Double-disk Test Research Articles

Molecular and Phenotypic Study of Carbapenem Resistant E. coli.

Carbapenem resistance is an emerging problem. The aim of the present study was to determine the prevalence of carbapenem resistance and ESBLs among clinical isolates of E. coli by phenotypic methods and to study the molecular bases of the resistance by polymerase chain reaction (PCR). The study was carried on 153 non repetitive E. coli strains collected from different clinical samples from Baghdad hospitals. The strains were isolated according to standard microbiological procedures and identified by Gram stain and biochemical reactions. E. coli strains were subjected to antimicrobial susceptibility tests by the disc diffusion method, carbapenem screening, ESBL screening tests, and molecular studies for genes responsible for such resistance. The study was carried out on 153 non repetitive E. coli. Detection of ESBLs by the double disc method reveals that 63 isolates (41.2%) of isolated E. coli had ESBL activity. Carbapenem resistance among E. coli reveals that 30 isolates (19.6%) had both metallo-β-lactamase and carbapenemase activity as detected by double disc synergy test and boronic acid disc. Among 63 isolates of E. coli with positive double disc tests for ESBL resistance, the most frequent gene detected by PCR was blaTEM (52.4%), followed by blaSHV (33.3%) and blaCTX-M (14.3%). Among 30 E. coli strains with metallo-β-lactamase activity and carbapenemase activity, the most frequent genes were IMP, VIM (30% for each), followed by NDM (20%), GIM, SIM, SPM, and KPC (10% for each). Isolated E. coli with carbapenem resistance represented 47.6% of E. coli with ESBL activity. The present study highlights the incidence of carbapenemase among clinical isolates of E. coli combined with extended β-lactamases activity. Phenotypic screening methods were valuable for detection of different types of resistance. The common genes responsible for carbapenem resistance were IMP, VIM, and NDM. Further studies are recommended with more included E. coli isolates.

Activity of Ceftolozane/tazobactam (C/T) and Ceftazidime/avibactam (CZA) against Extended-spectrum Β-lactamase (ESBL)-producing Enterobacteriaceae and Multidrug-resistant (MDR) Pseudomonas aeruginosa Isolates

BackgroundAntibiotic resistance among Gram-negative bacterial pathogens is a serious public health threat underscored by a diminishing antibiotic development pipeline. This study evaluated the in vitro activity of two new β-lactam/β-lactamase inhibitors, C/T and CZA, against the problematic pathogens ESBL-producing Enterobacteriaceae and MDR P. aeruginosa.MethodsA convenience sample of 74 ESBL-producing Enterobacteriaceae and 32 MDR P. aeruginosa clinical isolates (1 per patient episode), collected between 2012 and 2017 from 2 academic medical centers in Boston and Philadelphia, was used. MDR was defined as non-susceptibility to ≥1 agent in at least 3 antibiotic classes. Phenotypic confirmation of ESBL isolates was conducted via double disk testing. MICs were determined by broth microdilution in at least duplicate, on separate days, as recommended by CLSI. Klebsiella pneumoniae ATCC 700603 was used as a quality control strain with each experiment. Interpretive criteria for C/T per CLSI were: ≤2 (susceptible) / 4 (intermediate) / ≥8 (resistant) mg/L (Enterobacteriaceae) and ≤4 (susceptible) / 8 (intermediate) / ≥16 (resistant) mg/L (P. aeruginosa). Interpretive criteria for CZA per FDA were: ≤8 (susceptible) / ≥16 mg/L (resistant) (all organisms).ResultsCulture sites for the total isolate collection consisted of urine (n = 62), sputum (n = 30), wounds (n = 9), blood (n = 3), and bone (n = 2). ESBL-producing Enterobacteriaceae included Escherichia coli (n = 60), Klebsiella spp. (n = 12), Enterobacter cloacae (n = 1), and Citrobacter freundii (n = 1). ESBL Enterobacteriaceae isolates were 97.3% and 100% susceptible to C/T and CZA, respectively. The corresponding MIC50/90 values were 0.5/2 mg/L for C/T and 0.25/0.5 mg/L for CZA. MDR P. aeruginosa isolates were 90.6% and 96.9% susceptible to C/T and CZA, respectively. The corresponding MIC50/90 values were 1/4 mg/L for C/T and 2/8 mg/L for CZA.ConclusionC/T and CZA, two recently approved β-lactam/β-lactamase inhibitor combinations, maintained reliable activity against a collection of 106 MDR isolates from 2 geographically diverse medical centers.Disclosures E. B. Hirsch, Merck: Grant Investigator, Research grant The Medicines Company: Speaker’s Bureau, Speaker honorarium; T. Bias, Merck: Grant Investigator, Research grant The Medicines Company: Speaker’s Bureau, Speaker honorarium

Molecular epidemiology of macrolide resistant Group A streptococci from Puducherry, India.

In penicillin allergic patients, macrolides are the most commonly used antibiotics for treating streptococcal infections, irrespective of the higher resistance rates. The objective of this study was to evaluate the comparative prevalence, phenotypes, and genetic determinants of macrolide resistance and associated emm types among different clinical isolates of Streptococcus pyogenes. A total of 173 Streptococcus pyogenes isolates were examined for macrolide resistance phenotype by double-disc test, resistance determinants by multiplex PCR and emm genotyping. Erythromycin resistance was found in 51.4% of isolates, with MIC90 ≥ 256 µg/mL Inducible phenotype was commonly found (iMLS, 67.4%) followed by the M phenotype (32.5%). Among these isolates, 65.1% harboured ermB and 32.5% mefA as sole macrolide resistance gene, whereas presence of both, ermB plus mefA was observed in 2.2% cases. The most common types among resistant strains were emm63 (11.2%), emm44 (6.7%), emm42 (5.6%), and emm75.3, emm82, emm85, emm92, emm111.1 (4.4% each). Statistically significant association was observed between emm63, emm44 and erythromycin resistance (p ≤ 0.05). Association of these emm types and macrolide resistance have not been reported earlier. Higher macrolide resistance in this study can be attributed to overuse and misuse of this antibiotic. These findings indicate that macrolides should not be empirically used for treating severe streptococcal infections.

Molecular characterization of antibiotic-resistant Staphylococcus aureus from livestock (bovine and swine).

Aim:The aim of this study was to figure the prevalence, phenotypic and genotypic antibiotic resistance (AR) pattern of Staphylococcus aureus isolated from bovine and swine nares.Materials and Methods:Colonies with typical morphology on Baird-Parker agar supplemented with egg-yolk tellurite emulsion were selected and biochemically/genotypically identified as S. aureus. These strains were further subjected to epsilometer test for their sensitivity to various clinically important antibiotics and antibiotic susceptibility testing for amoxicillin/clavulanic acid, and double-disk diffusion testing was performed by the standard disc diffusion method following CLSI guidelines. S. aureus strains were also tested for the presence of AR genes, viz., blaZ, mecA, aacA-aphD, erm (ermA, ermB, ermC), tet (efflux genes tetK and tetL, tetM and tetO of the ribosomal protection family), and vanA.Results:The nasal cavities of 17 out of 47 randomly selected bovine and 20 out of 28 randomly selected swine were positive for S. aureus, representing the prevalence of 36.2% (95% confidence interval [CI]: 22.5-49.9) and 71.4% (95% CI: 54.7-88.1), respectively. Most of the S. aureus strains showed higher resistance to penicillin (94.6%, minimal inhibitory concentration [MIC] ≥1.5 µg/ml) followed by ciprofloxacin (56.7%, MIC ≥32 µg/ml) and tetracycline (18.9%, MIC ≥32 µg/ml). About 10-15% of the strains were resistant to gentamicin (MIC 16 µg/ml) and oxacillin (MIC 6-8 µg/ml). None of the strains were resistant to vancomycin (MIC 0.25-1.5 µg/ml). In this study, 32.4% strains were resistant to three or more than three antibiotics and prevalence of this multi-drug resistant S. aureus was 45% (95%CI: 26.6-63.4) and 17.6% (95%CI: 6.7-28.5) in swine and bovine nasal samples, respectively. Four strains from pigs were borderline oxacillin-resistant S. aureus MIC 6-8 µg/ml, but none were mecA positive. Two of these strains were β-lactamase hyperproducers. Among the resistance genes blaZ, tetK, tetL, tetM, ermB, and aacA-aphD were found.Conclusion:Our results demonstrated the absence of mecA and pvl gene, but the presence of multi-drug resistant S. aureus in the nares of healthy animals which has a potential to spread in a community.

Detection of extended-spectrum β-lactamase (ESBL) and plasmid-borne blaCTX-M and blaTEM genes among clinical strains of Escherichia coli isolated from patients in the north of Iran.

Escherichia coli is an important cause of hospital-acquired infections worldwide. Antimicrobial resistance leads to treatment failure of hospital infections caused by E. coli. Production of extended-spectrum β-lactamases (ESBLs) is one of the major causes of antibiotic resistance in these bacteria. This study aimed to investigate the frequency of blaTEM and blaCTX-M genes in ESBL-producing E. coli strains isolated from clinical specimens of patients admitted to six hospitals in the north of Iran. A total of 160 E. coli strains were isolated from various clinical samples of hospitalised patients. Antibiotic resistance patterns were determined by the Kirby-Bauer disk diffusion method. The double-disk phenotypic confirmatory test was carried out amongst β-lactam-resistant isolates to detect ESBL-producing strains. Plasmid DNA of ESBL-producing strains was extracted and subjected to PCR for detection of the blaTEM and blaCTX-M genes, and isolates were extensively verified by sequencing. The highest resistance rate was to amoxicillin; all E. coli isolates (100%) were susceptible to imipenem. Amongst the 160 clinical E. coli isolates, 83 (51.9%) were ESBL-positive, of which 27 (32.5%) and 72 (86.7%) were positive for blaTEM and blaCTX-M, respectively. This study is the first report of an ESBL phenotype disseminated in hospitals in the north of Iran. These findings showed that there was a direct relationship between the development of resistance to β-lactam antibiotics and production of TEM and CTX-M enzymes.

Erythromycin Resistance in Streptococcus Pyogenes Causing Pharyngotonsillitis in Egyptian Children and Its Association with the Presence of Fibronectin Binding Protein F1 (prtf1) Gene

Background: Erythromycin resistance among Streptococcus pyogenes (S. pyogenes) has been reported with alarming rates throughout the world. Fibronectin-binding proteins, especially F1 (PrtF1) encoded by prtF1 gene have been implicated in mediating internalization of S. pyogenes within epithelial cells. Objectives: the current study aimed to detect erythromycin resistance among S. pyogenes causing acute pharyngo-tonsillitis in Egyptian children and to explore the association between erythromycin resistance and the presence of prtf1 gene. Methodology: Throat swabs were collected from 100 pediatric patients with age ranging from 5 to 12 years and suffering from pharyngo-tonsillitis . s. pyogenes was recovered from samples and identified by conventional methods . In-vitro susceptibility of s. pyogenes isolates to different antimicrobials was done using disk diffusion method. Minimum inhibitory concentration for erythromycin was determined using E test strips. The phenotype of macrolide resistance was determined using the double-disk test. The presence of prtF1 gene was detected by polymerase chain reaction. Results: S. pyogenes were isolated from 45% (45/100) of the patients’ throat swabs. Erythromycin resistance was detected in 11 (24.4%) isolates , among which 5 (45.5%) exhibited the M phenotype of macrolide resistance; 4 isolates had the constitutive phenotype of resistance while 2 isolates had the induced phenotype. prtF1 gene was detected in 16/45 isolates (35.6%). There was statistically significant association between prtF1 gene presence and erythromycin resistance (OR: 4.8611; 95% CI: 1.14-20.63, p= 0.032). Conclusion : The present study provides a preliminary idea about the frequency of macrolide resistance and contribute to a better understanding of the pathogenesis of S. pyogenes causing pharyngo-tonsillitis in Egyptian children.

Detection of extended spectrum beta-lactamases and resistance in members of the Enterobacteriaceae family isolated from healthy sheep and dogs in Umuarama, Paraná, Brazil

Bacterial resistance is a primary public health concern worldwide. Within this context, pets and breeding animals act as reservoirs for multidrug-resistant bacteria (MR), such as those producing extended spectrum beta-lactamases (ESBL) and those presenting plasmid-mediated quinolone resistance (PMQR). The aim of this study was to detect the presence of ESBL and PMQR in members of the Enterobacteriaceae family, isolated from healthy sheep and dogs from non-intense farming rural properties in the Umuarama region of Paraná, Brazil. A total of 81 oral and rectal swabs from dogs and sheep from 11 small rural properties were analyzed. These swabs were inoculated in tubes containing brain heart infusion broth (BHI), and the resulting cultures were inoculated on MacConkey agar (MAC) supplemented with 10 ?g/mL cefotaxime for the selection of ESBL producers. The cells were also plated on MAC supplemented with 50 ?g/mL nalidixic acid for selecting quinolone-resistant enterobacteria. The bacterial isolates were subjected to biochemical identification tests, antibiograms, double-disk synergic tests, and polymerase chain reaction analysis for resistance-inducing genes (blaESBL, qnr, and genes encoding efflux pump and acetylases). Four (5.00%) bacterial isolates (3 Escherichia coli and 1 Morganella morganii) resistant to cephalosporins and/or quinolones were identified; of these, three (75%) isolates were from sheep and one (25%) from a dog. These findings indicate the presence of MR bacteria in the normal microbiota of the animals studied. Animals colonized with such bacteria can contribute to the dissemination of antimicrobial resistance to other animals, environment, and/or human beings and can harbor endogenous infections in unfavorable conditions, which have poor prognosis due to the limited therapeutic options.

Prevalence and antibiogram of extended spectrum β-lactamase producing Klebsiella pneumoniae and Proteus mirabilis in clinical isolates

Rapid emergence of ESBL production in bacteria like Klebsiella and Proteus species has put forward a major challenge in their detection as well as treatment options. This study was conducted from Oct 2012 to May 2015 to know the prevalence and antibiogram of ESBL producing Klebsiella pneumonia & Proteus mirabilis. Blood, sputum, pus, wound samples were collected and processed; identification of isolates was done as per standard methods. ESBL detection was done with double disk test and combination disk and confirmed with MIC reduction test. 208/578 (36.0%) &126/ 405 (31.1%) isolates of Klebsiella pneumoniae & Proteus mirabilis were ESBL producers respectively. The present study showed a high percentage of ESBL producers among clinical isolates of Klebsiella pneumoniae and Proteus mirabilis to frequently used antibiotics. Knowledge of antibiogram studies will help physicians in choosing an empirical treatment. To know the changing trends in resistance regular antibiotic susceptibility studies should be conducted. Key words: Extended spectrum beta lactamases (ESBL), Klebsiella pneumoniae, Proteus mirabilis, antibiogram, Resistance

Prevalence and Characterization of Carbapenem-Resistant Enterobacteriaceae Isolated from Mulago National Referral Hospital, Uganda.

IntroductionCarbapenemases have increasingly been reported in enterobacteriaceae worldwide. Most carbapenemases are plasmid encoded hence resistance can easily spread. Carbapenem-resistant enterobacteriaceae are reported to cause mortality in up to 50% of patients who acquire bloodstream infections. We set out to determine the burden of carbapenem resistance as well as establish genes encoding for carbapenemases in enterobacteriaceae clinical isolates obtained from Mulago National Referral Hospital, Uganda.MethodsThis was a cross-sectional study with a total of 196 clinical isolates previously collected from pus swabs, urine, blood, sputum, tracheal aspirates, cervical swabs, endomentrial aspirates, rectal swabs, Vaginal swabs, ear swabs, products of conception, wound biopsy and amniotic fluid. All isolates were subjected to phenotypic carbapenemase screening using Boronic acid-based inhibition, Modified Hodge and EDTA double combined disk test. In addition, all the isolates were subjected to PCR assay to confirm presence of carbapenemase encoding genes.ResultsThe study found carbapenemase prevalence of 22.4% (44/196) in the isolates using phenotypic tests, with the genotypic prevalence slightly higher at 28.6% (56/196). Over all, the most prevalent gene was blaVIM (21,10.7%), followed by blaOXA-48 (19, 9.7%), blaIMP (12, 6.1%), blaKPC (10, 5.1%) and blaNDM-1 (5, 2.6%). Among 56 isolates positive for 67 carbapenemase encoding genes, Klebsiella pneumonia was the species with the highest number (52.2%). Most 32/67(47.7%) of these resistance genes were in bacteria isolated from pus swabs.ConclusionThere is a high prevalence of carbapenemases and carbapenem-resistance encoding genes among third generation cephalosporins resistant Enterobacteriaceae in Uganda, indicating a danger of limited treatment options in this setting in the near future.

Antimicrobial Profile and Phenotypic Metallo-β-Lactamase Detection of Acinetobacter baumannii Isolated From Clinical and Environmental Specimens

Background:: One of the most challenging isolates in nosocomial infections is Acinetobacter baumannii which is rapidly changing towards multi-drug resistant. Resistance to the last resort antibiotics, carbapenems, is reported around the world. In particular, metallo-β-lactamases (MBL) are responsible and detection of MBLs is of interest. Objectives:: In this study we have evaluated the prevalence of MBLs in A. baumannii isolated during 2012 by two feasible described methods. Materials and Methods:: In this cross sectional study, during 2012, 40 A. baumannii non-replicate isolates gathered from clinical and environmental specimens from Afzalipur hospital of Kerman. Isolates were characterized by conventional biochemical tests as A. baumannii-calcoaceticus complex (ABC). For antimicrobial susceptibility testing, 17 different antibiotics were sought by agar diffusion method. Phenotypic MBL detection was assessed by double disk test and Modified Hodge test for carbapenem resistant isolates. Results:: During the study 40 isolates of ABC were collected. Of which, 23 (57%) isolates were resistant to carbapenems. Phenotypic MBLs detection was negative by two methods. Conclusions:: The rates of resistance of the isolated ABCs in our clinical setting were at high level. Though, carbapenems were the most efficient antibiotic, but, there was a pronounced rate of resistant. MBL genes were not responsible for carbapenem resistance in under study clinical setting.

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.

Study of Serotypes, Susceptibility to Macrolide and Virulence and Resistance Molecular Profiles in Invasive Strains of <i>Streptococcus agalactiae</i> in Two Argentine Provinces

A study of invasive strains of Streptococcus agalactiae (GBS) from Cordoba and Misiones, Argentina; was conducted to determine serotypes, the susceptibility to macrolides and molecular profiles of virulence and resistance. We studied 17 strains, recovered from cerebrospinal fluid, blood and cellulite and, a strain of trophoblastic remnants from Misiones. The serotypes were determined by agglutination with sera and phenotypes of resistance to macrolide-lincosamide-streptogramin B (MLSB), were determined with the double-disk test (D-test). The confirmation was performed by E-test by ERI and CLI respectively that determined the minimum inhibitory concentration (MIC). Results were interpreted as recommended by the Clinical and Laboratory Standards Institute (CLSI) 2013. Resistance genes: ermB, ermTR and mefA and the virulence genes: bac, bca, rib, lmb, hylB, scpB, fbsA, fbsB and cylB were investigated by conventional PCR. Serotype III (50%) and Ia (50%) were detected in Cordoba. One strain showed cMLSB phenotype, confirmed by MIC. The same strains showed a resistance gene ermB. All studied virulence genes were detected in 100% of these strains. In Misiones, serotypes were III (72.7%), Ia (18.2%) and Ib (9.1%). All strains were susceptible to CLI and ERI by D-test, confirmed by MIC. None of the strains showed resistance genes. Virulence genes bca, rib, hylB, lmb, fbsA, fbsB and cylB were detected in 100% of the strains, bac in 81.8% and scpB in 90.9%. Our results are in accordance with international data, associating higher frequency of serotype III of invasive neonatal disease followed by Ia. The presence of serotype Ib could indicate a regional difference for Misiones. We highlight the macrolides susceptibility in strains of Misiones and consistency in the results for D-test, MIC and PCR for the single strain resistant phenotype cMLSB from Cordoba. The virulence genes studied were presented with high frequency as expected for invasive strains.

Determination of antimicrobial resistance profile and inducible clindamycin resistance of coagulase negative staphylococci in pediatric patients: the first report from Iran.

Currently, coagulase negative staphylococci (CoNS) have got much attention as a serious health problem especially in neonates and children. High incidence of antibiotic resistance, in particular methicillin resistance, has complicated the treatment of these organisms. The aim of this study is to determine the susceptibility to different antimicrobial agents and the prevalence of macrolideslincosamides-streptogramins B (MLSB) resistance in CoNS isolates obtained from pediatric patients. Totally 157 CoNS isolates from various clinical samples were examined for antibiotic resistance using disk diffusion and E-test methods. Double-disk test was applied to detect constitutive and inducible MLSB resistance (cMLSB and iMLSB) phenotypes. Resistance to methicillin was seen in 98 (62.4%) isolates. All isolates were susceptible to vancomycin and linezolid. The prevalence of resistance to antibiotics tested was as follows: fusidic acid (n=58, 36.9%), gentamicin (n=73, 46.5%), ciprofloxacin (n=81, 51.6%), clindamycin (n=112, 71.3%), erythromycin (n=129, 82.2%) and trimethoprim/sulfamethoxazole (n=133, 84.7%). iMLSB phenotype was seen in 14 (8.9%) isolates, and 18 (11.5%) and 98 (62.4%) isolates showed MS and cMLSB phenotypes, respectively. We observed that high overall antibiotic resistance rates were associated significantly with methicillin resistance. Conversely, iMLSB phenotype was correlated neither with methicillin resistance nor with invasiveness. Given the similarity observed between the prevalence of iMLSB and MS phenotypes, the performance of disk diffusion induction test is strongly recommended in our region.

Erythromycin-resistant genes in group A β-haemolytic Streptococci in Chengdu, Southwestern China.

The management of Group A β-haemolytic Streptococci (Streptococcus pyogenes or GAS) infection include the use of penicillins, cephalosporins or macrolides for treatment. A general increase in macrolides resistance in GAS has been observed in recent years. Differences in rates of resistance to these agents have existed according to geographical location and investigators. To investigate the antibiotic pattern and erythromycin-resistant genes of GAS isolates associated with acute tonsillitis and scarlet fever in Chengdu, southwestern China. To assess the macrolide resistance, phenotype, and genotypic characterization of GAS isolated from throat swabs of children suffering from different acute tonsillitis or scarlet fever between 2004 and 2011 in the city of Chengdu, located in the southwestern region of China. Minimal inhibitory concentration with seven antibiotics was performed on 127 GAS isolates. Resistance phenotypes of erythromycin-resistant GAS isolates were determined by the double-disk test. Their macrolide-resistant genes (mefA, ermB and ermTR) were amplified by PCR. A total of 98.4% (125/127) of the isolates exhibited resistance to erythromycin, clindamycin and tetracycline. All isolates were sensitive to penicillin G and cefotaxime. Moreover, 113 ermB-positive isolates demonstrating the cMLS phenotype of erythromycin resistance were predominant (90.4%) and these isolates showed high-level resistance to both erythromycin and clindamycin (MIC 90>256 μg/ml); 12 (9.6%) isolates demonstrating the MLS phenotype of erythromycin resistance carried the mefA gene, which showed low-level resistance to both erythromycin (MIC 90=8 μg/ml) and clindamycin (MIC 90=0.5 μg/ml); and none of the isolates exhibited the M phenotype. The main phenotype is cMLS, and the ermB gene code is the main resistance mechanism against macrolides in GAS. Penicillin is the most beneficial for treating GAS infection, and is still used as first-line treatment. And macrolide antibiotics are not recommended for treatment of GAS infection in children because of the high rates of antimicrobial resistance in mainland China.

Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae.

BackgroundMultidrug resistance and, in particular, carbapenem resistance is spreading worldwide at an alarming rate, comprehending a variety of bacterial species and causing both nosocomial and community acquired outbursts. Early and efficient detection of infected patients or colonized carriers are mandatory steps in infection control and prevention of multidrug resistance diffusion. The latest EUCAST guidelines for detection of carbapenemase-producing Enterobacteriaceae have set low clinical breakpoints to ensure the maximum detection sensitivity of positive samples. Current workflows involve an initial screening step for species and resistance pattern detection, followed by phenotypic and/or genotypic confirmation. The aim of the present study was to assess the efficiency of six widely used and validated phenotypic assays for the detection of carbapenemases/AmpC in Enterobacteriaceae, to estimate the best workflow in the routine characterization of Enterobacteriaceae isolates.MethodsA panel of 108 non-repetitive Enterobacteriaceae isolates with reduced susceptibility to carbapenems was analyzed by means of 1) Modified Hodge Test, 2) Metallo Beta Lactamase Etest, 3) Double disk test with EDTA, 4) Rosco Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test. Confirmation and validation of results was achieved by genotypic analysis.ResultsThe most accurate identification of resistance determinants was obtained with the combined disc test (Rosco Diagnostica KPC and MBL confirm kit) which had to be coupled with the cloxacillin inhibition test for correct detection of AmpC enzymes. However, in general, phenotypic tests failed to characterize isolates harboring multiple carbapenem resistance determinants, which were successfully assessed only by PCR-based analysis.ConclusionsTo detect and control the spread of pathogens with complicated resistance patterns, both optimized phenotypic analysis (i.e. Rosco Diagnostica KPC and MBL confirm kit coupled with the cloxacillin inhibition test) and genotypic assays are recommended in the routine diagnostic of clinical laboratories.

Characterization of AmpC, CTX-M and MBLs types of β-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli producing Extended Spectrum β-lactamases in Kerman, Iran.

Background:Extended spectrum β-lactamases (ESBLs) and AmpC β-lactamases enzyme are major sources of resistance to β-lactam antibiotics especially in Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae. Increasing frequency of the co-existence of ESBLs with AmpC-β-lactamases in bacteria is a serious threat for treating bacterial infections.Objectives:The aim of this study was to determine the presence of AmpC and CTX-M types of β-lactamases in clinical isolates of E. coli and K. pneumoniae producing ESBLs.Materials and Methods:Resistance to different antibiotics was determined using the standard disk diffusion method. ESBLs, MBLs and AmpC-β-lactamases were detected by the combination double disk test (CDDT) method and polymerase chain reaction (PCR) was used to determine blaCTX-M genes in the ESBLs and AmpC positive isolates.Results:The prevalence of ESBLs and AmpC-β-lactamase producer isolates was 181 (43.8%) and 133 (37.2%), respectively. The prevalence of blaCTX-M among isolates was 61 (14.7%).Conclusions:Outbreak of isolates co-expressing AmpC-β-lactamases and ESBLs can cause serious problems in the future, regarding the treatment of infections caused by these common enteric pathogens.

Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in Menoufia University Hospitals

Objective The aim of the study was to evaluate the prevalence of extended-spectrum β-lactamase (ESBLs) in Enterobacteriaceae among patients conducted at Menoufia University Hospitals. Background Resistance to β-lactam antibiotics is an increasing problem, and β-lactamase production is the most common mechanism of drug resistance, especially in Enterobacteriaceae. The Clinical and Laboratory Standard Institute (CLSI) interpretive guidelines stated that Enterobacteriaceae that produce ESBLs are resistant to therapy with penicillin, cephalosporins, and aztreonam, despite apparent in-vitro susceptibility to some of these agents. Therefore, detection of ESBLs in Enterobacteriaceae is crucial for optimal treatment of patients and to control the spread of resistance. Patients and methods The isolated strains of Enterobacteriaceae are subjected to phenotype detection by screening for ESBLs by disk diffusion test then to confirmatory tests for ESBLs by double disk test. Results A total of 97 of 160 (60.6%) isolates were found to be ESBL producers by the CLSI confirmatory method. Of 97 ESBL producers, 45 (46.4%) were Escherichia coli , 28 (28.9%) were Enterobacter spp., and 22 (22.7%) were Klebsiella spp. Of the various clinical samples, the most frequent ESBLs isolates were from urine samples (27.8%), followed by sputum samples (18.6%). Conclusion High prevalence of ESBL producers in our hospital (60.6%) calls for strict policies regarding antibiotic usage and their screening methods, and hospital-based clinical laboratories should screen isolates following hospitalization in patients in need for antibiotics to formulate effective antibiotic strategy and plan a proper hospital infection control strategy to prevent the spread of these ESBL strains.

Metallo beta lactamase mediated resistance in carbapenem resistant gram-negative bacilli: A cause for concern

Introduction: The emergence of acquired metallo-β-lactamases (MBL) in Gram-negative bacilli is becoming a therapeutic challenge, as these enzymes usually possess a broad hydrolysis profile that includes carbapenems, extended-spectrum β-lactams. Aim: To detect Extended spectrum β-lactamases and metallo-β-lactamase in carbapenem resistant Gram negative clinical isolates from various clinical specimens and to evaluate their antibiotic susceptibility patterns. Material and Methods: A total of 100 non duplicates imipenem resistant isolates were tested for the presence of extended spectrum β-lactamases by phenotypic confirmatory test, metallo-β-lactamases by Double disk synergy test with various distances from edge to edge (10mm,15mm,20mm), between the IPM and EDTA and combined disc test. Result: Of the 100 IMP resistant isolates screened 30 (30%) were MBL positive by phenotypic methods, i.e., double disk synergy test and combined disc test. Co-existence of Extended spectrum β- lactamases and MBL were detected in 3 (30%). All the 30 MBL positive isolates had shown synergy at (100%) at 10 mm distance, 27 (90%) isolates had shown synergy at 15 mm distance and 13 (43.4%) isolates were shown synergy at 20 mm distance. All the 30 MBLs producers were multidrug resistant and 27 (90%) were sensitive to colistin (CL). All MBL positive Pseudomonas aeruginosa were sensitive to polymyxin B (100μg). Conclusion: Microbiologists are now facing a challenge of drug resistance due to MBL production. Although CLSI guidelines do not quote about the ESBL detection in Pseudomonas aeruginosa MBLs and ESBL have to be detected in them. The use of combination tests would increase the sensitivity to detect the presence of MBL among the clinical isolates of Gram-negative bacilli. The spread of MBL producing Gram negative organism can be prevented if they are detected in all isolates and routinely adopted in all laboratories.

Detection of extended-spectrum ß-lactamases in members of the family Enterobacteriaceae: comparison of the Combination Double Disc Test Method and the Etest ESBL.

Purpose: To evaluate the performance of the Combination Double Disc Method in the identification of ESBL-producing Enterobacteriaceae isolates. Materials and Methods: A total of 44 clinically relevant Enterobacteriaceae isolates were examined. The ESBL classification furnished by Combination Double Disc Test Method was concordant with that of the comparison method (Etest ESBL and molecular identification of beta-lactamase genes) for 18 (41%) of the 44 isolates evaluated. Results: ESBL production was correctly detected in 41% ESBL-producing organisms with at least one of the combination disks (sensitivity, 80%). Conclusion: In our study, Combination Double Disc Test Method appears to be a rapid and reliable tool for routine identification of ESBL-producing isolates of Enterobacteriaceae.

Resistance Pattern of Pseudomonas aeruginosa in a Tertiary Care Hospital of Kanchipuram, Tamilnadu, India.

This study was undertaken to analyze the extended spectrum of β lactamase (ESBL), metallo β lactamase (MBL) & AmpC production in Pseudomonas aeruginosa in various clinical samples. One hundred four non repetitive clinical specimens were inoculated onto nutrient agar, blood agar and incubated at 37(o)C overnight. The colonies were tested for oxidase test and other biochemical tests and antibiogram. ESBL screening was done using 3(rd) generation cephalosporins and confirmatory combined double disc test, imipenem-EDTA double disc synergy test for MBL enzyme and AmpC test using Cefoxitin disc. Results & Analysis: Out of 104 P.aeruginosa isolates, 42.30% were ESBL producer, 15.38 % MBL producer and none were AmpC producer. Imipenem, Ofloxacin, and aminoglycosides (amikacin (29.8%) tobramycin (29.8%) and netilmycin (13.46%) has got the better antipseudomonal activity in this study. 43 (41.35%) P.aeruginosa was found to be Multi Drug Resistant (MDR). This study highlights the prevalence of ESBL, MBL and MDR P.aeruginosa. Carbapenems and aminoglycosides are promising drugs with antipseudomonal activity in our study.