Inducible AmpC Beta-lactamase Research Articles

Phenotypic identification of different β-Lactamases in intrinsic and acquired colistin resistant uropathogenic gram negative bacteria.

Identification of MBL, AmpC and ESBLs in colistin intrinsic and acquired resistant uropathogenic gram negative bacteria. Urine samples were collected from Hayatabad Medical Complex, Peshawar during 17 January to 30 June 2019. Collected urine samples were aseptically transported microbiology lab of Health Research Institution (HRI), National Institute of Health (NIH), Khyber Medical College, Peshawar and streaked on different media. Positive growth was identified by API-10s. Antibiotic sensitivity profile was done by Modified Kirby Bauer disc diffusion method. Detection of metallo βlactamases (MBL) production by Imipenem EDTA synergy test, Double Disc Synergy Test (DDST) for detection of ESBLs and D-test for the detection of inducible AmpC beta lactamases test was used. Colistin resistance was identified via broth micro dilution according to CLSI manual. Colistin resistant bacteria was divided in two categories; acquired and intrinsic resistant bacteria according to CLSI manual. Out of 2000 urine samples, 281(14%) gram-negative bacteria were isolated. Among positive samples, acquired colistin resistant bacteria were 241 and intrinsic resistant bacteria were 40 isolates. MBL was produce by twenty one (11.7%) E.coli and seventeen (40.5%) Pseudomonas aeruginosa. E. coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Serratia Oderifora and Proteus Marblis were ESBLs producing bacteria. AmpC production was prevalent in fourteen (7.8%) E. coli and twelve (28.6%) Pseudomonas aeruginosa. Fifty-five samples showed resistance to colistin out of 241 samples. In colistin resistant bacteria, two E.coli were MBL, ESBLs, while one E.coli was ESBLs, AmpC co-producing bacteria. The most prevalent extended drug resistant bacteria were Pseudomonas aeruginosa (28.6%) and Escherichia coli (6.1%), While 155(86.6%) Escherichia coli, 25 (59.5%) Pseudomonas aeruginosa and 22 (95.7%) Serratia Oderifora was multi drug resistant bacteria. Current study concluded that ESBL, MBL AmpC enzymes and their co-expression was observed with colistin resistance in E.coli and Pseudomonas aeruginosa.

Development of a Candidate Multi-Epitope Subunit Vaccine against Klebsiella aerogenes: Subtractive Proteomics and Immuno-Informatics Approach.

Klebsiella aerogenes is a Gram-negative bacterium which has gained considerable importance in recent years. It is involved in 10% of nosocomial and community-acquired urinary tract infections and 12% of hospital-acquired pneumonia. This organism has an intrinsic ability to produce inducible chromosomal AmpC beta-lactamases, which confer high resistance. The drug resistance in K. aerogenes has been reported in China, Israel, Poland, Italy and the United States, with a high mortality rate (~50%). This study aims to combine immunological approaches with molecular docking approaches for three highly antigenic proteins to design vaccines against K. aerogenes. The synthesis of the B-cell, T-cell (CTL and HTL) and IFN-γ epitopes of the targeted proteins was performed and most conserved epitopes were chosen for future research studies. The vaccine was predicted by connecting the respective epitopes, i.e., B cells, CTL and HTL with KK, AAY and GPGPG linkers and all these were connected with N-terminal adjuvants with EAAAK linker. The humoral response of the constructed vaccine was measured through IFN-γ and B-cell epitopes. Before being used as vaccine candidate, all identified B-cell, HTL and CTL epitopes were tested for antigenicity, allergenicity and toxicity to check the safety profiles of our vaccine. To find out the compatibility of constructed vaccine with receptors, MHC-I, followed by MHC-II and TLR4 receptors, was docked with the vaccine. Lastly, in order to precisely certify the proper expression and integrity of our construct, in silico cloning was carried out. Further studies are needed to confirm the safety features and immunogenicity of the vaccine.

Early Treatment Outcomes for Bloodstream Infections Caused by Potential AmpC Beta-Lactamase-Producing Enterobacterales with Focus on Piperacillin/Tazobactam: A Retrospective Cohort Study.

The Gram-negative bacilli Serratia spp., Providencia spp., Morganella morganii, Citrobacter freundii complex, Enterobacter spp. and Klebsiella aerogenes are common Enterobacterales that may harbor inducible chromosomal AmpC beta-lactamase genes. The purpose of the present study was to evaluate treatment outcomes and identify predictors of early treatment response in patients with bloodstream infection caused by potential AmpC beta-lactamase-producing Enterobacterales (SPICE-BSI). This cohort study included adult patients with SPICE-BSI hospitalized between 01/2011 and 02/2019. The primary outcome was early treatment response 72 h after the start of active treatment, defined as survival, hemodynamic stability, improved or stable SOFA score, resolution of fever and leukocytosis and microbiologic resolution. Among 295 included patients, the most common focus was the lower respiratory tract (27.8%), and Enterobacter spp. (n = 155) was the main pathogen. The early treatment response rate was significantly lower (p = 0.006) in the piperacillin/tazobactam group (17/81 patients, 21.0%) than in the carbapenem group (40/82 patients, 48.8%). Independent negative predictors of early treatment response (p < 0.02) included initial SOFA score, liver comorbidity and empiric piperacillin/tazobactam treatment. In vitro piperacillin/tazobactam resistance was detected in three patients with relapsed Enterobacter-BSI and initial treatment with piperacillin/tazobactam. In conclusion, our findings show that piperacillin/tazobactam might be associated with early treatment failure in patients with SPICE-BSI.

The risk mitigation indices associated with some risk mitigation protocols performed on contaminated aqua-cultured catfish (Clarias gariepinus)

The present research quantified the risk mitigation indices associated with some risk mitigation protocols practiced by humans in Nigeria to mitigate the baseline risk of illness that were associated with Clarias gariepinus contaminated with multidrug-resistant AmpC beta-lactamase-producing Enterobacter cloacae pathogen. Identification of the multidrug-resistant AmpC beta-lactamase-producing E. cloacae pathogen was performed with phenotypic and molecular methods. The United States FDA-iRisk stochastic software quantified the baseline and residual risk of illness associated with the contamination and cross-contamination routes of the C. gariepinus chain. The fresh adult C. gariepinus was contaminated with the multidrug-resistant E. cloacae pathogen (median concentration = 4.49 CFU g-1), but this was significantly reduced by the risk mitigation protocols practiced by the human consumers. The risk mitigation practices of human consumers resulted in a risk mitigation index of 0.92, which was equivalent to a 92% reduction in the baseline risk of illness (baseline risk of illness = 7.58 × 10-4; residual risk of illness = 5.9 × 10-5). In spite of the significant risk mitigation, inducible AmpC beta-lactamase resistance genes was still found in the residual multidrug-resistant Enterobacter cloacae pathogen, which survived the inadequate risk mitigation protocols that accounted for 8% residual risk of illness.

A study on detection of extended-spectrum beta-lactamases (ESBLs) and comparison of various phenotypic methods of AmpC detection in Pseudomonas aeruginosa from various clinical isolates in a tertiary care teaching hospital

Introduction: Pseudomonas aeruginosa is one of the major etiological agents of nosocomial infection. They are difficult to treat as the majority of them exhibit varying degrees of innate resistance. Acquired resistance is mediated by the production of chromosomal and plasmid-mediated AmpC beta-lactamases, extended-spectrum beta-lactamases (ESBLs), and metallo-beta-lactamases (MBLs). Objectives: This study was conducted for the detection of ESBLs and the comparison of various phenotypic methods of AmpC beta-lactamases (both inducible and plasmid-mediated) from various clinical isolates of Pseudomonas aeruginosa. Materials and Methods: A prospective study was conducted for a period of 6 months from January 2014 to June 2014. Isolates of Pseudomonas aeruginosa were tested for ESBLs by combined disk diffusion method using ceftazidime/ceftazidime+clavulanic acid (CAZ+CAC). Inducible AmpC beta-lactamases were detected by both ceftazidime-imipenem antagonism test (CIAT) and cefotaxime/cefoxitin (CTX/CX) inducible disk diffusion test. Plasmid-mediated AmpC were detected using AmpC disk test and ethylenediaminetetraacetic acid (EDTA) disk test. All the isolates were tested for cefepime resistance. Results: Out of 116 Pseudomonas aeruginosa isolates, 70 were ceftazidime-resistant. While ESBLs were observed in 67 out of 70 isolates, 36 were positive for inducible AmpC by both the methods. Plasmid-mediated AmpC were detected in only 8 isolates. All the 70 strains showed resistance to cefepime. Conclusions: High prevalence of ESBL and chromosomal-mediated AmpC was observed in Pseudomonas aeruginosa. Both the inducible disk diffusion tests (CIAT, CTX/CX) were equally effective in the detection of inducible AmpC. As Pseudomonas is one of the major nosocomial pathogens, such high resistance poses a grave threat.

Characterization of ceftriaxone-resistant Aeromonas spp. isolates from stool samples of both children and adults in Southern India.

BackgroundAeromonas species can cause a wide spectrum of illnesses varying from intestinal to extra intestinal and vary in their susceptibility to different antibiotics. The current study was undertaken to characterize the third generation cephalosporin-resistant strains of Aeromonas spp. which were isolated from stool specimens.MethodsOut of a total of 2780 stool samples, 29 Aeromonas spp. were identified, out of which, 9 were resistant to ceftriaxone by the Kirby-Bauer antibiotic testing method. These strains were subjected to minimum inhibitory concentration (MIC) determination by agar dilution for ceftriaxone. Phenotypic and genotypic testing of AmpC beta-lactamase and extended spectrum beta-lactamase (ESBL) were performed. Gene transfer was carried out to demonstrate transmissibility of these genetic elements by conjugation experiments.ResultsOut of the 29 strains, 9 showed MIC of ≥4 μg/ml. Seven out of 9 showed presence of blaCTX-M, while 2 more strains showed the presence of inducible AmpC beta-lactamase and presence of MOX gene. Gene transfer experiments showed that these elements were transmissible to recipient (Escherichia coli J53 strain) in the presence of ceftriaxone.ConclusionsDissemination of these resistance determinants like plasmids is pivotal in the spread of these resistance genes into the aquatic environment into organisms like Aeromonas. This may further limit the future use of antibiotics for the treatment of diarrhoeal diseases. Hence, detection and antibiotic susceptibility testing of Aeromonas spp. should be performed when isolated from stool samples.

Identification of DHA-23, a novel plasmid-mediated and inducible AmpC beta-lactamase from Enterobacteriaceae in Northern Taiwan.

Objectives: AmpC β-lactamases are classified as Amber Class C and Bush Group 1. AmpC β-lactamases can hydrolyze broad and extended-spectrum cephalosporins, and are not inhibited by β-lactamase inhibitors such as clavulanic acid. This study was conducted to identify DHA-23, a novel plasmid-mediated and inducible AmpC β-lactamase obtained from Enterobacteriaceae.Methods: A total of 210 carbapenem-resistant Enterobacteriaceae isolates were collected from a medical center (comprising two branches) in Northern Taiwan during 2009–2012. AmpC β-lactamase genes were analyzed through a polymerase chain reaction using plasmid DNA templates and gene sequencing. The genetic relationships of the isolates were typed using pulsed-field gel electrophoresis following the digestion of intact genomic DNA by using XbaI.Results: Three enterobacterial isolates (one Escherichia coli and two Klebsiella pneumoniae) were obtained from three hospitalized patients. All three isolates were resistant or intermediately susceptible to all β-lactams, and exhibited reduced susceptibility to carbapenems. These three isolates expressed a novel AmpC β-lactamase, designated DHA-23, approved by the curators of the Lahey website. DHA-23 differs from DHA-1 and DHA-6 by one amino acid substitution (Ser245Ala), exhibiting three amino acid changes compared with DHA-7 and DHA-Morganella morganii; three amino acid changes compared with DHA-3; four amino acid changes compared with DHA-5; and eight amino acid changes compared with DHA-2 (>97% identity). This AmpC β-lactamase is inducible using a system involving ampR.Conclusion: This is the first report to address DHA-23, a novel AmpC β-lactamase. DHA-type β-lactamases are continuous threat in Taiwan.

Lab Based Surveillance of Multidrug Resistant Pseudomonas aeruginosa in Cairo University Hospitals, Egypt

Multidrug resistance is increasingly recorded among P. aeruginosa causing health care associated infections (HAI) due to the selective pressure by over-usage of antibiotics. The primary aim of the study was to perform a laboratory based surveillance to investigate the prevalence of β- lactamase and AmpC production and multi-drug resistance among clinical isolates of P. aeruginosa causing health care associated infections in Kasr Al Aini School of Medicine and National Cancer Institute, Cairo University, Egypt. β- lactamase and AmpC production was tested by the double disc test. Multidrug-resistant P. aeruginosa (MDRPA) were defined as demonstrating resistance to carbapenems, all anti-pseudomonal cephalosporins, amino glycosides and fluoroquinolones. In a six months period, out of 12,524 clinical samples, 2593 (20.7%) P. aeruginosa isolates were identified. Out of these isolates, 1138 (43.9%) were ceftazidime resistant, 138 (5.3%) were ESBL producers, and 661 (25.5%) isolates were confirmed positive for inducible AmpC beta-lactamase. The proportion of multidrug-resistant P. aeruginosa was 12.3%. In conclusion, high prevalence of AmpC indicates the necessity of performing routine tests to detect AmpC in ceftazidime resistant P. aeruginosa isolates. Lab based surveillance is important to identify the prevalence of MDRPA in order to guide empirical antibiotic therapy and prevent the spread of resistant strains.

Performance of extended spectrum beta lactamases (ESBL) screening agar in various clinical specimens

Performance of extended spectrum beta lactamases (ESBL) screening agar in various clinical specimens

English

Extended spectrum &beta;-lactamases (ESBLs) are frequently reported in Ghana, but AmpC bacteria reports are scanty. This study determines the prevalence of AmpC Pseudomonas aeruginosa and Proteus mirabilis clinical isolates at the Komfo Anokye Teaching Hospital (KATH), Kumasi, where ESBL and AmpC are not routinely tested. Non-duplicate 245 isolates comprising 187 (76.3%) P. aeruginosa and 58 (23.7%) P. mirabilis were tested for AmpC and ESBL production using the modified three-dimensional test method and the double disc synergy test (DDST) methods, respectively. The proportion of the 245 isolates producing AmpC &beta;-lactamase was 93 (38.0%) and 79 (32.2%) produced ESBL. AmpC producers confirmed 49 (52.7%) as inducible and 44 (47.3%) non-inducible AmpC producers. P. aeruginosa and P. mirabilis that produced AmpC were 84 (44.9%) and 9 (15.5%), respectively. ESBL production was 41 (21.9%) in P. aeruginosa and 38 (65.6%) in P. mirabilis. Co-producers of AmpC together with ESBL were 7.3%. Both enzyme were detectable in 13 (7.0%) of P. aeruginosa and 5 (8.6%) of P. mirabilis. AmpC and ESBL are detectable in high proportions among P. aeruginosa and P. mirabilis isolates at KATH and the Kumasi community. This emphasizes the need to start testing for both enzymes to guide therapeutics. Key words: AmpC beta-lactamase, extended spectrum beta-lactamase, inducible AmpC beta-lactamase, non-inducible beta-lactamase.

Evaluation of the βLacta Test, a Rapid Test Detecting Resistance to Third-Generation Cephalosporins in Clinical Strains of Enterobacteriaceae

For decades, third-generation cephalosporins (3GC) have been major drugs used to treat infections due to Enterobacteriaceae; growing resistance to these antibiotics makes the rapid detection of such resistance important. The βLacta test is a chromogenic test developed for detecting 3GC-resistant isolates from cultures on solid media within 15 min. A multicenter prospective study conducted in 5 French and Belgian hospitals evaluated the performance of this test on clinical isolates. Based on antibiotic susceptibility testing, strains resistant or intermediate to cefotaxime or ceftazidime were classified as 3GC resistant, and molecular characterization of this resistance was performed. The rates of 3GC resistance were 13.9% (332/2,387) globally, 9.4% in Escherichia coli (132/1,403), 25.6% in Klebsiella pneumoniae (84/328), 30.3% in species naturally producing inducible AmpC beta-lactamases (109/360), and 5.6% in Klebsiella oxytoca and Citrobacter koseri (7/124). The sensitivities and specificities of the βLacta test were, respectively, 87.7% and 99.6% overall, 96% and 100% for E. coli and K. pneumoniae, and 67.4% and 99.6% for species naturally producing inducible AmpC beta-lactamase. False-negative results were mainly related to 3GC-resistant strains producing AmpC beta-lactamase. Interestingly, the test was positive for all 3GC-resistant extended-spectrum beta-lactamase-producing isolates (n = 241). The positive predictive value was 97% and remained at ≥96% for prevalences of 3GC resistance ranging between 10 and 30%. The negative predictive values were 99% for E. coli and K. pneumoniae and 89% for the species producing inducible AmpC beta-lactamase. In conclusion, the βLacta test was found to be easy to use and efficient for the prediction of resistance to third-generation cephalosporins, particularly in extended-spectrum beta-lactamase-producing strains.

Accumulation of carbapenem resistance mechanisms in VIM-2-producing Pseudomonas aeruginosa under selective pressure

Pseudomonas aeruginosa has the potential to achieve resistance to carbapenems via the acquisition of carbapenemase-encoding genes, the downregulation of the OprD porin, the overexpression of efflux systems and the overproduction of cephalosporinases. One hundred and fifty carbapenem-non-susceptible isolates from 2008 to 2010 were screened for carbapenemase production, OprD porin loss, efflux pumps overexpression and inducible AmpC beta-lactamase production. For comparison reasons, the presence of the same mechanisms was also assessed in a previous collection of 30 carbapenem-non-susceptible P. aeruginosa isolated between 2003 and 2005. Results showed the accumulation of various resistance mechanisms among VIM-2 producers isolated between 2008 and 2010 with a parallel considerable increase in imipenem MIC90 and the geometric mean of the MIC values of imipenem and meropenem between the two study groups. The accumulation of carbapenem resistance mechanisms highlights the potential of this formidable pathogen for evolutionary success under antibiotic selective pressure.

AmpC beta lactamases among Gram negative clinical isolates from a tertiary hospital, South India

AmpC β-lactamases are cephalosporinases that hydrolyze cephamycins as well as other extended-spectrum cephalosporins and are poorly inhibited by clavulanic acid. Although reported with increasing frequency, the true rate of occurrence of AmpC β-lactamases in different organisms, including members of Enterobacteriaceae, remains unknown. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Pondicherry state, South India. A total of 235 Gram negative clinical isolates were tested for resistance to cefoxitin, third generation cephalosporin (3GC) antibiotics, ampicillin, amikacin, co-trimoxazole, gentamicin, meropenem and tetracycline by disc diffusion method. Isolates found resistant to 3GC and cefoxitin were tested for the production of AmpC β -lactamases by three dimensional extraction method and AmpC disc method. Isolates found to sensitive to 3GC were subjected to disc antagonism test for inducible AmpC production. One hundred and thirty four (57%) strains were resistant to 3GC, among which 63(47%) were positive for plasmid-mediated AmpC beta lactamases production. Among the 101 strains sensitive to 3GC, 23 (22.7%) revealed the presence of inducible AmpC beta lactamases by disc approximation test. A total of 80.9% (51/63) of screen positive isolates were detected by Amp C disc test and 93.6% (59/63) by three dimensional extraction method. Out of the 86 AmpC producers, 67 (77.9%) were cefoxitin resistant .Inducible AmpC was not found in Esch.coli and Klebsiella spp. The AmpC producers also concurrently showed multidrug resistance pattern. AmpC producers were found to be prevalent in our hospital and though three dimensional extraction test detects AmpC better, the disk test is easier to perform routinely and is user- friendly.

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo-N-acetyl-beta-glucosaminidase, found within Gram-negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N-acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC beta-lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo-N-acetyl-beta-glucosaminidases: O-GlcNAcase and the beta-hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring-group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2-N-acyl derivatives of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram-negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N-valeryl-PUGNAc, the most selective known inhibitor of NagZ over both the human beta-hexosaminidases and O-GlcNAcase. The selectivity stems from the five-carbon acyl chain of N-valeryl-PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O-GlcNAcase homologue bound to a related inhibitor N-butyryl-PUGNAc, which bears a four-carbon chain and is selective for both NagZ and O-GlcNAcase over the human beta-hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O-GlcNAcase homologue. A comparison of these complexes, and with the human beta-hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2-N-acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated beta-lactam resistance.

Extended-spectrum beta-lactamases screening agar with AmpC inhibition

The serious increase in the prevalence of extended-spectrum beta-lactamases (ESBLs) worldwide creates a need for effective and easy to perform screening methods for detection [1]. The use of an ESBL screening agar would allow rapid recognition and isolation of ESBL-producing bacteria. The currently available screening agars have low specificity, mainly due to growth of species with inducible AmpC beta-lactamases [2, 3]. The inhibition of AmpC beta-lactamases by cloxacillin is used in ESBL confirmation tests [4]; however, to the best of our knowledge, cloxacillin for ESBL screening has not been described. We developed an ESBL screening agar (ESA), which contains cloxacillin to inhibit growth of AmpC-producing species and vancomycin to inhibit growth of Enterococci, and compared it with the commercially available, selective medium for screening of presumptive ESBL Enterobacteriaceae, namely, BLSE agar (AES Laboratory, France). The ESA consists of two MacConkey agars: one containing ceftazidime 1.0 mg/l, and the other cefotaxime 1.0 mg/l, cloxacillin 400 mg/l, and vancomycin 64 mg/l. The BLSE agar is a commercial double-plate agar (MacConkey with ceftazidime 2 mg/l and Drigalski with cefotaxime 1.5 mg/l). The combined screening with cefotaxime and ceftazidime has been shown to yield the best sensitivity and specificity for ESBL detection [5]. The concentration of cefotaxime and ceftazidime in ESA is consistent with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) for screening of ESBL-producing Enterobacteriaceae [6] and with the guideline of the Dutch society for medical microbiology for screening and confirmation of ESBLs in Enterobacteriaceae (www.nvmm.nl). The inhibition of AmpC with cloxacillin depends on the concentration of cloxacillin in the agar (Fig. 1). Therefore, we evaluated the performance of ESA with different cloxacillin concentrations (400, 600, 800 and 1000 mg/l), whereby 400 mg/l yielded the most optimal sensitivity. Fig. 1 AmpC inhibition with cloxacillin. Citrobacter freundii, AmpC positive, ESBL negative. The increase in the zone around AM+CL and CFOXT with addition of cloxacillin to the MH indicates the inhibition of AmpC. R, resistant; I, intermediate susceptible; S, ... The ESA and the BLSE agar were evaluated with 208 Enterobacteriaceae isolates, 70 of them were ESBL-producers (37 Escherichia coli, 10 Klebsiella spp., 1 Proteus mirabilis, 20 Enterobacter spp., 2 Citrobacter freundii) and 138 were ESBL negative (43 E. coli, 17 Klebsiella spp., 9 Proteus mirabilis, 55 Enterobacter spp., 6 Citrobacter freundii, 4 Morganella morganii, 1 Providencia spp., 3 Serratia spp.). These isolates had previously been genotypically characterized with PCR and subsequent sequence analysis with SHV, TEM and CTX-M primers for the blaSHV,blaTEM, and blaCTX-M genes [7, 8]. The media were incubated in air at 37°C for 18 to 24 h. For negative cultures the incubation was prolonged for 48 h. The sensitivity and specificity of the ESA and the BLSE agar tested with the 208 Enterobacteriaceae isolates were 100% (70/70) and 84.7% (117/138), respectively, for the ESA, and 100% (70/70) and 57.2% (79/138) for the BLSE agar. The prolongation of incubation did not improve the sensitivity of the ESA or BLSE agars. The better performance of the ESA was mainly due to less false positive results due to AmpC-producing strains, especially Enterobacter spp. (false positive results for Enterobacter spp. with ESA was 29% or 16/55 and with BLSE was 89% or 49/55). The specificity of ESA for screening of ESBL-producing strains was significantly better than the specificity of BLSE agar, which reduced the number of unnecessary confirmations. A quick and easy to use screening method to facilitate the detection of ESBL-producing Enterobacteriaceae in clinical settings is very important for optimal therapy and early application of appropriate infection control measures. The ESA is, however, intended as a screening tool; therefore, it is important to note that any growth on the plates should not be taken as definitive proof of ESBL production, which can only be achieved by use of appropriate confirmatory tests. Despite the limitations of this preliminary study, our results show that ESA is a sensitive and convenient method to screen for ESBL-producing organisms. Further evaluation should be made with clinical specimens originating directly from human carries.

A simple disc diffusion method for detecting AmpC and extended-spectrum -lactamases in clinical isolates of Enterobacteriaceae

We sought to determine whether extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (derepressed and inducible), alone and in combination, could be detected in unidentified members of the Enterobacteriaceae using a simple, overnight disc diffusion test. The genetic basis of antibiotic resistance in cephalosporin-resistant wild-type (n = 140) and culture collection (n = 140) isolates of Enterobacteriaceae was determined using PCR. A scheme for detecting these resistance mechanisms phenotypically was devised using five antibiotic discs: cefpodoxime +/- clavulanate; cefepime +/- clavulanate and cefoxitin. AmpC beta-lactamases (derepressed and inducible) and ESBLs, alone and in combination, could reliably be detected using a disc diffusion method. ESBLs alone could be detected on the basis of a difference of >5 mm between cefpodoxime/clavulanate and cefpodoxime (10 microg) discs. ESBLs, in the presence of AmpC beta-lactamases, could be detected using a difference of >5 mm between cefepime/clavulanate and cefepime (30 microg) discs. AmpC beta-lactamases could be detected using a difference of >14 mm between cefepime/clavulanate and cefpodoxime/clavulanate discs. Inducible AmpC beta-lactamases could be discerned by observing the blunting of the cefpodoxime or cefpodoxime/clavulanate zones in proximity to cefoxitin (30 microg) discs.

Identification of AmpC Beta-Lactamases Using Phenotypic Tests and PCR in Clinical Isolates of Klebsiella spp. and Escherichia coli

Background: Indiscriminate use of beta-lactam antibiotics has resulted in emergence of AmpC Beta-lactamases. Considering the lack of comprehensive studies from India the present study was undertaken to detect and characterize AmpC Beta-lactamases in clinical isolates of Escherichia coli and Klebsiellaspp. Methods: One hundred non-repeat clinical isolates each of Escherichia coli and Klebsiella spp recovered from pus, urine, blood and sputum, were collected from various hospitals of Delhi. These were screened for susceptibility to cefoxitin (30 μg) by disc diffusion method. The screen positive isolates were subjected to Modified 3 Dimensional test (M3D), AmpC disk tests 1 and 2 and Inhibitor (Boronic acid) based detection method. AmpC positive isolates were subjected to Polymerase chain reaction (PCR) using family specific primers and also tested for production of ESBLs and inducible AmpC Beta-lactamases. Results: 34/100 (34%) of Escherichia coli and 21/100 (21%) of Klebsiella spp. were resistant to cefoxitin. Of the screen positive isolates 12 (35%) of Escherichia coli, and 5 (24%) of Klebsiella spp. were positive for AmpC by various phenotypic tests. Out of the AmpC positive isolates as many as 8/12 of Escherichia coli and 3/5 of Klebsiella isolates were positive for ESBLs. In 3/12 (25%) of Escherichia coli and 3/5 (60%) of Klebsiella isolates AmpC were inducible. PCR identified the AmpC of Escherichia coli as MIR/ACT- I and CIT types. Amongst Klebsiella isolates, the AmpC were found to be of MIR/ACTI, CIT, FOX and DHA families. Conclusion: The incidence of AmpC production was higher (12%) in Escherichia coli than in Klebsiella (5%) isolates. However, co-production of ESBLs was much higher in Klebsiella (60%) than in Escherichia coli (25%) isolates. The AmpC Beta-lactamases in Indian isolates were of common families reported from different parts of the world.

Utility of the ceftazidime-imipenem antagonism test (CIAT) to detect and confirm the presence of inducible AmpC beta-lactamases among enterobacteriaceae

Detection of AmpC beta-lactamase production by enterobacteria has been problematic. Contrary to ESBLs, no specific guidelines are available for detection and confirmation of AmpC production by clinical relevant microorganisms. Moreover, some bacterial species may produce inducible AmpC beta-lactamases that can be easily overlooked by routine susceptibility tests. We reported here a new test based on the strong inducible effect of imipenem on AmpC genes and the consequent antagonism with ceftazidime. This test is very simple and proved to be helpful in detecting AmpC-inducible enzymes among several species of clinical isolates.

EARLY ONSET MORGANELLA MORGANII SEPSIS IN A NEWBORN INFANT WITH EMERGENCE OF CEPHALOSPORIN RESISTANCE CAUSED BY DEREPRESSION OF AMPC ??-LACTAMASE PRODUCTION

A preterm infant with early onset Morganella morganii sepsis was treated with cefotaxime and gentamicin after confirmation of antimicrobial susceptibility. The infant developed persistent ventriculitis caused by the emergence of a cefotaxime-resistant Morganella variant with derepression of its AmpC beta-lactamase. When choosing antibiotic therapy, the risk of development of resistance to cephalosporins should be considered in infections caused by M. morganii and other Gram-negative organisms with inducible AmpC beta-lactamases.

Pharmacodynamic study of beta-lactams alone and in combination with beta-lactamase inhibitors against Pseudomonas aeruginosa possessing an inducible beta-lactamase.

The antimicrobial efficacies of beta-lactams alone and in combination with beta-lactamase inhibitors were investigated by applying a rabbit tissue cage model against a strain of Pseudomonas aeruginosa with an inducible AmpC (iAmpC) beta-lactamase. Two sterilized golf Wiffle balls were surgically implanted in the rabbit dorsal cervical area. After 4 weeks, Wiffle balls had filled with tissue cage fluid (TCF), in which 2 mL of 10(6) cfu/mL of the test isolate were inoculated. To achieve the same T > MIC as in humans, 400 mg/kg of the beta-lactams alone and in combination was administered twice a day via subcutaneous injection. The dosing regimens were as follows: piperacillin alone, 4 g piperacillin/0.5 g tazobactam; ticarcillin alone, 3 g ticarcillin/0.1 g clavulanate; and 3 g ticarcillin/ 0.3 g clavulanate. The changes in bacterial counts (log cfu/mL) after the 3 day treatments were as follows: 1.03 +/- 0.97 (control), -1.31 +/- 0.61 (piperacillin), -2.81 +/- 0.53 (4 g piperacillin/0.5 g tazobactam), -1.61 +/- 0.68 (ticarcillin), -3.42 +/- 0.75 (3 g ticarcillin/0.1 g clavulanate) and -1.65 +/- 1.47 log cfu/mL (3 g ticarcillin/0.3 g clavulanate). AmpC induction by high-dose clavulanate was observed in rabbit TCF, and was confirmed by the in vitro induction study. The study indicated that tazobactam significantly enhanced the antibacterial activity of piperacillin against iAmpC P. aeruginosa; clavulanate had synergy with the antibacterial activity of ticarcillin at low concentration, but had no effect on ticarcillin at high concentration due to AmpC induction by clavulanate.