ABSTRACT
Background: Resistance due to New Delhi metallo-?-lactamase (NDM) and OXA-48/181 continues to emerge as a threat which is associated with nosocomial outbreaks and is a serious healthcare concern. Phenotypic detection being laborious and time-consuming requires rapid detection of NDM and OXA-48/181, which is achieved through real-time polymerase chain reaction (RT-PCR). Materials and Methods: In this study, RT-PCR assay was developed to simultaneously detect NDM and OXA-48/181. The assay was validated on 102 non-duplicate, phenotypically characterised clinical samples. Results: The assay showed a sensitivity and specificity of 97% and 100% for the detection of carbapenemases in comparison to conventional PCR. The in-house developed multiplex RT-PCR would help to rule-in the presence of NDM and OXA-48/181. Conclusions: Rapid detection of these carbapenemases would be assist in better patient management, in terms of accurate antimicrobial treatment, help in cohorting infected from uninfected patient to prevent spread.
ABSTRACT
Background & objectives: New Delhi metallo-?-lactamase 1 (NDM-1) cleaves the beta-lactam ring, and confers bacterial resistance against most of the beta-lactam antibiotics, except tigecycline and colistin. Among these two antibiotics, colistin is considered toxic, and therefore, its clinical use and dosage need cautious approach. In the present study, six organic acids were screened individually and in combination of two acids for their effectiveness against NDM-1 Escherichia coli and a combination of colistin and oxalic or succinic acid was tested to find out the potential of combination therapy for reducing the dose of toxic colistin. Methods: Antibacterial activity of the organic acid and their combinations was tested by disc diffusion method against NDM-1 E. coli, and minimum inhibitory concentration (MIC) was determined by broth dilution method. Synergistic effect between organic acids and colistin was tested by checkerboard method. Results: Oxalic acid showed the highest zone of inhibition (15�mm) followed by succinic acid, tartaric acid, fumaric acid, citric acid and malic acid. The combination of two acids did not increase the zone of inhibition significantly. MIC was found to be the lowest with oxalic acid and succinic acid (320 ?g/ml). In the presence of 160 ?g/ml oxalic acid or succinic acid, MIC of colistin was reduced from 8 to 4 ?g/ml, indicating synergistic effect. Interpretation & conclusions: Our findings showed that combination therapy using colistin and oxalic acid or succinic acid might find safe clinical application of this antibiotic in controlling infections due to NDM-1 bacteria.
ABSTRACT
ABSTRACT During the last 30 years there has been a dissemination of plasmid-mediated -lactamases in Enterobacteriaceae in Brazil. Extended spectrum -lactamases (ESBL) are widely disseminated in the hospital setting and are detected in a lower frequency in the community setting. Cefotaximases are the most frequently detected ESBL type and Klebsiella pneumoniae is the predominant species among ESBL producers. Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae became widely disseminated in Brazil during the last decade and KPC production is currently the most frequent resistance mechanism (96.2%) in carbapenem resistant K. pneumoniae. To date KPC-2 is the only variant reported in Brazil. Polymyxin B resistance in KPC-2-producing K. pneumoniae has come to an alarming rate of 27.1% in 2015 in São Paulo, the largest city in Brazil. New Delhi metallo--lactamase was detected in Brazil in 2013, has been reported in different Brazilian states but are not widely disseminated. Antimicrobial resistance in Enterobacteriaceae in Brazil is a very serious problem that needs urgent actions which includes both more strict adherence to infection control measures and more judicious use of antimicrobials.
ABSTRACT
Pseudomonas aeruginosa es un bacilo gramnegativo capaz de adquirir genes que codifican para la producción de metalo β-lactamasas (MBLs), las cuales son activas contra carbapenemos e inhibidas por agentes quelantes, como el ácido etilendiaminotetraacético (EDTA). Se determinó la efectividad del EDTA y del ácido mercaptoacético de sodio (SMA) en la detección de MBLs en 25 cepas de P. aeruginosa procedentes de diferentes hospitales y centros de investigación, empleando el método de discos combinados. Se evaluó el efecto de los antibióticos solos y combinados con EDTA, SMA o ambos. Se encontró que, mediante el aumento del diámetro de los halos de inhibición de los carbapenemos combinados con la mezcla EDTA/SMA (0,5 M/300 mg/mL) o sólo con EDTA (0,5 M), se pudieron diferenciar de manera clara y confiable las cepas productoras de MBLs de las no productoras, estableciéndose como punto de corte una diferencia de 5 mm o más entre los halos de inhibición del antibiótico sólo y combinado con el o los agentes quelantes.
Pseudomanas aeruginosa is a gram negative bacillus capable of acquiring genes which code for metallo β-lactamase production (MBLs) which are active against carbapenems and inhibited by chelating agents such as ethylenediaminetetraacetic acid (EDTA). We determined the effectiveness of EDTA and sodium mercaptoacetic acid (SMA) in the detection of MBLs on 25 P. aeruginosa strains from different hospitals and research centers, using the combined disk technique. We evaluated the effect of the antibiotics alone and combined with EDTA, SMA, or both. We found that through the increase of the diameter of the inhibition halos of the carbapenems combined with an EDTA/SMA mixture (0.5 M/300 mg/mL) o with EDTA alone (0.5 M), we could differentiate in a clear and reliable way the MBLs producing from the non producing strains, establishing as cutoff point a difference of 5 mm or more between the antibiotic alone and the antibiotic combined with the chelating agent(s).
ABSTRACT
OBJECTIVE To evaluate the spectrum of imipenem-resistant Pseudomnas aeruginosa and the production of metallo-?-lactamase.METHODS The clinical strains of P.aeruginosa were collected from Jan to Dec 2007.The results of antimicrobial susceptibility tests and detection of metallo-?-lactamase were analyzed.Antimicrobial susceptibility tests were performed by K-B methods;the production of metallo-?-lactamase was tested by CAZ-EDTA synergy method.RESULTS Sixty strains were isolated,imipenem-sensitive and resistant strains were 40(66.7%) and 20(33.3%),respectively,and 7 strains with metallo-?-lactamase were detected.Among imipenem-resistant strains,at least 90.0% strains were resistant to meropenem,gentamicin,tobramycin,ciprofloxacin and SMZ-TMP;at least 80.0% strains were resistant to piperacillin and piperacillin/tazobactam;50.0% strains were resistant to ceftazidime and cefepime;polymyxin E was less resistant than others.Twenty strains were resistant to at least 3 antimicrobial agents,which was obviously higher than 27.5% of imipenem-resistant strains.CONCLUSIONS The resistance rate of imipenem-resistant P.aeruginosa is higher than imipenem-sensitive ones.The production of metallo-?-lactamase is one of the mechanisms of P.aeruginosa resistance to imipenem and shou1d be detected carefully,which could help us medicate reasonably in clinic and avoid using medicine which could induce and strengthen the resistance.
ABSTRACT
OBJECTIVE To establish a simpler method for screening bacterial metallo-?-lactamase(MBL) by comparing Hodge test and double-disk synergy tests(DDSTs),and using 5 inhibitors such as sodium citrate,EDTANa2,EDTAK2,sodium dimercaptopropane sulfonate and mercaptoethanol.METHODS In Hodge test,5 inhibitors of different concentration were added to the imipenem(IMP) disks,the sizes of the inhibition zones were compared.Disk containing IMP and disk containing a metallo-?-lactamase inhibitor were used in DDSTs.The optimal inhibitors concentration and edge-to-edge distance between the two disks were selected.The performances of the Hodge test and the DDSTs were also compared.RESULTS Among the metallo-?-lactamase inhibitors used in this study,1∶15 diluted mercaptoethanol gave the most reproducible and the clearest results when a filter disk containing mercaptoethanol was placed near the IMP disk with the edge distance of 3 mm.CONCLUSIONS Mercaptoethanol-IMP DDSTs are simple,convenient and sensitive methods for screening MBL.
ABSTRACT
OBJECTIVE To study the drug resistance of Stenotrophomonas maltophilia in order to direct clinical drug application.METHODS The S.maltophilia was identified and its sensitivity was analyzed with VITEK2-AMS.The extended-spectrum beta-lactamases(ESBLs) and metallo ?-lactamase(MBL) were screened by double-disk synergy test and double-disk confirmatory test.RESULTS The resistance rate of S.maltophilia to cefoperazone-sulbactam,levofloxacin and SMZ-TMP was the lowest,accounted for 12.50%,13.64% and 14.77%,respectively.The ESBLs positive rate was 69.32%,the MBL positive rate was 69.32%.CONCLUSIONS The multidrug resistance of S.maltophilia is serious and full dose use of two or more antibacterials is recommended.
ABSTRACT
OBJECTIVE To study the resistant characteristics and the resistant mechanisms of Gram-negative bacilli(G-) to ?-lactam antibiotics in the local nosocomial infections.METHODS The effects of efflux pump inhibitor on MICs were determined.Phenotypes and isoelectric points of ?-lactamases(Bla) were determined.Bla Genes were amplified and sequenced.RESULTS Of all tested isolates,the resistant rates to the most antibiotics were high.Besides 10.5% isolates with the efflux pumps,all tested isolates produced Bla,among which extended spectrum ?-lactamases(ESBLs),cephalosporin ?-lactamase(AmpC Bla) and metallo-?-lactamase were responsible for 42.1%,17.5% and 7.0% isolates,respectively.The complete nucleotide sequences of the ampC genes in 8 Enterobacter cloacae isolates had very high homology with the ampC gene in E.cloacae ECLC074.CONCLUSIONS The production of Bla is the main resistant mechanism of G-to ?-lactam antibiotics.ESBLs are the most frequent Bla.All of the ampC genes of AmpC Bla-producing E.cloacae originate from the ampC gene in E.cloacae ECLC074.Imipenen is the best choice for the treatment of the infections caused by multidrug resistant G-.Piperacillin/tazobactam and cefoperazone/sulbactam can be used to treat the infections caused by drug-resistant non-fermentative bacilli.Amikacin is effective to treat the infections caused by AmpC Bla-producing E.cloacae.
ABSTRACT
OBJECTIVE To analyze homology,antimicrobial susceptibility and metallo-?-lactamase gene type of metallo-?lactamase producing Pseudomonas aeruginosa in burn wards.METHODS Antimicrobial susceptibility tests were performed with E-tests.Using 2-mercaptoethanol disc synergy test to screen metallo-?-lactamase(positive) strains from imipenem-resistant P.aeruginosa in burn wards.Metallo-?-lactamase gene and integrase gene were amplified and sequenced.Resistance plasmid transfer and curing experiments were implemented to study the transfer of imipenem resistance and plasmid DNA was extracted and purified with Qiagen Plasmid Mini Kit.Random amplified polymorphic DNA(RAPD) typing was carried out to analyze the homology of the isolates.(RESULTS) Six strains of P.aeruginosa were suggested to produce metallo-?-(lactamase) by 2(-mercaptoethanol) disc synergy test.Using primers described for bla(VIM),the amplifications were observed among all 6 isolates and a VIM-2 metallo-?-(lactamase) gene was identified by sequencing.All isolates which producing VIM-2 metallo-?-(lactamase) had class 1 integrase gene and derived from a same clonal origin.CONCLUSIONS(VIM-2) metallo-?-(lactamase) producing P.aeruginosa is prevalent in burn wards,all isolates which producing VIM-2 metallo-?-(lactamase) have class 1 integrase gene.
ABSTRACT
OBJECTIVE To study the existence of integron and metallo-?-lactamase in Stenotrophomonas maltophilia in our hospital.METHODS The antibiotic susceptibility was tested by K-B method.The genomic DNA and their plasmids were extracted.The integron and metallo-?-lactamase gene were amplified by polymerase chain reaction(PCR).RESULTS L1 and L2 genes were amplified in chromosomes and plasmids.Integrons Ⅰ and Ⅱ were detected in genomic DNA.CONCLUSIONS Existence of metallo-?-lactamase is one of reasons of multi-drug resistance in S.maltophilia.S.maltophilia can receive integrons and gain its multi-drug resistance from other strains.
ABSTRACT
OBJECTIVE To investigate the prevalence of metallo-?-lactamase in carbapenem-resistant clinical Pseudomonas aeruginosa isolates.METHODS Sixteen strains of P.aeruginosa isolated from Tianjin General Hospital during 2005-2006 which were resistant to all tested antibiotics besides carbapenem were studied.The general PCR was adopted to amplify the resistance genes of matallo-?-lactamae.Restriction endonucleases performed restriction analysis were used to certify the type of genes of matallo-?-lactamase by different endonuclease recognition site.RESULTS Two of the 16 clinical strains of P.aeruginosa(named P02 and P16) were confirmed bearing IMP-1 metallo-?-lactamase gene detected by PCR amplification and restriction analysis.CONCLUSIONS P.aeruginosa producing IMP-1 metallo-?-lactamase is the major type in our hospital.The hydrolyzing effect of the metallo-?-lactamase is not the main machanism in these carbapenem-resistant P.aeruginosa isolates.
ABSTRACT
OBJECTIVE To find out the imipenem-resistan metallo-?-lactamase in P.aeruginosa to provide the proof of treatment for clinic.METHODS A multi-disk was used to detect the metallo-?-lactamase of P.aeruginosa and the K-B disk method was used for monitoring of the antibiotic-resistance to 15 antibiotics.RESULTS Fifteen strains producing metallo-?-lactamase were isolated from 93 imipenem-resistant P.aeruginosa strains.The positive rate with metallo-?-lactamase was 16.1%(15/93).The susceptibility tests showed the lower resistance was to cefoperazone sulbactam(Sulperazone),amikacin and piperacillinl tazobactam(Tazocin).their resistance rate respectively was 16.8%,24.8% and 26.6%.The resistance rate to ciprofloxacin and ceftazidime was 43.7% and 33.6%.The resistance rate over 90.0% was to ampicillin,ampicilillin/sulbactam,cefazolin,cefpodoxime,cefuroxime and so on.CONCLUSIONS P.aeruginosa with imipenem-resistance shows seriou multidrug-resistance.The metallo-?-lactamase is the main reason for P.aeruginosa resistance to imipenem and cephalosporins.The doctors should choose antibiotics reasonably according to the susceptibility test when taking effective treatment to P.aeruginosa infection.
ABSTRACT
OBJECTIVE To investigate the antibiotical resistance and to understand the phenotype and genotype of metallo-?-lactamase in Chryseobacterium indologenes.METHODS The MIC to 25 C.!indologenes strains was detected by agar dilution method.Phenotype of metallo-?-lactamase was detected by three-disc synergy test and modified three dimension test.Polymerase chain reaction(PCR)amplification for both metallo?-lactamase and integrase gene was conducted for all isolates.Complete coding gene of metallo-?-lactamase and their DNA sequence analysis was conducted.Conjugation experiment was used to study the transmission of metallo-?-lactamase encoding gene.pIs Of ?-lactamase was measured by isoelectric focusing assay.RESULTS The antibiotical resistance of C.indologenes to imipenem and meropenem was 88.0%,respectively.However,gatifloxacin,levofloxacin and rifampin had better antimicrobiotial ability to C.indologenes compared with other antibiotics in MIC Nineteen strains were identified to produce metallo-?-lactamase using three-disc synergy test and modified three dimension test.Twenty strains were detected to have metallo-?-lactamase genotype by PCR amplification using IND-like common prime sets and four prime sets of complete coding gene.Among them,9 strains were detected to have blaIND-1 genotype and 10 strains to have blaIND-2 genotype,strain CI-25 was identified to have blaIND-like genotype.The mutation of base sequences and amino acid sequences in 5 strains were found simultaneously.Conjugation experiment showed that metallo-?-lactamase encoding gene couldn′t transfer.Nineteen strains had 1 or 2 bands on isoelectric focusing electrophoresis.Strain CI-5 was proved to have blaIND-1,but phenotype of metallo-?-lactamase was negative.CONCLUSIONS C.indologenes has high rate of metallo-?-lactamase producing,and thus it is difficult to treat.The major genotype of metallo-?-lactamase for C.indologenes is blaIND-1 and blaIND-2 in Hefei.The expression of blaIND could exist negative or low level occasionally.