Disc Diffusion Methodology Research Articles

Reconsidering azithromycin disc diffusion interpretive criteria for Salmonellae in view of azithromycin MIC creep among typhoidal and nontyphoidal salmonella.

PURPOSE:Enteric fever continues to be an important public health challenge for the developing world. With the emergence of fluoroquinolone resistance in Salmonellae spp. azithromycin is increasingly being used for oral treatment of enteric fever. We investigated the antibiotic susceptibility pattern of azithromycin in Salmonellae spp. isolates from a tertiary care hospital to detect emerging resistance.METHODS:The study assessed the reliability of disc diffusion as a screening test to detect azithromycin resistance by comparing it with the minimum inhibitory concentrations (MICs) of the drug in 100 Salmonellae spp. strains. The strains of Salmonellae spp. showing resistance to azithromycin were further investigated for resistance markers – mphA, mphB, and mef B genes.RESULTS:This study was conducted on 100 Salmonella enterica strains recovered from blood culture samples between 2013 and 2017. Among these isolates, 18 showed resistance to azithromycin by disc diffusion methodology with zones of inhibition <13 mm. MIC of 6 of these isolates were ≥32 mg/L. The mean MIC of azithromycin increased from 5 mg/L in 2013 to 24 mg/L in 2017. Azithromycin consumption as defined daily doses per 1000 patient days also showed an increase over the past 4 years.CONCLUSION:Azithromycin disc diffusion diameter interpretations as recommended by Clinical and Laboratory Standards Institute can mislabel a few sensitive strains as resistant. Azithromycin resistance is emerging in typhoidal and nontyphoidal Salmonella. MphA gene is associated with high MICs in nontyphoidal Salmonella spp.

Discrepancies in fosfomycin susceptibility testing of KPC-producing Klebsiella pneumoniae with various commercial methods

Fosfomycin susceptibility testing with Sensititre, Vitek2, Etest, Mic Strip Test and disk diffusion methodologies was compared versus reference agar dilution method (AD) with 78 clinical isolates of KPC-producing Klebsiella pneumoniae. All methodologies showed a Categorical Agreement and Essential Agreement of ≤69% and ≤72%, respectively, revealing a very low concordance with AD.

Evaluation of the Shift in Antimicrobial Resistance Due to Extended Spectrum Beta-Lactamase and AmpC Producing Enterobacteriaceae in Hampshire England

The aim of this study was to identify shifts in antimicrobial resistance in the Hampshire region including correlations between patient demographic and antibiotic prescribing to inform safe and effective antimicrobial stewardship. 475 ESBL and AmpC producing bacteria, from various infection sites, were obtained from four hospital laboratories in Hampshire, UK, during 2010 and 2012. All isolates were identified to species level. ESBL production and antimicrobial susceptibility testing was performed using disc diffusion methodology. Multiplex PCR and gel electrophoresis was used to detect the BlaCTX, BlaTEM and BlaSHV resistance determinants. Corresponding patient data included patient age, gender, location, clinical details and previous antibiotic therapy. Patient information revealed mean ages of 60 and 62 for the 2010 and 2012 cohorts respectively, with ages ranging from 3 months to 96. ESBL production was the most prevalent resistance mechanism (65% in 2010, 79% in 2012), produced mostly by E.coli (85% in 2010, 84% in 2012). While 9 of the 13 antibiotics demonstrated increased resistance, 4 demonstrated a decrease. CTX was the most prevalent resistance determinant (38% in 2010 and 27% in 2012), followed by dual expression of CTX &amp; TEM, TEM, TEM &amp; SHV, SHV, SHV &amp; CTX and all three resistance determinants. A significant correlation between patient age and joint expression of TEM &amp; SHV was observed in 2010, in 2012, patient age significantly correlated with joint CTX and SHV expression. Significant differences could also be determined between resistance determinant type and antimicrobial resistance. This study shows that the incidence of ESBL and AmpC infections and resistance to commonly used antibiotics within Hampshire is increasing both within the hospital and community setting. This emphasises the need for judicious antibiotic prescribing to safeguard this valuable medical commodity.

Aerococcus urinae and Aerococcus sanguinicola: Susceptibility Testing of 120 Isolates to Six Antimicrobial Agents Using Disk Diffusion (EUCAST), Etest, and Broth Microdilution Techniques.

Background: Aerococcus urinae and Aerococcus sanguinicola are relatively newcomers and emerging organisms in clinical and microbiological practice. Both species have worldwide been associated with urinary tract infections. More rarely cases of bacteremia/septicemia and infective endocarditis have been reported. Treatment options are therefore important. Just recently, European recommendations on susceptibility testing and interpretive criteria have been released.Objective:In this investigation 120 A. urinae and A. sanguinicola isolates were tested for susceptibility to six antimicrobial agents: Penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin.Methods:Three susceptibility testing methods were used; disk diffusion according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) standardized disk diffusion methodology and MIC determination with Etest and broth microdilution (BMD). All testing was performed with EUCAST media for fastidious organisms.Results:Data obtained in this study were part of the background data for establishing EUCAST breakpoints. MIC values obtained by Etest and BMD were well correlated with disk diffusion results.Conclusion:All isolates were found susceptible to all six antimicrobial agents: penicillin, cefotaxime, meropenem, vancomycin, linezolid, and rifampicin.

Bioactive Potential of 3D-Printed Oleo-Gum-Resin Disks: B. papyrifera, C. myrrha, and S. benzoin Loading Nanooxides-TiO2, P25, Cu2O, and MoO3.

This experimental study investigates the bioactive potential of filaments produced via hot melt extrusion (HME) and intended for fused deposition modeling (FDM) 3D printing purposes. The oleo-gum-resins from benzoin, myrrha, and olibanum in pure state and also charged with 10% of metal oxide nanoparticles, TiO2, P25, Cu2O, and MoO3, were characterized by ultraviolet-visible (UV-Vis) and Fourier transform infrared (FTIR) spectroscopy, energy-dispersive X-ray microanalysis (EDXMA), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). Disks were 3D-printed into model geometries (10 × 5 mm) and the disk-diffusion methodology was used for the evaluation of antimicrobial and antifungal activity of materials in study against the clinical isolates: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. Due to their intrinsic properties, disks containing resins in pure state mostly prevent surface-associated growth; meanwhile, disks loaded with 10% oxides prevent planktonic growth of microorganisms in the susceptibility assay. The microscopy analysis showed that part of nanoparticles was encapsulated by the biopolymeric matrix of resins, in most cases remaining disorderly dispersed over the surface of resins. Thermal analysis shows that plant resins have peculiar characteristics, with a thermal behavior similar to commercial available semicrystalline polymers, although their structure consists of a mix of organic compounds.

In vitro susceptibility of Burkholderia cepacia complex isolates: Comparison of disk diffusion, Etest®, agar dilution, and broth microdilution methods

Broth microdilution, agar dilution, Etest® and disk diffusion techniques were compared to evaluate the susceptibility profile of 82 Bcc clinical isolates against six antimicrobials as recommended by CLSI. Broth microdilution was considered the “gold standard” method. The regression analysis was applied to determine the essential (EA) and categorical (CA) agreement rates. STX (MIC50, 1 mg/L) was the most potent antimicrobial tested against Bcc isolates. The worst in vitro activity was observed for chloramphenicol (MIC50, 16 mg/L) and ticarcillin-clavulanic acid (MIC50, >256 mg/L). The EA among broth microdilution and agar dilution results was good for the majority of antimicrobial tested. When comparing broth microdilution and Etest®, ceftazidime, SXT and chloramphenicol exhibited EA rates below 90%. SXT showed an excellent CA (100%) when dilution methodologies were compared. However, a low CA rate was found for this agent between dilution and disk diffusion methodologies resulting in unacceptable very major and minor error rates.

Antimicrobial susceptibility testing of Arcobacter butzleri and Arcobacter cryaerophilus strains isolated from Belgian patients.

To evaluate the feasibility of different methods for susceptibility testing of human Arcobacter isolates, to assess susceptibility to antibiotics commonly used to treat diarrhoeal illness and to obtain MIC distribution data. One-hundred-and-six unique Arcobacter strains were collected during an epidemiological study on pathogens in gastroenteritis. Strains were identified by multiplex PCR and PCR-RFLP, and characterized by PFGE. Susceptibility to ampicillin, erythromycin, tetracycline, doxycycline, gentamicin and ciprofloxacin was determined using gradient strip and disc diffusion methodology. Optimal conditions for growth and incubation were tested. Azithromycin was tested with gradient strip diffusion on a subset of 40 strains. Sequence analysis of the quinolone resistance-determining region of gyrA was performed for a subset of 18 strains. Based on gradient diffusion results, most Arcobacter strains were susceptible to gentamicin (99%) and tetracycline (89%). Erythromycin (78%), ciprofloxacin (72%) and doxycycline (76%) retained moderate activity against Arcobacter spp. Only 9% of the strains were susceptible to ampicillin. Most Arcobacter butzleri strains were susceptible to ciprofloxacin (87%), whereas half of the Arcobacter cryaerophilus isolates (51%) showed high-level resistance (MIC >32 mg/L). MIC50 values were comparable for both macrolide antibiotics. Ciprofloxacin-resistant strains possessed an identical mutation in gyrA. Overall, categorical agreement between gradient and disc diffusion results was 60%. Gradient diffusion showed superior readability. Gradient diffusion methodology is preferred for routine susceptibility testing. Acquired resistance to fluoroquinolones was observed in A. cryaerophilus. Macrolides are not first-choice empirical antibiotics for Arcobacter infections. Tetracyclines can be suggested for treatment of documented Arcobacter-related gastrointestinal infections.

Prospective evaluation of an algorithm for the phenotypic screening of carbapenemase-producing Enterobacteriaceae.

The objective of this study was to assess the performance of an algorithm based on the disc diffusion method for the screening of carbapenemase-producing Enterobacteriaceae (CPE) referred to the French National Reference Centre for Antibiotic Resistance. From April to June 2014, all isolates of Enterobacteriaceae referred to the French National Reference Centre for Antibiotic Resistance were included. The inhibition zone diameters of imipenem, ticarcillin/clavulanate and temocillin using EUCAST disc diffusion methodology were recorded. All isolates were subjected to the algorithm proposed by the Antibiogram Committee of the French Society of Microbiology (CA-SFM) for the screening of carbapenemase producers. Phenotypic, biochemical and molecular detection of carbapenemases was performed for all isolates. A total of 621 consecutive enterobacterial isolates with decreased susceptibility to carbapenems were tested. They included 213 CPE [OXA-48-like (n = 183), NDM (n = 22), VIM (n = 3), KPC (n = 3) and OXA-48-like + NDM (n = 2)] and 408 non-carbapenemase producers. The CA-SFM algorithm combining cut-off values of 15 mm, 15 mm and 22 mm for ticarcillin/clavulanate, temocillin and imipenem, respectively, perfectly detected 204 isolates (32.8%) as non-carbapenemase producers, leading to a negative predictive value of 100% for this algorithm. Implementation of the CA-SFM algorithm in clinical microbiology laboratories may avoid additional testing for CPE in one-third of the enterobacterial isolates with decreased susceptibility to carbapenems.

ANTIBIOTIC RESISTANCE IN ENTEROBACTERIACEAE STRAINS ISOLATED FROM CHICKEN AND MILK SAMPLES

Antibiotic resistance and identification of strains in Enterobacteriaceae genera isolated from milk, milk products and rectal swabs of chicken was examined in this experiment. After samples collection cultivation and identification of bacterial strain was done. MALDI TOF MS Biotyper for identification of Enterobacteriaceae strains was used. For susceptibility testing disc diffusion methodology was used according by EUCAST. Results showed high level of ampicillin resistance in isolates from milk and milk samples. The highest streptomycin resistance was detected in isolates from rectal swabs of chicken. After identification, we determined that S. enterica ser. Typhimurium, which was isolated from rectal swabs of chicken showed the most multi-resistance from all identificated strains of Enterobacteriaceae. The most isolates bacterial strain was E. coli, which showed resistance against four antibiotics from rectal swabs of chicken. Also our results showed that the higher resistance level is in rectal swabs of chicken like in milk samples.

Preparation and characterization of Trimethoprim inclusion complex with Methyl-β-Cyclodextrin and determination of its antimicrobial activity

The present study focuses on the formation and characterization of inclusion complexes between Trimethoprim (TMP), an inhibitor of bacterial dihydrofolate reductase, and cyclodextrins, namely, methyl-βeta-cyclodextrin (MBCD) and hydroxylpropyl-βeta-cyclodextrin (HBCD) in aqueous solution. MBCD was selected to prepare inclusion complexes in the solid state. These complexes were prepared by different methods: spray drying, kneading and freeze drying. Physical mixtures were prepared as reference. The prepared systems were then characterized by different techniques: Differential scanning calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM). The dissolution profile and the antimicrobial activity of Trimethoprim and the inclusion complexes were evaluated using the dissolution test, and the disk diffusion methodology, respectively. An increase of TMP solubility was observed in phase solubility studies. The obtained apparent stability constants (Ks) showed that MBCD formed an inclusion complex more stable with the drug than HPBCD, so it was decided to prepare inclusion complexes in solid state with MBCD. The results obtained with DSC, FTIR and SEM proved the formation of inclusion complexes in solid state. The dissolution profile and the antibacterial activity increased with the complexation process.

Virulence Gene Profiles of Multidrug-Resistant Pseudomonas aeruginosa Isolated From Iranian Hospital Infections.

Background:The most common hospital-acquired pathogen is Pseudomonas aeruginosa. It is a multidrug resistant bacterium causing systemic infections.Objectives:The present study was carried out in order to investigate the distribution of virulence factors and antibiotic resistance properties of Pseudomonas aeruginosa isolated from various types of hospital infections in Iran.Patients and Methods:Two-hundred and seventeen human infection specimens were collected from Baqiyatallah and Payambaran hospitals in Tehran, Iran. The clinical samples were cultured immediately and samples positive for P. aeruginosa were analyzed for the presence of antibiotic resistance and bacterial virulence genes using PCR (polymerase chain reaction). Antimicrobial susceptibility testing was performed using disk diffusion methodology with Müeller–Hinton agar.Results:Fifty-eight out of 127 (45.66%) male infection specimens and 44 out of 90 (48.88%) female infection specimens harbored P. aeruginosa. Also, 65% (in male specimens) and 21% (in female specimens) of respiratory system infections were positive for P. aeruginosa, which was a high rate. The genes encoding exoenzyme S (67.64%) and phospholipases C (45.09%) were the most common virulence genes found among the strains. The incidences of various β-lactams encoding genes, including blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, and blaVEB were 94.11%, 16.66%, 15.68%, 18.62%, 21.56%, and 17.64%, respectively. The most commonly detected fluoroquinolones encoding gene was gyrA (15. 68%). High resistance levels to penicillin (100%), tetracycline (90.19%), streptomycin (64.70%), and erythromycin (43.13%) were observed too.Conclusions:Our findings should raise awareness about antibiotic resistance in hospitalized patients in Iran. Clinicians should exercise caution in prescribing antibiotics, especially in cases of human infections.

Virulence genotype and phylogenetic groups in relation to Chinese herb resistance among Escherichia coli from patients with acute pyelonephritis.

Clinical isolates of herb-resistant uropathogenic E. coli were isolated. It was possible that the virulence genotypes and phylogenetic background of E. coli differed between Chinese herb-resistant E. coli and -susceptible isolates. For this purpose, the prevalence of virulence factors (VFs) and phylogenetic background, with regard to Chinese herb resistance, among E. coli strains causing acute pyelonephritis from China were investigated. E. coli isolates from patients with acute pyelonephritis were used in this study. Standard disc diffusion methodology was used to test the susceptibility of Chinese herbal concoction against E. coli strains. Multiplex PCR amplifications employed three markers (chuA, yjaA, and TSPE4.C2) to classify E. coli isolates into one of four phylogenetic groups (group A, B1, B2, or D). The isolates were also tested for 14 virulence-associated traits (VFs) of uropathogenic E. coli. A total of 115 E. coli strains were isolated. 79 (68.7%) were susceptible and 36 (31.3%) were resistant to the herbal concoction. 20.9% of the isolates encoded three or more of VFs for which they were screened, with 13.9% in susceptible isolates and 36.1% in resistant isolates. The key VFs (fyuA and/or iutA siderophores) present in >80% of isolates. The papA and papC adhesins were detected in the majority of resistant isolates (72.2% and 63.9% respectively). 78.5% of susceptible isolates belong to phylogenetic groups A, while 83.3% of resistant isolates belong to group B2. PapA and papC are significant VFs with an essential role in contributing to Chinese herb-resistance. Chinese herb-resistance is associated with a shift towards more virulent strains and B2 phylogenetic group.

Electrospun DOXY-h loaded-poly(acrylic acid) nanofiber mats: in vitro drug release and antibacterial properties investigation

Electrospun DOXY-h loaded-poly(acrylic acid) (PAA) nanofiber mats (PAA/DOXY-h nanofiber mats) were prepared by the electrospinning technique and post-spinning sorption method at various doses: PAA/DOXY-h125, PAA/DOXY-h250, PAA/DOXY-h500, and PAA/DOXY-h1000. The morphology, drug content, release characteristics, and antibacterial activities of the PAA/DOXY-h nanofiber mats were investigated with scanning electron microscopy, UV–vis spectrophotometry, and disc diffusion methodology. The PAA/DOXY-h nanofiber mats had a diameter range of 285–340 nm, and a smooth surface without beads. Adsorption isotherms of DOXY-h could be described well with the Freundlich model. The amounts of DOXY-h, after the post-spinning sorption process, in the PAA/DOXY-h nanofiber mats ranged between 27.57 and 101.71 mg/g. All of the PAA/DOXY-h nanofiber mats exhibited an initial burst release characteristic with cumulative releasing percentages between 37.14 and 45.97%, which followed the Fickian diffusion mechanism. Based on the antibacterial investigation, the tested gram-positive bacteria, Staphylococcus aureus and Streptococcus agalactiae, seemed to be more sensitive to PAA/DOXY-h nanofiber mats than the tested gram-negative bacteria, Pseudomonas aeruginosa. These PAA/DOXY-h nanofiber mats could be used as an antibacterial wound dressing.

Virulence factors, serogroups and antimicrobial resistance properties of Escherichia coli strains in fermented dairy products

BackgroundFrom a clinical perspective, it is essential to know the microbial safety of fermented dairy products. Doogh and kashk are fermented dairies. These products are used by millions of people but their microbial qualities are unknown. Shiga toxin producing Escherichia coli (STEC) is one of the most commonly detected pathogens in the cases of food poisoning and food-borne illnesses. The present investigation was carried out in order to study the molecular characterization and antimicrobial resistance properties of STEC strains isolated from fermented dairy products.MethodsSix hundred fermented dairy samples were collected and immediately transferred to the laboratory. All samples were cultured immediately and those that were E. coli-positive were analyzed for the presence of O157 , O26, O103, O111, O145, O45, O91, O113, O121 and O128 STEC serogroups, tetA, tetB, blaSHV, CITM, cmlA, cat1, aadA1, dfrA1, qnr, aac (3)-IV, sul1 and ereA antibiotic resistance genes and stx1, stx2, eaeA, ehly, cnf1, cnf2, iutA, cdtB, papA, traT, sfaS and fyuA virulence factors using PCR. Antimicrobial susceptibility testing was performed also using disk diffusion methodology with Mueller–Hinton agar.ResultsFifty out of 600 (8.33%) dairy samples harbored E. coli. In addition, yoghurt was the most commonly contaminated dairy. O157 (26%) and O26 (12%) were the most commonly detected serogroups. A significant difference was found between the frequency of Attaching and Effacing E. coli and Enterohaemorrhagic E. coli (P <0.05). Stx1 (44%), eae (36%), papA (32%) stx2 (30%), and ehly (28%) were the most commonly detected virulence factors. The genes encode resistance against tetracycline (tetA and tetB) (76% and 70%, respectively), cephalothin (blaSHV) (38%), ampicillin (CITM) (36%) and gentamicin (aac (3)-IV) (32%) were the most commonly detected. High resistance levels to tetracycline (84%), penicillin (46%), ampicillin (38%) and streptomycin (36%) were observed.ConclusionFermented dairy products can easily become contaminated by antibiotic resistant STEC strains. Our findings should raise awareness about antibiotic resistance in Iran. Clinicians should exercise caution when prescribing antibiotics, especially in veterinary treatments.

Performance of the EUCAST Disk Diffusion Method, the CLSI Agar Screen Method, and the Vitek 2 Automated Antimicrobial Susceptibility Testing System for Detection of Clinical Isolates of Enterococci with Low- and Medium-Level VanB-Type Vancomycin Resistance: a Multicenter Study

Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n=28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n=12) and Enterococcus faecium (n=18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n=5), Norwegian (n=13), and Swedish (n=10) laboratories using the EUCAST disk diffusion method (n=28) and the CLSI agar screen (n=18) or the Vitek 2 system (bioMérieux) (n=5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P=0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P<0.0001) or Merck Mueller-Hinton (MH) agar (P=0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P=0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges.

Prevalence and characterization of human mecC methicillin-resistant Staphylococcus aureus isolates in England

ObjectivesThere are limited data available on the epidemiology and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the human population that encode the recently described mecA homologue, mecC. To address this knowledge gap we undertook a prospective prevalence study in England to determine the prevalence of mecC among MRSA isolates.Patients and methodsThree hundred and thirty-five sequential MRSA isolates from individual patients were collected from each of six clinical microbiology laboratories in England during 2011–12. These were tested by PCR or genome sequencing to differentiate those encoding mecA and mecC. mecC-positive isolates were further characterized by multilocus sequence typing, spa typing, antimicrobial susceptibility profile and detection of PBP2a using commercially available kits.ResultsNine out of the 2010 MRSA isolates tested were mecC positive, indicating a prevalence among MRSA in England of 0.45% (95% CI 0.24%–0.85%). The remainder were mecA positive. Eight out of these nine mecC MRSA isolates belonged to clonal complex 130, the other being sequence type 425. Resistance to non-β-lactam antibiotics was rare among these mecC MRSA isolates and all were phenotypically identified as MRSA using oxacillin and cefoxitin according to BSAC disc diffusion methodology. However, all nine mecC isolates gave a negative result using three different commercial PBP2a detection assays.ConclusionsmecC MRSA are currently rare among MRSA isolated from humans in England and this study provides an important baseline prevalence rate to monitor future changes, which may be important given the increasing prevalence of mecC MRSA reported in Denmark.

Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers

To assess the performance of the agar disc diffusion method for the detection of carbapenemase-producing Enterobacteriaceae (CPE) referred to the national reference laboratories (NRLs) in Belgium and France. All Enterobacteriaceae isolates referred to the NRLs for the confirmation of CPE in 2012 were included. The inhibition zone diameters of meropenem, piperacillin/tazobactam and temocillin using CLSI disc diffusion methodology were recorded. Phenotypic and molecular detection of carbapenemases was performed on all isolates. A total of 1354 Enterobacteriaceae isolates, including 435 (32.1%) confirmed CPE isolates [OXA-48 (n = 323), KPC (n = 60), VIM (n = 32) and NDM (n = 20)] and 919 carbapenemase-negative isolates, were tested. Using recommended interpretative criteria, non-susceptibility to meropenem had poor sensitivity (52.0% by CLSI susceptibility breakpoint and 80.0% by EUCAST screening breakpoints), while non-susceptibility to piperacillin/tazobactam (according to CLSI breakpoint) or to temocillin (according to Fuchs, Barry, Thornsberry et al. Eur J Clin Microbiol 1985; 4: 30-3) was highly sensitive (99.8% and 98.2%, respectively) but poorly specific (29.4% and 42.9%, respectively) for the detection of CPE. Temocillin diameters <12 mm alone had high specificity (90.0%) and the combination of temocillin diameters ≥12 mm with piperacillin/tazobactam diameters ≥16 mm observed in 40% of all referred isolates displayed excellent negative predictive value (99.2%). In geographical areas with a high prevalence of OXA-48 producers, recommended meropenem susceptibility or screening breakpoints failed to detect CPE in a large proportion of isolates. The combination of modified zone diameter cut-offs for piperacillin/tazobactam (≥16 mm) and temocillin (≥12 mm) can be used to rule out the presence of carbapenemase and avoid unnecessary additional testing for confirmation of CPE.

Chinese Herb-Resistant Clinical Isolates ofEscherichia coli

Many Chinese herbal formulations are used in the treatment of urinary tract infection. Clinical isolates of Escherichia coli (E. coli) resistant to Chinese herbs have been identified rarely. Report here is a case in which Chinese herbs-resistant E. coli isolate was identified. The strain of E. coli C16 was isolated from urine of a 72-year-old woman with a diagnosis of pyelonephritis. Standard disc diffusion methodology was used to test the Chinese herbal decoction against E. coli C16. The minimum inhibitory concentration value was 0.1 g/mL. The minimum bactericidal concentration value was 0.2 g/mL. The decoction was orally administered for 4 weeks. The therapeutic regimen was well tolerated initially. At the end of 4 weeks, the disease relapsed. E. coli C16 was isolated again, which was resistant to herbal solution. This case illustrates that excessive exposure to Chinese herbs used in unchanging standard formulations can also lead to bacterial resistance. Appropriate clinical use of Chinese herbs is imperative.

Evaluation of bacterial growth inhibition by mercaptopropionic acid in metallo-β-lactamase detection on multidrug-resistant Acinetobacter baumannii

Metallo-β-lactamase (MBL) has been reported all over the world. The inhibitory effect of mercaptopropionic acid (MPA) on bacterial growth was evaluated by comparison between disk diffusion and broth dilution methodology with determination of the minimum inhibitory concentration (MIC) for multidrug-resistant Acinetobacter baumanni strains. MPA significantly inhibited growth of the strains. The use of MPA can affect the results in phenotypic methods of MBL detection.

Increasing Trend of Resistance to Penicillin, Tetracycline, and Fluoroquinolone Resistance inNeisseria gonorrhoeaefrom Pakistan (1992–2009)

Emergence and spread of drug resistant Neisseria gonorrhoeae is global concern. We evaluated trends of antimicrobial resistance in Neisseria gonorrhoeae over years 1992–2009 in Pakistan. Resistance rates were compared between years (2007–2009) and (1992–2006). Antimicrobial susceptibility testing was performed and interpreted according to Clinical Laboratory Standards Institute (CLSI) criteria using the disk diffusion methodology against penicillin, ceftriaxone, tetracycline and ofloxacin. Additional antibiotics tested in 100 strains isolated during 2007–2009, included cefotaxime, cefoxitin, cefuroxime, cefipime, ceftazidime, ceftizoxime, cefixime, cefpodoxime, spectinomycin and azithromycin. Neisseria gonorrhoeae ATCC 49226 was used as control. Chi-square for trend analysis was conducted to assess resistance trend over the study period. During study period significant increase in combined resistance to penicillin, tetracycline and ofloxacin was observed (P value <0.01). Resistance rates during the two study period also increased significantly (P value <0.01). Ceftriaxone resistance was not observed. None of the isolates were found to be resistant or with intermediate sensitivity to additional antibiotics. Our findings suggest that penicillin, ciprofloxacin, tetracycline should not be used in the empirical treatment of gonorrhea in Pakistan. Ceftriaxone and cefixime should be the first line therapy; however periodic MICs should be determined to identify emergence of strains with reduced susceptibility.