coli) identified by standard laboratory methods were studied. Antibiotic susceptibility profile was performed for all the isolates and the suspected isolates were phenotypically tested for the production of extended spectrum β-lactamase (ESBL), metallo β-lactamase (MBL) and AmpC β-lactamase (AmpC) by recommended methods.ResultsAround three-fourth (78%) of the total isolates were multi-drug resistant. ESBL, MBL and AmpC production was found in 24%, 15% and 9% of isolates respectively. Amikacin, chloramphenicol and colistin were found to be the most effective antibiotics.ConclusionsHigh percentage of MDR was observed. β-lactamase mediated resistance was also high. Thus, regular surveillance of drug resistance due to β-lactamases production and infection control policy are of utmost importance to minimize the spread of resistant strains.

Mechanism of Meropenem Hydrolysis by New Delhi Metallo β-Lactamase

New Delhi metallo β-lactamase (NDM-1) is a recent addition to the metallo-β-lactamases family that is capable of hydrolyzing most of the available antibiotics, including the new generation carbapenems. Here, we report the mechanism of Meropenem hydrolysis catalyzed by NDM-1 based on hybrid quantum-mechanical/molecular-mechanical metadynamics simulations. Our work elicits the molecular details of the catalytic mechanism and free energy profiles along the reaction pathway. We identified the ring opening step involving the nucleophilic addition of the bridging hydroxyl group on the β-lactam ring of the drug as the rate-determining step. Subsequent protonation of β-lactam nitrogen occurs from a bulk water molecule that diffuses into the active site and is preferred over proton transfer from the bridging hydroxyl group or from the protonated Asp124. The roles of important active site residues of NDM-1 and change in the coordination environment of Zn ions during the hydrolysis are also scrutinized.

Biofilm Formation and β-Lactamase Production in Burn Isolates of Pseudomonas aeruginosa.

Background:Pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents. Plasmid-mediated drug resistance also occurs by the production of extended-spectrum β-lactamases (ESBL), metallo β-lactamases (MBL), and AmpC β-lactamases. Another important factor for establishment of chronic infections by P. aeruginosa is biofilm formation mediated by the psl gene cluster.Objectives:The aim of this study was to evaluate biofilm formation and presence of the pslA gene in burn isolates of P. aeruginosa as well as the association of antibiotic resistance, MBL, ESBL and AmpC β-lactamase production with biofilm formation among the isolates.Materials and Methods:Sixty-two burn isolates of P. aeruginosa were obtained from Shahid Motahari Hospital in Tehran from August to October 2011. Antibiotic susceptibility was determined by the disc diffusion assay. MBL, AmpC and ESBL production were screened using the double disc synergy test, AmpC disc test and combined disc diffusion assay, respectively. The potential to form biofilm was measured using the microtiter plate assay and pslA gene was detected using specific primers and PCR.Results:Biofilm formation was observed in 43.5% of the isolates, of which 66.7% produced strong and 33.3% formed weak biofilms. All biofilm-positive and 14.2% of biofilm-negative isolates harbored the pslA gene. MBL, AmpC and ESBL production were significantly higher in the biofilm-positive isolates (70.3%, 62.9% and 33.3%, respectively) compared to the biofilm-negative strains (31.4%, 34.2% and 20%, respectively). Overall, 19 isolates (30.6%) co-produced MBL and AmpC, among which the majority were biofilm-positive (63.1%). Finally, four isolates (6.4%) had all three enzymes, of which 3 (75%) produced biofilm.Conclusions:Biofilm formation (both strong and weak) strongly correlated with pslA gene carriage. Biofilm formation also correlated with MBL and AmpC β-lactamase production. More importantly, multiple-β-lactamase phenotype was associated with formation of strong biofilms.

Nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of Japanese Society of Chemotherapy, the Japanese Association for Infectious Diseases, and the Japanese Society for Clinical Microbiology in 2010: General view of the pathogens' antibacterial susceptibility.

The nationwide surveillance on antimicrobial susceptibility of bacterial respiratory pathogens from patients in Japan, was conducted by Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases and Japanese Society for Clinical Microbiology in 2010. The isolates were collected from clinical specimens obtained from well-diagnosed adult patients with respiratory tract infections during the period from January and April 2010 by three societies. Antimicrobial susceptibility testing was conducted at the central reference laboratory according to the method recommended by Clinical and Laboratory Standard Institutes using maximum 45 antibacterial agents. Susceptibility testing was evaluable with 954 strains (206 Staphylococcus aureus, 189 Streptococcus pneumoniae, 4 Streptococcus pyogenes, 182 Haemophilus influenzae, 74 Moraxella catarrhalis, 139 Klebsiella pneumoniae and 160 Pseudomonas aeruginosa). Ratio of methicillin-resistant S.aureus was as high as 50.5%, and those of penicillin-intermediate and -resistant S.pneumoniae were 1.1% and 0.0%, respectively. Among H.influenzae, 17.6% of them were found to be β-lactamase-non-producing ampicillin (ABPC)-intermediately resistant, 33.5% to be β-lactamase-non-producing ABPC-resistant and 11.0% to be β-lactamase-producing ABPC-resistant strains. Extended spectrum β-lactamase-producing K.pneumoniae and multi-drug resistant P.aeruginosa with metallo β-lactamase were 2.9% and 0.6%, respectively. Continuous national surveillance of antimicrobial susceptibility of respiratory pathogens is crucial in order to monitor changing patterns of susceptibility and to be able to update treatment recommendations on a regular basis.

Genetic acquisition of NDM gene offers sustainability among clinical isolates of Pseudomonas aeruginosa in clinical settings.

New Delhi metallo β-lactamases are one of the most significant emerging resistance determinants towards carbapenem drugs. Their persistence and adaptability often depends on their genetic environment and linkage. This study reports a unique and novel arrangement of bla NDM-1 gene within clinical isolates of Pseudomonas aeruginosa from a tertiary referral hospital in north India. Three NDM positive clonally unrelated clinical isolates of P. aeruginosa were recovered from hospital patients. Association of integron with bla NDM-1 and presence of gene cassettes were assessed by PCR. Genetic linkage of NDM gene with ISAba125 was determined and in negative cases linkage in upstream region was mapped by inverse PCR. In which only one isolate’s NDM gene was linked with ISAba125 for mobility, while other two reveals new genetic arrangement and found to be inserted within DNA directed RNA polymerase gene of the host genome detected by inverse PCR followed by sequencing analysis. In continuation significance of this novel linkage was further analyzed wherein promoter site detected by Softberry BPROM software and activity were assessed by cloning succeeding semi-quantitative RT-PCR indicating the higher expression level of NDM gene. This study concluded out that the unique genetic makeup of NDM gene with DNA-dependent-RNA-polymerase favours adaptability to the host in hospital environment against huge antibiotic pressure.

Isolation and identification of Acinetobacter species with special reference to antibiotic resistance.

Background:Acinetobacter is clinically important pathogen with widespread resistance to various antibiotics. We assessed the incidence of Acinetobacter infection at a tertiary care hospital, analyze their resistance pattern and identify the production of extended spectrum β-lactamases (ESBLs) and metallo β-lactamases (MBLs).Materials and Methods:The study was conducted in tertiary care hospital, India over a period of 2 years. Acinetobacter species were isolated from various clinical samples received in Department of Microbiology. After identification, Acinetobacter isolates were speciated and antibiotic susceptibility was determined by the standard disc diffusion method. ESBL and MBL production was detected by the double disc synergy test and combined disc diffusion test respectively.Results:Of 3298 infected samples, 111 (3.36%) were found to be Acinetobacter. The most predominant species was Acinetobacter calcoaceticus-A. baumannii (Acb) complex (72%). High incidence of resistance was recorded for piperacillin (55%), followed by ceftriaxone (46%) and ceftazidime (46%). Isolation rate and antibiotic resistance was higher in the Intensive Care Units (ICUs) of the hospital. ESBL and MBL production was detected in 31.5% and 14.4% of the isolates respectively.Discussion and Conclusion:A high level of antibiotic resistance was observed in our study and maximum isolation rate of Acinetobacter was in the ICUs. Acb complex was the most predominant and most resistant species. The analysis of susceptibility pattern will be useful in understanding the epidemiology of this organism in our hospital setup, which will help in treating individual cases and controlling the spread of resistant isolates to other individuals.

Klebsiella Pneumoniae in Septicemic Neonates with Special Reference to Extended Spectrum β-lactamase, AmpC, Metallo β-lactamase Production and Multiple Drug Resistance in Tertiary Care Hospital.

Background:β-lactamases viz., extended spectrum β-lactamase (ESBL), AmpC, and metallo β-lactamase (MBL) production in Klebsiella pneumoniae has led to a serious concern about septicemic neonates in Neonatal Intensive Care Units due to high resistance against commonly used antimicrobials.Purpose:To study the prevalence of ESBL, AmpC, and MBL production in K. pneumoniae isolates in neonatal septicemia, to check antimicrobial susceptibility to various drugs including tigecycline; and to assess burden of multiple drug resistance (MDR).Materials and Methods:Total 24 clinical isolates of K. pneumoniae isolated from 318 blood samples of suspected cases of neonatal septicemia were studied. Isolates were screened for ESBL, AmpC, and MBL production by Clinical and Laboratory Standards Institute (CLSI) disk method, AmpC cefoxitin screen, and imipenem, meropenem, ceftazidime disk screen respectively; and confirmation was done by CLSI phenotypic disk confirmatory test, AmpC sterile disk method, and imipenem ethylenediamine tetracetic acid double disk synergy test respectively. Antimicrobial susceptibility was determined by Kirby–Bauer's disk diffusion method. Efficacy of tigecycline was evaluated using United States Food and Drug Administration guidelines.Results:Of the 24 K. pneumoniae isolates, co-production of AmpC + MBL was found in more number of isolates (67%) (P < 0.0001) compared to single enzyme production (ESBL and MBL 8% both, AmpC 12.5%). Rate of resistance for penicillins and cephalosporins was highest. Susceptibility was more for imipenem, co-trimoxazole, and meropenem. Nonsusceptibility to tigecycline was low (21%). A total of 23 (96%) isolates were MDR.Conclusions:Routine detection of ESBL, AmpC, and MBL is required in laboratories. Carbapenems should be kept as a last resort drugs. Trend of tigecycline susceptibility has been noted in the study. Continued monitoring of susceptibility pattern is necessary to detect true burden of resistance for proper management.

In vitro Antibacterial Activity of Aqueous and Ethanol Extracts of Aristolochia indica and Toddalia asiatica Against Multidrug-Resistant Bacteria.

Bacteria have developed multidrug resistance against available antimicrobial agents. Infectious diseases caused by these multidrug-resistant bacteria are major causes of morbidity and mortality in human beings. Synthetic drugs are expensive and inadequate for the treatment of diseases, causing side effects and ineffective against multidrug-resistant bacteria. The medicinal plants are promising to have effective antimicrobial property due to presence of phytochemical compounds like alkaloids, flavanoids, tannins and phenolic compounds. The present study aimed to find the antimicrobial activity of medicinal plants against multidrug-resistant bacteria. Multidrug-resistant bacteria were identified by Kirby-Bauer disc diffusion method. Production of β-lactamases (extended spectrum β-lactamases, metallo β-lactamase and AmpC β-lactamase) were identified by combination disc method. Antibacterial activity of aqueous and ethanol extract of Aristolochia indica and Toddalia asiatica were detected by agar well diffusion assay and minimum inhibitory concentration. All bacteria used in this study showed antibiotic resistance to ≥3 antibiotics. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Vibrio cholerae were found to be positive for β-lactamase production. Ethanol extract of Aristolochia indica showed more significant antibacterial activity against multidrug-resistant bacteria than Toddalia asiatica. Ethanol extracts of Aristolochia indica and Toddalia asiatica showed minimum inhibitory concentration values of 50-100 μg/ml and 100-200 μg/ml, respectively against multidrug-resistant bacteria. From this study, it was concluded that Aristolochia indica has more potential to treat multidrug-resistant bacteria than Toddalia asiatica.

A prospective study on aetiological agents of acute and chronic suppurative otitis media

Otitis media is a persistent, insidious, and dangerous disease because of multiple aetiology and serious complications. Hence, a study was conducted to find out the bacterial and fungal aetiology of suppurative otitis media. Aim: The aim was to find the prevalence of bacterial and fungal pathogens in acute and chronic otitis media. Materials and Methods: A total of 260 patients having otitis media belonging to all age groups and both sexes who attended the ENT outpatient department were selected for study for a period of 1-year. Ear discharge was collected under aseptic precautions using sterile cotton swabs. Both bacterial and fungal culture were done. The antimicrobial susceptibility of bacterial isolates was done including detection of extended-spectrum β-lactamase and detection of metallo β-lactamase for resistant strains. Results: In acute suppurative otitis media, Gram-positive organisms 48 (55.18%) were predominant compared to Gram-negative organisms 37 (42.52%). No fungal isolates were obtained in the present study. In the case of chronic suppurative otitis media (CSOM), bacteria were isolated in 129 (74.57%) cases and fungus in 41 (23.7%) cases. Polymicrobial infection was found only in 14 (10.85%) cases. Among the bacterial isolates in CSOM, Gram-negative organisms (65.35%) were more compared to Gram-positive organisms (35.65%).

Simplified Protocol for the Phenotypic Identification of Carbapenem Resistance Mechanism in Enterobacteriaceae.

Dear Editor, Carbapenems are often one of the last resorts available for the treatment of multi-drug resistant Enterobacteriaceae infections. However, in the recent past, reports were accumulating on the emergence and dissemination of carbapenem resistance [1]. Therefore, carbapenem resistance in Enterobacteriaceae is considered as one of the major public health problems worldwide. According to recent report [2], there are three major mechanisms involved in carbapenemase production, which confers carbapenem resistance among Enterobacteriaceae. They are, production of Klebsiella pneumoniae carbapenemase (blaKPC), production of metallo β-lactamase (MBL) encoded by blaNDM, blaIMP and blaVIM genes, and production of Oxacillinase-48(blaOXA-48) or its derivatives. However, the other mechanisms of carbapenem resistance often observed among Enterobacteriaceae are loss of porin channel along with the co-production of Extended Spectrum β-lactamase (ESBL) or Ambler Cass C (AmpC) β-lactamase [1]. Identification and documentation of carbapenem resistance in Enterobacteriaceae is imperative to contain their spread. Based on the literature review and minor modifications from the previously published protocols [1,3] herewith we present a simplified protocol for the easy detection and differentiation of the mechanism of resistance in Carbapenem Resistant Enterobacteriaceae (CRE) by combined Disk Test (CDT), a phenotypic method. In CDT, the synergism is identified as the significant enlargement (≥5 mm) of the Zone of Inhibition (ZOI) around the antibiotic disk with specific inhibitor, compared to the ZOI around the antibiotic disk without inhibitor. All clinical isolates of Enterobacteriaceae could be initially screened with ertapenem (ETP) disk (10μg), which is reportedly having high sensitivity in detecting carbapenemases among all carbapenems [4]. Then, the clinical isolates showing ZOI <25mm around ETP disk [3] could be further tested with following disks in single 150mm petri plate: ETP (10 μg), ETP+clavulanate (10 + 10μg), ETP+cloxacillin (10 + 750μg), ETP+boronate (10 + 400μg), ETP+EDTA (10 + 585μg), and temocillin (30 μg); optionally aztreonam (30 μg) disk may be included for susceptibility testing. Then the mechanism of carbapenem resistance can be identified based on the synergy observed with specific inhibitor and/or resistance to temocillin as described in [Table/Fig-1]. [Table/Fig-1]: Combined disk tests for the detection of common mechanisms of carbapenem resistance in Enterobacteriaceae (important test for each mechanism is shaded with blue colour) (*ZOI<10mm around the disk) Although the above protocol is highly reliable in detecting most isolates expressing any one of the major carbapenem resistance mechanisms, it may fail in detecting the isolates expressing more than one type of resistance mechanism. That is, production of more than one type of carbapenemases and/or ESBLs or AmpC enzymes with porin loss results in the loss of synergism. Further, this may lead to the false identification of KPC or MBL as OXA-48. Therefore, again we are dependent to molecular tools for the confirmation of OXA-48. However, this protocol would be helpful to reduce the need of molecular diagnosis to selective isolates especially in resource-limited settings. In conclusion, this protocol facilitates timely detection of CRE and identification of the mechanism of carbapenem resistance in Enterobacteriaceae; thus, it could help timely implementation of infection control measures.

Isolation of a carbapenem-resistant K1 serotype Klebsiella pneumonia strain and the study of resistance mechanism

To study the virulence and mechanism of carbapenem resistance of a clinical isolate of carbapenem-resistant K1 serotype Klebsiella pneumonia strain. Identification of isolate was carried out with VITEK-2 compact system. Antimicrobial susceptibility was determined by E-test; Metallo β-lactamases and carbapenemases screening were conducted by imipenem-EDTA double disc synergy test and modified Hodge test, respectively.Specific polymerehse chain reaction (PCR) and DNA sequencing were preformed to detect the virulence genes including K1, K2, K5, K20, K54, K57, magA, rmpA, wcaG and a series of β-lactamase resistence genes. Conjunction experiment was also performed. The plasmids of transconjugants were submitted to PCR-based replicon typing (PBRT) method. Molecular typing was performed by multilocus sequence typing (MLST). Antimicrobial susceptibility testing revealed that the Klebsiella pneumonia strain was resistant to most of the antibiotics used in clinic. Phynotype confirmary rest revealed the production of carbapanemases, while Metallo β-lactamases were negative; PCR and DNA sequencing confirmed the isolate was positive for blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-1 and virulence genes K1, magA, rmpA, wcaG simultaneously; blaKPC-2 was transferred from donor to Escherichia EC600 by conjunction experiment, while no virulence genes were found in the transconjugants. PBRT revealed that Frep plasmid was found in transconjugants. MLST analysis revealed that this strain belonged to ST23. K1 serotype Klebsiella pneumonia strain carries virulence genes and carbapenem resistance gene blaKPC-2, noteworthily the carbapenem resistance genes can be transferred through horizontal transmission on plasmids.

Association of a Zn(2+) containing metallo β-lactamase with the anticancer antibiotic mithramycin.

Pathogenic bacteria that are resistant to β-lactam antibiotics mostly utilize serine β-lactamases to degrade the antibiotics. Current studies have shown that different subclasses of metallo β-lactamases (E[MBL]) are involved in the defense mechanism of drug resistant bacteria. Here we report that the Zn(2+) containing subclass B1 E[MBL] from Bacillus cereus binds to a naturally occurring anti-cancer drug mithramycin (MTR). Spectroscopic (CD and fluorescence) and isothermal titration calorimetry studies show that MTR forms a high affinity complex with the Zn(2+) ion containing E[MBL]. Abolished interaction of MTR with apo E[MBL] suggests that the formation of this high affinity complex occurs due to the potential of MTR to bind bivalent metal ions like Zn(2+). Furthermore, CD spectroscopy, dynamic light scattering and differential scanning calorimetry studies indicate that the strong association with sub-micromolar dissociation constant leads to an alteration in the enzyme conformation at both secondary and tertiary structural levels. The enzyme activity decreases as a consequence to this conformational disruption arising from the formation of a ternary complex involving MTR, catalytic Zn(2+) and the enzyme. Our results suggest that the naturally occurring antibiotic MTR, a generic drug, has the potential as an E[MBL] inhibitor.

Prevalence of Extended-Spectrum and Metallo β-Lactamase Production in AmpC β-Lactamase Producing Pseudomonas aeruginosa Isolates From Burns.

Background:Pseudomonas aeruginosa is one of the most common causes of nosocomial infections. Resistance of P. aeruginosa to β-lactam antibiotics may be the result of acquired resistance through mutation and over production of various antibiotic inactivating enzymes. This research aimed to determine the prevalence of extended-spectrum β-lactamases (ESBL) and metallo β-lactamase (MBL) production as well as the presence of their related genes among AmpC β-lactamase producing P. aeruginosa isolated from burns.Objectives:The current study aimed to determine the prevalence of class A ESBL and MBL production in relation to the presence of their related genes among AmpC β-lactamase producing P. aeruginosa isolated from burns.Materials and Methods:The antimicrobial susceptibility of 51 P. aeruginosa isolates from patients with burns was examined against 13 antibiotics by the disc diffusion method. Minimum inhibitory concentrations (MIC) for imipenem and ceftazidime were measured by the microdilution method. AmpC production was detected by AmpC disc and the modified three-dimensional extract tests. ESBL phenotype was confirmed by the double disc synergy test (DDST). Presence of β-lactamase genes was detected by specific primers and polymerase chain reaction (PCR).Results:All isolates were multidrug resistant. AmpC, ESBL and MBL production were observed in 35 (68.6%), 20 (39.2%) and 19 (37.3%) isolates, respectively. Overall, 43 isolates (84.3%) carried β-lactamase genes, out of which 31 (60.8%) harbored blaAmpC, 20 (39.2%) had blaTEM and 11 (21.6%) carried blaPER-1 genes. Among the AmpC producers, two isolates (6.5%) carried blaAmpC + blaESBL, 13 (41.9%) had blaAmpC + blaMBL and six (19.4%) produced the three enzymes.Conclusions:A high prevalence of multiple β-lactamase production was observed among the AmpC producers (60%), of which the majority co-produced AmpC and MBL. The current study results showed correlation between β-lactamase production and the presence of antibiotic resistance genes in the isolates.

Carbapenemase NDM-1, Structural Analysis of the Catalytic Mechanism

The New Delhi Metallo β-lactamase (NDM-1), first identified in Klebsiella pneumoniae has been shown to hydrolyze nearly all clinical β-lactam antibiotics including carbapenems, considered "last resort" antibiotics. Its gene resides on mobile plasmids that move between different strains of bacteria posing a serious global threat to human health. There have also been reports of several variants, up to NDM-9, some with increased carbapenemase activity. As part of the NIGMS PSI:Biology effort, the Midwest Center for Structural Genomics (MCSG) together with the Structures of Mtb Proteins Conferring Susceptibility to Known Mtb Inhibitors partnership, made significant progress in investigating the enzyme atomic structure and catalytic mechanism. A large number of protein constructs as well as mutants were made and a number of high-resolution structures of NDM-1 (no Zn, one Zn, two Zn, two Mn or Cd, and complexed with antibiotics) and NDM-1 variants, NDM-2, NDM-3, NDM-4, NDM-5 and NDM-6 have been determined. We have determined the two structures of Michaelis complex: NDM-1 with two cadmium ions and a mixture of hydrolyzed and unhydrolyzed ampicillin (1.50 Å) and one with two cadmium ions and partly hydrolyzed faropenem (2.00 Å). The crystal structures revealed a ligand-binding pocket consisting of several flexible loops capable of accommodating many β-lactam substrates of different sizes and shapes. The structures with various metals suggest that the distance between the two metal atoms is closely correlated with substrate binding efficiency and hydrolysis and the pH-dependency of catalytic activity. For better understanding of catalytic mechanism of NDM-1, particularly the dynamics of substrate binding and the energy surfaces along the suggested reaction pathways, molecular dynamics calculations and hybrid classical/quantum (QM/MM) calculations were performed. This work was supported by NIH Grant GM094585 and by the U.S. DOE, OBER contract DE-AC02-06CH11357

Molecular characterization of VIM1 isolates of Pseudomonas aeruginosa isolated from cases of sinusitis

Background Acquired metallo-b-lactamases (MBLs) are emerging resistance determinants in clinically relevant Gram negative species. These enzymes confer broad spectrum b lactam resistance, including resistance to carbapenems. Their potential for rapid and wide dissemination makes them of great concern. Methods This study describes the molecular characterization of 15 Pseudomonas aeruginosa strains, isolated from endoscopic pus specimens, obtained from sinusitis cases. Susceptibility testing was performed by disk diffusion assay. The MIC of ceftazidime was determined by E test. Ceftazidime (CDM) - EDTA combined disc test was used to screen for class B b lactamase production. Plasmid DNA extraction was performed by alkali lysis method. The carriage of the MBL gene, blaVIM and IMP1 gene were screened by PCR. PCR products of 7 positive strains for MBL were sequenced and analysed using bioinformatics tools. Results Metallo-beta-lactamase was present in 9 strains. These results correlated with the presence of VIM1 gene by PCR. IMP1 gene was absent in all the 15 strains. Plasmid was present in 10 strains only. The nucleotide and deduced protein sequences were analyzed with the software available over the Internet. The nucleotide sequences reported in the study has been submitted to the Genbank and accession numbers were obtained (KF975367, KF975368, KF975369, KF975370, KF975371, KF975372, KF975373). A Phylogenetic tree was constructed and it consisted of 2 clades. Both the clades had evolved from the common ancestor VIM-28. Conclusion Direct detection of MBL genes may circumvent the problems of the phenotypic detection of carbapenemase producing microorganisms especially those exhibiting low carbapenem MICs.