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.

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.

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.

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.