Detection of Extended-Spectrum Beta Lactamases and AmpC Beta Lactamases Producing Uropathogenic Escherichia coli in a Tertiary Care Hospital

Background: The multidrug resistant among uropathogenic E. coli has become a potential threat to global health. The aim of the current study to evaluate the antimicrobial activities of nitrofurantoin and fosfomycin along with other antimicrobials against Extended Spectrum β-Lactamases (ESBL) and AmpC producer isolates from the most common organism E. coli. Methods: A total of 6046 clean catch midstream urine samples were collected and processed in Microbiology department of tertiary care hospital. The antimicrobial susceptibility of E. coli isolates was initially screened by Kirby-Bauer disk diffusion method. The resistant isolates were confirmed to be ESBL and AmpC producers by their respective phenotypic confirmatory tests of combined disc method. Results: Out of 6046 patients there were 1855 E. coli positive patients. Maximum patients in the age group of 21-30 years were 51.5% followed by 31-40 years where patients were 26%. 64.4% E. coli were isolated from female patients and 35.6% from male patients. E. coli showed higher sensitivity towards, fosfomycin (100%), imipenem (100%), nitrofurantoin (84.1%), piperacillin and tazobactam (77.3%), amikacin (76.1%) and while they showed high degree resistance pattern against Penicillin, cotrimoxazole, ciprofloxacin, norfloxacin and 2nd and 3rd generation cephalosporin. Out of 1855 E. coli, multi drug resistance was seen in 520 E. coli isolates. ESBL production was observed among 50% of E. coli isolates by combined disk method. Out of 520 isolates, 150 isolates showed resistance to one or more extended-spectrum cephalosporins and cefoxitin by Kirby-Bauer disk diffusion method. These were selected and screened for ESBL and AmpC production. Among 150 cefoxitin-resistant isolates, AmpC phenotype was detected in 100 isolates (66.6%) by AmpC disc method. The overall occurrence of AmpC in the study was found to be 19.2%. Susceptibility of ESBL and AmpC producers to fosfomycin, imipenem, nitrofurantoin and amikacin were found to be 100%, 98.5%, 89% and 75% respectively. Conclusions: There is increased prevalence of ESBL and AmpC producing E. coli. Thus, early detection of ESBL and AmpC producer E. coli by simple phenotypic methods is necessary to avoid treatment failure, where molecular techniques are not available.

Extended-spectrum β-lactamase and AmpC β-lactamase Production among Gram-negative Bacilli Isolates Obtained from Urinary Tract Infections and Wound Infections.

Extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases continue to be a major problem in healthcare settings. Due to the scarcity of information regarding the antibiotic susceptibility patterns particularly from urinary tract infections (UTIs) and wound infections, the current study was carried out to assist the clinicians to prescribe appropriate antibiotics against gram-negative clinical isolates. In the current study, urine (n = 620) and pus (n = 228) samples were collected from different sites (at various clinical departments) and subjected to direct microscopic examination, culture and antibiotic susceptibility testing (AST). In the AST testings, the isolates that exhibited reduced zone of inhibition to one or more of the antibiotics such as cefotaxime (≤27 mm), ceftriaxone (≤25 mm), ceftazidime (≤22 mm), cefpodoxime (≤17 mm) and aztreonam (≤27 mm) were considered as potential ESBL producers and the ESBL production was confirmed using phenotypic screening test (doubledisk synergy test) and phenotypic confirmatory test (combined-disk test). However, isolates showing resistance or decreased sensitivity to cefoxitin, cefotaxime, ceftriaxone, ceftazidime, cefpodoxime or aztreonam and sensitive to cefepime were considered as a screen positive AmpC producer and subjected to AmpC disk tests. The current study concluded that 72.41% and 21.76% of ESBL and AmpC producers were detected, respectively in our hospital. It was also observed that the double-disk synergy and combined-disk tests were equally effective for ESBL detection. Further, AmpC disk test is simple, easy to perform and interpret, requiring less expertise for the rapid detection of AmpC isolates.

Characterization of plasmid mediated AmpC producing Escherichia coli clinical isolates from a tertiary care hospital in South India.

Context: Plasmid mediated AmpC (pAmpC) β-lactamase producing Escherichia coli are an emerging problem worldwide as they are now exhibiting resistance to multiple classes of antibiotics and are a major cause of therapeutic failure. Aims: The aim of this study was to characterize pAmpC β-lactamase producing extraintestinal E. coli, their phylogenetic distribution, resistance pattern, treatment options, and impact on patient’s clinical outcome. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Materials and Methods: A total of 300 clinically signifi cant, non-repeat isolates were studied. AmpC disk test was used for phenotypic AmpC-β- lactamase detection. Molecular types of pAmpC were determined by a multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Metallo-beta-lactamase (MBL) detection was done by E test. Antibiogram, treatment, and clinical outcome were collected in a structured proforma. Results: Although 95 isolates (32%) were phenotypically positive for AmpC, PCR detected CIT type of AmpC gene in only 37 isolates. Majority of strains were from phylogroup A (85%) and B1 (58%) which are considered as commensal groups. Co-production of ESBL’s was observed in 33 strains and 5 strains were found to be MBL producers. Most widely prescribed antibiotics were 3rd generation cephalosporins (30%), carbapenems (19%) and aminoglycosides (16%). Conclusions: Plasmid mediated AmpC producing isolates were found to exhibit a high degree of drug resistance, and they mainly belonged to commensal strains possibly due to misuse of antibiotics. Proper antibiotic policy is required to limit the spread of pAmpC producers or else it will lead to a therapeutic dead end in the near future.

Antimicrobial resistance in bacteria causing ventilator-associated pneumonia in a tertiary care hospital: one year prospective study.

Background: Ventilator-associated pneumonia (VAP) is the most common infection diagnosed in intensive care units (ICUs). The causative organisms of VAP vary among different populations and are increasingly associated with resistance against various antimicrobial agents. Objective of current study was to determine the bacteriological etiology of VAP, antimicrobial susceptibility pattern of the isolates and detect the presence of extended-spectrum -lactamases (ESBL), metallo β-lactamases (MBL) and AmpC -lactamases in multidrug resistant isolates causing VAP in the medical ICU. Methods: A prospective study was carried out over a year to know the various etiological agents of VAP and their drug susceptibility patterns. ESBL, MBL and AmpC -lactamases were detected in various isolates by combination disk method, imipenem-EDTA combined disk method and AmpC disk method respectively. Results: The majority of bacterial isolates causing VAP were found to be gram negative bacilli. Acinetobacter spp accounted for 34.28% of VAP cases followed by Pseudomonas aeruginosa which was responsible for 25.71% cases. Other gram negative bacilli isolated were Klebsiella pneumoniae, Citrobacter freundii, Enterobacter spp, and Escherichia coli. Out of the total 70 isolates, 67 (95.7%) were multidrug resistant and not even a single isolate was sensitive to all the drugs tested. Conclusions: Most of the pathogens causing VAP in our institute were multidrug resistant and in many isolates this resistance was due to production of ESBL, MBL, and AmpC β-latamases. Polymixin-B and colistin were found to be highly effective against multidrug resistant Acinetobacter spp and P. aeruginosa.

Prevalence of Plasmid-Mediated ampC Genes in Clinical Isolates of Enterobacteriaceae from Cairo, Egypt.

Aims: To determine the prevalence of acquired pAmpCs in clinically important and relevant enterobacterial species and to characterize the molecular types of pAmpC present in our geographic area. Methodology: Sixty Enterobacterial clinical isolates resistant to third generation cephalosporins and to cephamycins were included in the study. Samples were collected for a period of 6 months between July 2008 and December 2008 from Theodor Bilharz Research Institute (TBRI), Egypt. Bacterial species were identified using API E20. AmpC genes clusters: (bla ACC, bla EBC, bla FOX, bla CMY, bla MOX, and bla DHA) were tested by PCR and DNA sequencing. Clonal relatedness of AmpC-producing Klebsiellae isolates was determined by Pulsed Field Gel Electrophoresis (PFGE). Results: AmpC genes were detected in 28.3% (17/60) of the study population including E. coli, Klebsiella and Proteus mirabilis (P mirabilis). CMY-2 enzyme was found disseminating in all 6 AmpC-positive Escherichia coli (E. coli) and in 6/10 of Klebsiellae species. Only one Klebsiella pneumonia (K. pneumonia) isolate harbored CMY-4 while DHA-1 was detected in 3 Klebsiellae and in one P. mirabilis isolate. PFGE patterns showed no clonal relatedness among the 6 CMY-2-positive Klebsiella isolates. Conclusion: Plasmid-mediated AmpC enzymes are important mechanisms of resistance to ß- lactam drugs. CMY-2 and DHA-1 are the most common gene clusters of pAmpC in our region. AmpC-type resistance in our hospital setting is not due to the dissemination of clonal strains but due to the spread of resistant genes. This is the first report from Egypt identifying DHA-1 and CMY-4 in enterobacterial isolates.

Co-existence of Pseudomonas-derived cephalosporinase among plasmid encoded CMY-2 harbouring isolates of Pseudomonas aeruginosa in north India.

Context: In Pseudomonas aeruginosa, AmpC β-lactamases are often responsible for high-level resistance to β-lactam antibiotics. The co-production of plasmid-mediated AmpC along with chromosomal Pseudomonas-derived cephalosporinases thus remain a serious clinical concern owing to high resistance spectrum towards antibiotics. Aim: The present study was performed to investigate the co-existence of both chromosomally-encoded and plasmid-mediated AmpC β-lactamase among clinical isolates of P. aeruginosa. Setting and Design: It is a cross-sectional study carried out in the Department of Microbiology in a tertiary referral hospital of northern India. Methods and Methods: A total of 329 consecutive, non-duplicate clinical isolates of P. aeruginosa, were selected for the detection of AmpC β-lactamases and confirmed for AmpC production by modified three dimensional (M3D) test. Ceftazidime -imipenem antagonism test was used to detect inducible AmpC producers. Molecular characterisation of chromosomally-encoded blaPDC and plasmid-mediated AmpC gene was studied by performing polymerase chain reaction (PCR). Result: A total of 214 (65%) isolates were confirmed for AmpC production by M3D test. On performing multiplex PCR, 27 isolates were detected posessing blaCMY type of plasmid-mediated AmpC gene. While 48 isolates were found to harbour chromosomally-encoded blaPDC gene co-production of both chromosomal and plasmid-encoded AmpC was reported in eleven isolates. Conclusions: Although these chromosomally-encoded cephalosporinases might spread more slowly than mobilised AmpC, but it is likely that in the present scenario of intense antibiotic pressure, this will become an increasing problem and may further limit our antibiotic choices.

Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae.

Background: Detecting plasmid-mediated AmpC (pAmpC) β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA) disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82) and Escherichia coli (n: 191) were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX) screening test, modified three dimensional test (M3DT), and multiplex polymerase chain reaction (PCR). Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL) in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.

High occurrence of blaCMY-1 AmpC lactamase producing Escherichia coli in cases of complicated urinary tract infection (UTI) from a tertiary health care centre in north India.

AmpC beta lactamase producing Gram-negative bacteria have emerged worldwide. It is important to distinguish plasmid mediated AmpC β lactamases from chromosomally mediated enzymes for surveillance, epidemiology and hospital infection control as plasmid mediated genes can spread to other organisms. Occurrence of blaCMY-1 AmpC β-lactamase, a plasmid mediated cephamycinase was studied in 100 consecutive isolates of Escherichia coli from cases of complicated urinary tract infection (UTI). Screening for AmpC production was done by modified Hodge test, three dimensional test and AmpC disk test. All isolates showing a positive result by 2 out of 3 tests were then tested for blaCMY-1 gene by PCR. Fifty nine isolates were positive for AmpC β lactamase production, 56.6 per cent were positive by PCR. Eight out of 13 isolates which were negative by EDTA disk method were positive by PCR, whereas none of the isolates negative by 3D and modified Hodge test was positive by PCR. Among admitted patients urinary catheterisation was the major risk factor followed by obstructive uropathy, three patients developed urosepsis. High occurrence of blaCMY-1 AmpC β-lactamase warrants health care workers to endorse good hospital practices.

Phenotypic & molecular characterization of AmpC β-lactamases among Escherichia coli, Klebsiella spp. & Enterobacter spp. from five Indian Medical Centers.

Background & objectives: AmpC β-lactamases which are often plasmid mediated hydrolyze all β-lactam antibiotics except cefepime and carbapenems. We evaluated the presence of AmpC β-lactamases among Enterobacteriaceae strains recovered prospectively from patients at five Indian tertiary care centres. Methods: The study included 909 consecutive Gram-negative isolates recovered from clinically significant specimens during June 2007 - May 2008 as part of an ICMR-ESBL study. Among the study isolates, 312 were found to be cefoxitin resistant by disc diffusion test (DDT). Minimum inhibitory concentration (MIC) determination by E test was done against amikacin, levofloxacin, impinem, meropenem, ertapenem, tigecycline and piperacillin-tazobactam. Combined DDT using phenyl boronic acid as inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. The common Amp C genotypes ACC, FOX, MOX, DHA, CIT and EBC were detected by multiplex PCR. Results: Plasmid mediated Amp C phenotype was confirmed in 114 of the 312 (36.5%) cefoxitin resistant isolates with 255 (81.7%) showing multidrug resistance. Susceptibility to tigecycline was highest (99%) followed by imipenem, meropenem (97%), ertapenem (89%), amikacin (85%), and piperacillin-tazobactam (74.6%). Levofloxacin resistance was 82 per cent. ESBL co carriage was observed among 92 per cent of Amp C producers. Among 114 Amp C producers, 48 could be assigned a genotype, this included CIT- FOX (n=25), EBC (n=10), FOX (n = 4), CIT (n=3), EBC-ACC (n=2) and one each of DHA, EBC-DHA, FOX -DHA and FOX-EBC-DHA. Interpretation & Conclusions: Overall, AmpC phenotypes were found in 12.5 per cent isolates, multidrug resistance and ESBL co-carriage among them was high suggesting plasmid mediated spread. The study results have implications in rational antimicrobial therapy and continued surveillance of mechanisms of resistance among nosocomial pathogens.

Molecular description of plasmid-mediated AmpC β-lactamases among nosocomial isolates of Escherichia coli & Klebsiella pneumoniae from six different hospitals in India.

Background & objectives: Plasmid mediated AmpC β-lactamase (PMABL) resistance in Escherichia coli and Klebsiella spp. is an emerging problem worldwide. Phenotypic methods are commonly used for detection of PMABL production in Gram-negative isolates, but molecular data about the prevalence of plasmid-mediated AmpC-type resistance at the national level are needed. Hence, a prospective study was undertaken to determine the occurrence of PMABL gene and its types among clinical isolates of E. coli and K. pneumoniae obtained from six different hospitals in India. Methods: A total of 241 nosocomial isolates of K. pneumoniae (n=109) and E.coli (n=132) from six geographically distant hospitals in India were included. These were screened for cefoxitin resistance. AmpC disk test and modified three dimensional extraction test were used for phenotypic detection of PMABL production. Molecular types were determined by a multiplex PCR. Results: Among the 241 isolates, 187 (77.5%) were found to be cefoxitin resistant (K. pneumoniae n=83, E. coli n=104). AmpC activity was detectable in 153 (63.4%) isolates, (K. pneumoniae n=69, E. coli n=84). By PCR, the plasmid encoded AmpC genes were found in 92 (38.1%) isolates and the molecular types of the genes detected predominantly were DHA, CIT followed by MOX and ACC types. Interpretation & conclusions: A high percentage of plasmid-encoded AmpC enzymes was noted in E. coli and K. pneumonia isolates obtained from different parts of the country. Phenotypic methods alone may not reflect the true number of PMABL producers. Genotypic methods need to be employed in national surveillance studies.

Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL among AmpC-producing Enterobacteriaceae.

Purpose: Extended spectrum β-lactamase (ESBL) and AmpC β-lactamase are important mechanisms of betalactam resistance among Enterobacteriaceae . The ESBL confirmation test described by Clinical Laboratory Standards Institute (CLSI) is in routine use. This method fails to detect ESBL in the presence of AmpC. Therefore, we compared two different ESBL detection methods against the CLSI confirmatory test. Materials and Methods: A total 200 consecutive clinical isolates of Enterobacteriaceae from various clinical samples were tested for ESBL production using (i) CLSI described phenotypic confirmatory test (PCT), (ii) boronic acid disk potentiation test and (iii) cefepime-CA disk potentiation method. AmpC confirmation was done by a modified three-dimensional test. Results: Among total 200 Enterobacteriaceae isolates, 82 were only ESBL producers, 12 were only AmpC producers, 55 were combined ESBL and AmpC producers, 14 were inducible AmpC producers and 37 isolates did not harboured any enzymes. The CLSI described PCT detected ESBL-producing organisms correctly but failed to detect 36.3% of ESBLs among combined enzyme producers. The boronic acid disk potentiation test reliably detected all ESBL, AmpC, and combined enzyme producers correctly. The cefepime-CA method detected all ESBLs correctly but another method of AmpC detection has to be adopted. Conclusion: The use of boronic acid in disk diffusion testing along with the CLSI described PCT enhances ESBL detection in the presence of AmpC betalactamases.

Multi-drug resistance induced by β-1actamases and class Ⅰ integrons with deficiency of Omp genes in Enterobacter cloacae / 中华临床感染病杂志

Objective To investigate the drug resistance and its distribution induced by β-lactamases and class Ⅰ integrons with the deficiency of Omp genes in Enterobacter cloacaes.Methods Totally 112 strains of Enterobacter cloacaes were isolated during January 2008 and May 2011.The identification of strains was performed by using Vitek-2 Compact automatic system; and antibiotic susceptibility was determined by K-B method.Isolates of E.cloacae were screened for carbapenemases by modified Hodge test,improved three dimensional test and EDTA-meropenem synergy test.Genes encoding AmpC β-lactamase,metallo-β-1actamases (MBLs) and OXA-like β-1actamases were screened by multiple PCR.Single PCR was used to detect ISEcpl,OmpK35/36,NDM-1 and OXA-48.The variable regions of class Ⅰ integrons were amplified and sequenced.Results Among 112 isolates,6 (5.4％) demonstrated positive in the modified Hodge test and 14 ( 12.5％ ) were positive in the improved three-dimensional test.No carbapenemases gene was found.There were 29(25.9％ ) strains positive for ESBLs genes,ISEcpl was found in the upstream of all the CTX-M-type ESBLs; OXA-1 ESBLs were detected in 2 isolates.AmpC β-lactamase genes were positive in 45 (40.2％) strains,and 82.2％ (37/45) were MIR-3 type.Twenty two isolates carried class Ⅰ integrons,and four different cassettes arrangements were identified within 16 strains:9 isolates harbored aadB-aadA2 ( 1 000 bp),5 isolates with dfrAl5 (700 bp),2 isolates with aadAl ( 1 000 bp).One isolate harbored all the above gene cassettes.The deficiency of OmpK35/36 was found in all strains.Conclusion ESBL,AmpC β-lactamase and the deficiency of OmpK35/36 are correlated with the resistance to carbapenems in Enteobacter clocace,and class Ⅰ integrons may also partly account for the multidrug-resistance.

Preliminary study on AmpC β-lactamase and related drug-resistant mechanism of pathogenic bacteria in blood stream / 上海交通大学学报（医学版）

Objective To investigate the characteristics of strains of AmpC β-lactamase(AmpC enzyme)production in Dathogenic bacteria in blood stream and clinical presentations of the cases, and study the related ampC and ampD genes.Methods One hundred and eighty-one strains of gram negative bacillus in blood stream were collected,Cefoxitin screening test and three-dimensional test were performed for screening of strains of AmpC enzyme,production and those of AmpC enzyme hyperproduction retrospective analysis was condected in the strains with positive results.ampC and ampD gene PCR ampliftcation, sequencing and sequence analysis of positive strains were performed, and gene homology of ampC positive strains was analysed bv Rep-PCR. Results Among 181 strains in blood stream,strains of AmpC enzyme production were detected in 39 isolates by Cefoxitin screening test,with the detection rate of 21.5%(39/181).The detection rate of strains of AmpC enzyme hyperproduction by three-dimensional test was 43.6%(17/39).PCR revealed that the positive rates for ampC and ampD genes were 41%(16/39)and 56.4%(22/39),respectively.The ampC gene sequencing of 16 positive strains indicated that the homology was 98%to 100%by comparison with the GenBank,while the ampD gene sequencing of 2 strains of Enterobacter cloacae demonstrated that the suspected gene mutations existed in the carboxy-terminal of ampD gene. Conclusion The prevalence of drug-resistant pathogenic bacteria in blood stream in this study is due to nosocomial infection.The mutation of ampC gene is rare in the pathogenic bacteria in blood stream with production of AmpC enzyme,while the rate of gene mutation in Enterobacter cloacae is higher, and the deletion and amino acid substitutions in the carboxy-terminal of ampD is highly relevant to the depressed expression of AmpC enzyme.

The mechanism of AmpC β-lactamase change from inducible type to constitutive type / 复旦学报（医学版）

Objective To investigate the influence of plasmid spread and ampD mutation to Enterobacter cloacae that leads to the AmpC β-lactamase change from inducible type to constitutive type. Methods The Enterobacter cloacae were isolated from the patients with nosocomial infection. The inducible type isolations and their constitutive type changers were put into the same group. The plasmid ampC gene and chromatin ampD gene in pairs in each group were amplified, sequenced and compared. Results Of 195 patients infected by Enterobacter cloacae of inducible type, 25 (12.82%) were changed to the ones of constitutive high type. In these 25 changed groups, 10 were caused by plasmid spread, 10 by ampD mutation, 1 by both, and 4 by neither. Twelve changed constitutive type strains had ampD significant mutations, in which 7 were frame-shift mutations and 5 were spot mutations. Conclusions The change ratio of Enterobacter cloacae from inducible type to constitutive type is rather high. Both plasmid spread and ampD mutation are possibly the mechanism of such change. Plasmid mediated AmpC β-lactamase spreads among different species and interregionally. The mutation rate of chromatin ampD gene is also higher than the natural mutation rate. These two mechanisms should be considered in clinical treatment.

Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria.

Background & objectives: The production of carbapenemases is an important mechanism responsible for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase producers is essential. A prospective study was undertaken to detect metallo-β-lactamases (MBLs) and AmpC β-lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for detection of carbapenemases and MBLs. Methods: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. lwoffi i) and 140 Pseudomonas spp. (103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby- Bauer disc diffusion method. Modifi ed Hodge test, EDTA disk synergy (EDS) test and AmpC disk test were used for the detection of carbapenemases, MBLs and AmpC β-lactamases, respectively. Results: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffi i, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC β-lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9 (28.1%) found positive for carbapenemases by modifi ed Hodge test. Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC β-lactamase producers, 3 (6.5%) MBL producers, but only 1 (14.3%) was positive for carbapenemases by modifi ed Hodge test. One P. aeruginosa was positive for carbapenemase by modifi ed Hodge test, but was negative for MBL and AmpC β-lactamase. Interpretation & conclusions: MBL production is an important mechanism of carbapenem resistance among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection of MBLs than modifi ed Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance. AmpC β-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

Genotype identifications of ESBLs and AmpC β-Iactamase of a Enterococcus gallinarum isolated from chloebia gouidiae / 中国兽医学报

The genotypes of extended spectrum β-laetamases(ESBLs) and AmpC β-lactamases produced by Enterococcus gallinarum isolated from chloebia gouldiae were determined to elucidate the evolution mechanism of the resistant genes.The minimum inhibitory concentrations (MICs) of 18 antibacterial drugs against the Enterococcus gallinarum were detected with two dilution method,and the ESBLs and AmpC β-lactamases from the bacterium were amplified by PCR using the primers of TEM,SHV,CTX-M,ACC,CIT,DHA,EBC,FOX and MOX,respectively.The PCR products were cloned and then the cloned fragments were sequenced to identify their genotypes and subtypes.The bacterium was proved to be a ESBL-producing and AmpC β-lactamase-producing bacterium,showing severe resistant to the other drugs,except the third and forth cephalosporins,carbopenems and fosfomycin.Compared with that of AJ847364 (TEM-116),the sequence of the TEM-type was characterized by two nucleotide mutations (512T→A and 695A→C),which led to two mutations of amino acids(17111e→Lys and 232Lys→Thr),showing that the detected TEM-type was a new genotype,the sequence of the AmpC β-lactamase was similar to that of EF078894 (ACT-like type)with a 97% homology.The genotype of ESBLs of Enterococcus gallinarum was a new TEM-type derived from the TEM-type ESBLs of klebstella pneumoniae isolated from the same avian.The genotype of AmpC lactamase was ACT-type,which probably concerned with β-1actam antibiotics used.

Analysis of the multi-resistance to antibiotics of clinical isolated klebsiella pneumoniae / 中国基层医药

Objective To investigate the multi-resistance to antibiotics of clinical isolated klebsiella pneumoniae.Methods The resistance to antibiotics of clinical isolated klebsiella pneumoniae were monitored.The discconfirmatory test was used to detect extended-spectrum β-lactamases(ESBLs) and cefoxitin three-dimension was used to detect AmpC β-lactamases.Results Among the isolates there were 53 strains of ESBLs-producing bacteria (49.5% ), 30 strains of AmpC-producing bacteria(28.0%), 24 strains of ESBLs + AmpC-producing bacteria (22.46%).They were high resistance to aminoglycosides,quinolones and cephalosporins.Conclusion The multi-resistance to antibiotics of clinical isolated klebsiella pneumoniae were widespread.It is important to control nosocomial infection to strengthen the detection of the epidemiology of ESBLs and AmpC β-lactamases in clinical isolates.

Analysis of AmpC β-lactamase Gene in Pseudomonas aeruginosa / 华中科技大学学报（医学）（英德文版）

The gene and the amino acid sequence of the structural and regulatory region of the Pseudomonas aeruginosa with different resistance patterns were analyzed. Six strains with different resistance patterns were selected and the AmpC β-lactamase was identified. The objective gene fragment was amplified by colonies PCR. The sequences of the PCR-products were analyzed. The DNA sequence of the structural gene ampC and the regulatory genes ampR, ampD and ampE was detected. The 6 strains and the wild-type Pseudomonas aeruginosa are highly homogeneous in structural and regulatory region. Some new mutant points were found.