Agar Dilution Results Research Articles

Prevalence of antibiotic resistance in Clostridium difficile: A meta-analysis

In recent years, the resistance of Clostridium difficile to various common mainstream antibiotics cannot be ignored, the incidence of CDI in many areas is also increasing year by year, posing a great threat to public safety. Literature collection was carried out on 6 open databases, and high-quality literature data conforming to classification criteria were screened and extracted. The WPR and WPF of each antibacterial drug were calculated using the random effects model. Subgroup analysis was used to investigate the influence of regional and experimental methods. Results: A total of 40 studies were included. The WPR of vancomycin was 0.0372, 95%CI: [0.0148; 0.0671], I: 93.7%, WPF was 0.2195, I: 94.0%. The WPR of fidaxomicin was 0.0051, 95%CI: [0.0000; 0.0205], I: 82.7%, WPF was 0.1757, I: 93.6%. The WPR of metronidazole was 0.0318, 95%CI: [0.0056; 0.0727], I: 91.6%. Among the three antibiotics, the highest resistance of C. difficile to metronidazole were mainly distributed in the Middle East and Africa. The conclusion seems to indicate that C. difficile is most sensitive to Fidaxomicin, and the clinical cure rate is better than vancomycin. However, the clinical cure failure rate increased significantly from 2007 to 2019, while the drug resistance rate did not change significantly, suggesting that other factors besides drug resistance may affect the therapeutic effect of fidaxomicin on CDI. There was no significant difference in the results of AGAR dilution and e test, the main methods for drug susceptibility testing.

An ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance

Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR, an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI’s AMRFinderPlus, as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.

Evaluation of Antibacterial Effect of Propolis and its Application in Mouthwash Production.

Objectives:Our purpose was to determine the antibacterial properties of propolis and to evaluate its use as an antibacterial mouthwash with minimal complications.Materials and Methods:In this experimental laboratory study, an alcoholic propolis extract was prepared. The minimum inhibitory concentration (MIC) was calculated for four bacterial species including Staphylococcus aureus (S. aureus), Streptococcus mutans (S. mutans), Lactobacillus acidophilus (L. acidophilus), and Enterococcus faecalis (E. faecalis) using agar dilution. According to the MIC, a propolis antibacterial mouthwash was produced and compared to water, chlorhexidine (CHX), and Listerine using laboratory rats for clinical examination. Salivary specimens of rats were collected at 12 hours, 1 week, and 2 weeks after using the mouthwash and examined by real-time polymerase chain reaction (RT-PCR). Data were analyzed using one-way analysis of variance (ANOVA) and repeated measures ANOVA (α=0.05).Results:The results of agar dilution by the number of colony-forming units showed the lowest MIC for S. aureus and the highest for L. acidophilus. Our RT-PCR findings indicated that water alone had no effect on the level of oral bacteria. Propolis mouthwash showed a significant difference with CHX and Listerine (P<0.05) in terms of the number of S. mutans, E. faecalis, and L. acidophilus colonies, while CHX and Listerine were less efficient. There was no significant difference between CHX and propolis (P=0.110) regarding S. aureus colonies, but Listerine had a lower efficacy than either (P<0.05).Conclusion:According to the results, propolis mouthwash was more efficient against the studied oral bacteria compared to CHX and Listerine.

Assessment of Etest as an Alternative to Agar Dilution for Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae

We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturer's recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest.

Frequency distribution of genes encoding aminoglycoside modifying enzymes in uropathogenic E. coli isolated from Iranian hospital.

BackgroundEscherichia coli is considered as the most common cause of urinary tract infection (UTI) and acquired multiple resistances to a wide range of antibiotics such as aminoglycosides. Enzymatic alteration of aminoglycosides (AMEs) by aminoglycoside- modifying enzymes is the main mechanism of resistance to these antibiotics in E. coli. The aim of this study was detection and investigation of frequency of genes encoding aminoglycoside modifying enzymes (aac(3)-IIa and ant(2′′)-Ia) in UPEC isolated from hospitalized patients in teaching hospital of Tehran, Iran.FindingsA total of 276 UPEC were obtained from Urine samples in a hospital from Tehran. Antibiotic susceptibility to aminoglycosides was determined by disk diffusion method according CLSI guidelines in UPEC isolates. MICs of target antibiotics were determined by agar dilution method. All isolates were screened for the presence of the AMEs genes using the PCR. The results of disk diffusion showed 21%, 24.6%, 23.18%, 3.62% and 6.15% of isolates were resistant to Gentamicin, Tobramycin, Kanamicin, Amikacin and Netilmicin respectively. The agar dilution’s results (MICs) were high, 66.19% for Gentamicin. The aac (3)-IIa and ant(2″)-Ia genes were detected in (78.87%) and 47.88% of isolates respectively.ConclusionsThis study shows the high frequency of genes encoding (AMEs) aac(3)-IIa and ant(2”)-Ia genes and their relationship between different aminoglycoside resistance phenotypes.

In VitroActivity of Fosfomycin against a Collection of Clinical Pseudomonas aeruginosa Isolates from 16 Spanish Hospitals: Establishing the Validity of Standard Broth Microdilution as Susceptibility Testing Method

The broth microdilution method for fosfomycin and Pseudomonas aeruginosa was assessed and compared with the approved agar dilution method in 206 genetically unrelated P. aeruginosa clinical isolates. Essential agreement between the two methods was 84%, and categorical agreement was 89.3%. Additionally, Etest and disk diffusion assays were performed. Results validate broth microdilution as a reliable susceptibility testing method for fosfomycin against P. aeruginosa. Conversely, unacceptable concordance was established between Etest and disk diffusion results with agar dilution results.

Antimicrobial Susceptibility Testing of Helicobacter pylori Determined by Microdilution Method Using a New Medium

Antibiotic susceptibility testing of Helicobacter pylori isolates was performed by broth microdilution method with MegaCell RPMI-1640 Medium (SIGMA). Fifty five clinical isolates of H. pylori were tested against metronidazole, tinidazole, amoxicillin, and clarithromycin. The results were compared to those obtained by standard agar dilution method. The microdilution method performed with new medium, showed excellent correlation with agar dilution results, with 100% agreement for metronidazole, 96.3% for amoxicillin, 90.7% for clarithromycin, and 92.8% for tinidazole. MICs determined by proposed method were highly reproducible: replicate results were variable within one-two-fold dilution by using different inocula and different batches of medium.

Determination of antimicrobial susceptibilities of clinically isolated anaerobic bacteria by E-test, ATB-ANA and agar dilution

A total of 60 anaerobic strains were isolated from 322 clinical specimens. These isolates were tested for susceptibility to seven antibiotics (penicillin G, amoxicillin/clavulanic acid, cefoxitin, imipenem, chloramphenicol, metronidazole, clindamycin) by using ATB-ANA and Epsilometer test (E-test) strips and the results were compared with the gold standard agar dilution method. Imipenem was found as the most effective agent in vitro among the agents tested (100%). Susceptibility to penicillin G, amoxicillin/clavulanic acid, cefoxitin, chloramphenicol, metronidazole and clindamycin are 36.7%, 83.3%, 88.3%, 96.6%, 85% and 90%, respectively. E-test has showed a good correlation ( r=0.62, p=0.001) statistically with the results of agar dilution (total agreement for all antibiotics changing between 90.01% and 98.45%) and a moderate correlation ( r=0.45, p=0.048) with the results of ATB-ANA method (total agreement for all antibiotics changing between 75.46% and 98.76%). However, the routine use of agar dilution procedure is concluded to be cumbersome, whereas E-test method offers a reliable alternative.

Activity of Retapamulin against Streptococcus pyogenes and Staphylococcus aureus Evaluated by Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methodologies

The in vitro activity of retapamulin against 106 Staphylococcus aureus isolates and 109 Streptococcus pyogenes isolates was evaluated by the agar dilution, broth microdilution, E-test, and disk diffusion methodologies. Where possible, the tests were performed by using the CLSI methodology. The results of agar dilution, broth microdilution, and E-test (all with incubation in ambient air) for S. aureus yielded similar MICs, in the range of 0.03 to 0.25 microg/ml. These values corresponded to zone diameters between 25 and 33 mm by the use of a 2-microg retapamulin disk. Overall, 99% of the agar dilution results and 95% of E-test results for S. aureus were within +/-1 dilution of the microdilution results. For S. pyogenes, the MICs obtained by the agar and broth microdilution methods (both after incubation in ambient air) were in the range of 0.008 to 0.03 microg/ml, and E-test MICs (with incubation in ambient air) were 0.016 to 0.06 microg/ml. For S. pyogenes, 100% of the agar dilution MIC results were within +/-1 dilution of the broth microdilution results. E-test MICs (after incubation in ambient air) were within +/-1 and +/-2 dilutions of the broth microdilution results for 76% and 99% of the isolates, respectively. E-test MICs for S. pyogenes strains in CO(2) were up to 4 dilutions higher than those in ambient air. Therefore, it is recommended that when retapamulin MICs are determined by E-test, incubation be done in ambient air and not in CO(2), due to the adverse effect of CO(2) on the activity of this compound. Diffusion zones (with incubation in CO(2)) for S. pyogenes were 18 to 24 mm. Retapamulin MICs for all strains by all methods (with incubation in ambient air) were < or =0.25 microg/ml. These results demonstrate that S. pyogenes (including macrolide-resistant strains) and S. aureus (including methicillin-resistant and vancomycin-nonsusceptible strains) are inhibited by very low concentrations of retapamulin and that all four testing methods are satisfactory for use for susceptibility testing.

Assessment of metronidazole susceptibility in Helicobacter pylori: statistical validation and error rate analysis of breakpoints determined by the disk diffusion test.

Metronidazole susceptibility of 100 Helicobacter pylori strains was assessed by determining the inhibition zone diameters by disk diffusion test and the MICs by agar dilution and PDM Epsilometer test (E test). Linear regression analysis was performed, allowing the definition of significant linear relations, and revealed correlations of disk diffusion results with both E-test and agar dilution results (r2 = 0.88 and 0.81, respectively). No significant differences (P = 0.84) were found between MICs defined by E test and those defined by agar dilution, taken as a standard. Reproducibility comparison between E-test and disk diffusion tests showed that they are equivalent and with good precision. Two interpretative susceptibility schemes (with or without an intermediate class) were compared by an interpretative error rate analysis method. The susceptibility classification scheme that included the intermediate category was retained, and breakpoints were assessed for diffusion assay with 5-microg metronidazole disks. Strains with inhibition zone diameters less than 16 mm were defined as resistant (MIC > 8 microg/ml), those with zone diameters equal to or greater than 16 mm but less than 21 mm were considered intermediate (4 microg/ml < MIC </= 8 microg/ml), and those with zone diameters of 21 mm or greater were regarded as susceptible (MIC </= 4 microg/ml). Error rate analysis applied to this classification scheme showed occurrence frequencies of 1% for major errors and 7% for minor errors, when the results were compared to those obtained by agar dilution. No very major errors were detected, suggesting that disk diffusion might be a good alternative for determining the metronidazole sensitivity of H. pylori strains.

Use of the polymerase chain reaction for rapid detection of high-level mupirocin resistance in staphylococci.

The minimum inhibitory concentrations (MICs) of mupirocin were determined by the E test (AB Biodisk, Sweden) and the agar dilution method for 107 staphylococci. The organisms consisted of 34 coagulase-negative staphylococci and 73 methicillin-resistant Staphylococcus aureus. Polymerase chain reaction (PCR) primers designed to amplify a 456 bp region of the plasmid-borne isoleucyl tRNA synthetase gene (ileS-2), responsible for high-level mupirocin resistance in staphylococci, were used on DNA preparations from these isolates. Isolates with high-level mupirocin resistance due to the ileS-2 gene should be PCR positive. There was close correlation between the E test and agar dilution MIC values, with only two strains differing by more than two serial dilutions. Most (51 of 54 strains) of the high-level resistant strains (MIC>256 microg/ml) were resistant to the highest concentration of mupirocin tested (1024 microg/ml). PCR correctly classified all but four (96%) of the isolates in accordance with the results of agar dilution. All four isolates that gave discrepant results were methicillin-resistant Staphylococcus aureus. Two of these were PCR positive, yet the MIC of mupirocin for these strains was <0.06 microg/ml; on prolonged incubation they produced halos within the inhibition zone on agar diffusion testing, suggesting that the phenotypic results may have been erroneous. One of 54 isolates for which the MIC exceeded 256 microg/ml was PCR negative when tested by the original methodology, but a 456 bp product was produced when retested using a lowered annealing temperature. One isolate for which the MIC of mupirocin was 16 microg/ml by agar dilution and 8 microg/ml by the E test was positive by PCR. PCR of the ileS-2 gene is a useful, rapid method for detecting high-level mupirocin resistance in staphylococci.

In-vitro activity of fosfomycin against vancomycin-resistant enterococci.

The effect of fosfomycin against 69 vancomycin-resistant isolates of Enterococcus faecium (VanA), five of E. faecium (VanB), 11 of Enterococcus faecalis (VanA), three of E. faecalis (VanB), 10 of Enterococcus gallinarum (VanC1) and two of Enterococcus casseliflavus (VanC2) and glycopeptide-sensitive E. faecium (n = 8) and E. faecalis (n = 10) was tested in vitro. Fosfomycin inhibited 97%, 94% and 96% of the vancomycin-resistant strains, according to results of agar dilution, broth microdilution, and a disc diffusion method (DIN 58940). The disc diffusion test by the NCCLS method does not include fosfomycin; using breakpoints suggested by Andrews et al. (< or = 11 mm, resistant; > or = 18 mm, susceptible), 5% of the vancomycin-resistant strains tested would have been considered fosfomycin resistant. Minimal inhibitory concentrations of most vancomycin-resistant isolates were in the intermediate sensitivity range, yielding an MIC50 of 32 mg/L and an MIC90 of 64 mg/L. Moreover the majority of inhibitory zone sizes by the disc diffusion method (DIN 58940) corresponded to intermediate susceptibility. These results suggest that fosfomycin at a high dosage and possibly used in combination with other drugs could be a potentially useful drug for the treatment of infections caused by vancomycin-resistant enterococci.

Critical appraisal of E test for the detection of fluoroquinolone resistance.

The ability of E test to accurately detect fluoroquinolone resistance was compared with an agar dilution reference method. Nearly 300 isolates belonging to 26 different species (62.5% with documented ciprofloxacin resistance) were tested with ciprofloxacin, fleroxacin, levofloxacin, norfloxacin, ofloxacin, and sparfloxacin. In contrast to earlier reports, E test MIC values for pneumococci and all rapid growing aerobes were routinely higher (approximately 0.5 log2 dilution step) than agar dilution results. The E test stable-gradient method also efficiently identified fluoroquinolone-resistant subpopulations which were not detected by the reference procedure with the standard inoculum. Categorical agreement for 1710 test comparisons was approximately 90% with no very major, false-susceptible errors. We found the E test to be a valid, reproducible method for fluoroquinolone susceptibility testing that provided quantitative results and produced a conservative (1.6% false-resistant results) assessment of susceptibility of bacteria including isolates of Streptococcus pneumoniae and Pseudomonas aeruginosa to compounds in this antimicrobial class.

Validation of a modified Kirby-Bauer disk diffusion method for metronidazole susceptibility testing of Helicobacter pylori

Triple antimicrobial therapy that includes metronidazole has been recommended as a first-line therapy for Helicobacter pylori because it has the highest eradication rates. However, resistance in H. pylori to metronidazole has been reported worldwide and its presence may reduce the efficacy of triple therapy. Various methods for testing H. pylori against metronidazole have been used including agar dilution, disk diffusion and the Etest but there has been little standardization of methods. One hundred isolates of H. pylori from different patients were tested for susceptibility to metronidazole by agar dilution, Etest and disk diffusion (5 μg disk). The agar dilution results confirmed the MIC susceptibility breakpoint to be ⩽8 μg/ml. Using this breakpoint there was close agreement (98%) between Etest and agar dilution results. For susceptible strains, MICs by E-test were generally one twofold dilution lower. Using the error-rate bounded method, agreement between disk diffusion zome diameter and MIC was 98% for agar dilution with breakpoints of ⩾12 mm and ⩽8 μg/ml and 100% for Etest with breakpointsof ⩾12 mm and ⩽8 μg/ml. The Etest discriminated better than agar dilution between susceptible and resistant strains and was simple to perform. The disk diffusion test is a reliable and cheaper alternative to the Etest with susceptibility being a zone diameter ⩾12 mm with a 5 μg disk. The prevalence of metronidazole resistance in this study was 40% by Etest.

Detection of a genetic locus encoding resistance to rifampin in mycobacterial cultures and in clinical specimens

The polymerase chain reaction (PCR) and automated DNA sequencing were used to detect a genetic locus, rpoB, associated with rifampin resistance in Mycobacterium tuberculosis (TB) in clinical isolates and directly in clinical specimens. Primers derived from the sequence of a TB rpoB gene fragment were used to amplify DNA from bacterial and mycobacterial isolates. An rpoB-specific PCR product was obtained for five of five TB, seven of eight other mycobacterial species. Nocardia sp. , Corynebacterium sp. , Streptomyces sp. , Actinomyces sp. , and Rhodococcus sp. , but not for 15 isolates (eight genera) representing usual bacterial flora. Sequence comparison of the amplified rpoB region revealed the occurrence of TB-specific “signature nucleotides” at three positions. PCR yielded amplification products for seven of 16 clinical specimens. Five of the seven contained TB-specific DNA, as well as sequences that predicted rifampin susceptibility in accord with agar dilution results. None of ten specimens that were culture negative for TB yielded TB-specific PCR products. These results with a limited number of clinical specimens demonstrate the feasibility of direct detection by PCR of rifampin-resistant TB in clinical specimens. Such testing may serve as a rapid surrogate test for multidrug-resistant TB in laboratories with PCR and automated sequencing capability.

Influence of cation supplements on activity of netilmicin against Pseudomonas aeruginosa in vitro and in vivo.

In vitro studies were performed with 74 Pseudomonas aeruginosa isolates which were collected during a multicenter trial. The isolates were obtained from 70 patients who had been treated with netilmicin as the only antipseudomonal antibiotic. Clinically, 83% of the patients were cured or improved, and 64% of the Pseudomonas isolates were eliminated by chemotherapy. The 74 clinical isolates and 38 additional isolates with known mechanisms of aminoglycoside resistance were tested in three separate laboratories by disk diffusion methods and by microdilution tests with three broth media (Mueller-Hinton broth with full, half, and no cation supplements). Isolates that responded to netilmicin therapy and those that failed to respond were all susceptible by the disk test, and most were susceptible by microdilution tests with unsupplemented broth. However, over half of the clinical isolates appeared to be resistant when cations were added to the broth medium. Strains capable of producing enzymes that inactivate netilmicin were resistant by all methods tested. Broth dilution and agar dilution results were most comparable when half of the recommended cation supplements was added to Mueller-Hinton broth. Further consideration should be given to reducing the concentration of cations that are added to Mueller-Hinton broth when netilmicin susceptibility tests are being performed. However, additional studies with other aminoglycosides are needed before appropriate testing conditions can be standardized.