Etest Minimal Inhibitory Concentrations Research Articles

Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms

BackgroundColistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation.ResultsOf 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L.ConclusionsThe Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.

Evaluation of the in vitro interaction of fosfomycin and meropenem against metallo-β-lactamase–producing Pseudomonas aeruginosa using Etest and time-kill assay

Pseudomonas aeruginosa is a nosocomial pathogen containing various resistance mechanisms. Among them, metallo-β-lactamase (MBL)–producing Pseudomonas are difficult to treat. Fosfomycin is an older antibiotic that has recently seen increased usage...

High abundance of the colistin resistance gene mcr-1 in chicken gut-bacteria in Bangladesh

Colistin is considered a last-resort reserved drug for the treatment of critical human infections by Gram-negative bacteria. Phenotypic colistin-resistance is strongly associated with plasmid-mediated mobile colistin resistance (mcr) genes. The mcr-bearing Enterobacteriaceae have been detected in many countries from environments, animals, and humans. This study investigated phenotypic colistin-resistance and the distribution of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes in chicken-gut bacteria in Bangladesh. Bacteria were isolated from poultry- and native-chicken droppings, and their susceptibilities to colistin were determined by agar dilution and E-test minimal inhibitory concentration (MIC) measurements. Multiplex polymerase chain reactions detected mcr-1 to mcr-5 genes. Overall, 61.7% (92/149) of the isolates showed colistin resistance by agar dilution assessment (MIC > 2.0 μg/mL). The phenotypic resistance was observed considerably higher in poultry-chicken isolates (64.6%, 64/99) than in native-chicken isolates (56%, 28/50; p = 0.373). All the resistant isolates showed MIC levels between > 2 and > 128 μg/mL. The mcr-genes (mcr-1and mcr-2 combined) were detected more in poultry gut bacteria (36.4%) than native-chicken isolates (20%, p = 0.06). Despite bacteria sources, mcr-genes appeared to be significantly associated with phenotypic colistin-resistance phenomena (p < 0.001). Prior colistin usage led to a substantial increase in the proportion of bacteria with mcr-genes and phenotypic resistance (p < 0.001).

Methicillin-Resistant Staphylococcus Strains Isolated from Adult Intensive Care Units with E-test MIC Values of Different Antibiotic Research

Objective: Nasocomial infections are major health problems due to their high morbidity and mortality, prolonged hospital duration and higher treatment costs. Methicillin-resistant staphylococcus species became one of the leading bacteria causing nasocomial infections especially in intensive care units, recently. The minimum inhibitory concentration value of an antibiotic gives the concentration of antibiotic needed to inhibit the bacteria in the infection area. Careful monitoring of minimal inhibitory concentration (MIC) values is necessary especially during long-term treatments of meticillin-resistant Staphylococcus aureus (MRSA) and meticillin-resistant coagulase-negative staphylococci (MRCoNS) infections 1,2 . Increasing antibiotic resistance in methicillin-resistant staphylococci, has led to the need for different antibiotics. Methods: A total of 60 meticillin-resistant staphylococci strains isolated in Microbiology Laboratory of Dicle University Hospital, from clinical specimens of patients in adult Intensive Care Units (ICUs) between April 2013 and March 2014 were included in this study. After identification with conventional and automated system, the antibiotic susceptibility rates of vancomycin, teicoplanin, daptomycin, linezolid, quinupristin/dalfopristin, tigecycline, ceftaroline were determined by E-test method. Results: The majority of the samples (26.7%) were sent from Pulmonary Diseases and Tuberculosis intensive care unit and the blood samples were the most common materials (80%) . All staphylococcal strains in our study were determined as susceptible to vancomycin, daptomycin, linezolid, teicoplanin and tigecycline. One (1.6%) MRCoNS isolate was resistant to quinupristin/dalfopristin while 11 (36.6%) of the MRSA isolates were resistant to ceftaroline. In comparison with the MIC values of MRSA and MRCoNS, only tigecycline was significantly different. Thirty MRSA strains were evaluated in terms of vancomycin-intermediate Staphylococcus aureus /heteroresistant vancomycin-intermediate Staphylococcus aureus (VISA/hVISA) with macro E-test method; any VISA/hVISA isolate was not detected. Antibiotic concentrations below the MIC level, not only leads to treatment failure but also causes mutant bacteria to appear. In order to control the resistance to antibiotics in the treatment of infections due to MRSA and MRCoNS agents, the clinician should be notified of the MIC values of the drugs and the treatment should be planned accordingly. VISA/hVISA isolates should be considered in treatment failures of infections due to MRSA which are in vitro susceptible to vancomycin. Further testing is needed to detect these isolates. Despite the fact that ceftaroline is not a drug used in our country, the high resistance rate in our study is remarkable. This situation may be due to the intensive use of other beta-lactam antibiotics. Therefore, antibiotic susceptibility results should be taken into consideration during planning the treatment of infections. The high average MIC values of tigecycline in MRCoNS infections should also be monitored carefully

Rapid spread of Neisseria gonorrhoeae ciprofloxacin resistance due to a newly introduced resistant strain in Nuuk, Greenland, 2012–2015: a community-based prospective cohort study

ObjectivesTo determine the antimicrobial susceptibility and genotype distribution of Neisseria gonorrhoeae strains isolated from a cohort of patients in Nuuk, Greenland in order to assess the risk of rapid spread...

Relationship of teicoplanin MICs to treatment failure in teicoplanin-treated patients with methicillin-resistant Staphylococcus aureus pneumonia

The objective of this study was to determine the predictive value of teicoplanin minimal inhibitory concentrations (MICs) for treatment failure among patients with methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. In this study, all patients with ≥1 tracheal aspirates or sputum cultures positive for MRSA admitted to the hospital between April 2011 and September 2011 were reviewed. We enrolled patients who are ≥18 years of age, with a diagnosis of pneumonia, and with a receipt of teicoplanin therapy throughout the course. The relationship between teicoplanin Etest MICs and treatment outcomes of MRSA pneumonia was analyzed to identify the breakpoint of teicoplanin MICs influencing treatment outcomes. Of the 80 patients enrolled, 31 had a lower teicoplanin MIC level (<2.0mg/L) and 49 had a higher MIC level (≥2.0mg/L) for MRSA. The lower MIC group had a higher clinical resolution rate in 14 days [24 (77.4%) vs. 23 (46.9%), p=0.007] and a lower treatment failure rate at the end of teicoplanin treatment [4 (12.9%) vs. 18 (36.7%), p=0.020]. A comparison between the treatment success and failure groups showed that the former had a longer duration of teicoplanin use (18.76±10.34vs.12.41±5.65 days; p=0.014). Results of a multivariate analysis showed that teicoplanin MICs≥2.0mg/Land shorter duration of teicoplanin therapy were independent risk factors for treatment failure. A higher teicoplanin MIC value (≥2.0mg/L) may predict the treatment failure among patients with teicoplanin-treated MRSA pneumonia.

Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae: a 6-year study

BackgroundA variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae.ObjectiveThe aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect...

Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study

BackgroundThe frequency of primary resistance to antibiotics in H. pylori isolates is increasing worldwide. In Tunisia, there are limited data regarding the pattern of H. pylori antibiotic primary resistance.AimTo evaluate the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and to detect the mutations involved in clarithromycin resistance.Materials and methods273 strains isolated from adults and children were enrolled. The primary resistance to clarithromycin, metronidazole and amoxicillin was evaluated by means of E-test minimal inhibitory concentration (MIC). The real-time PCR using Scorpion primers was performed in all cases to assess clarithromycin primary resistance and point mutations involved.ResultsNo resistance to amoxicillin was detected. For adults, resistance to clarithromycin and metronidazole was found respectively in 14.6% and 56.8%, and respectively in 18.8% and 25% in children. Overall, the rates of global primary resistance to clarithromycin and metronidazole in Tunisia were respectively determined in 15.4% and 51.3%.By the use of Scorpion PCR, the A2143G was the most frequent point mutation observed (88.1%), followed by the A2142G (11.9%); the A2142C was not found and 18 of 42 patients (42.8%) were infected by both the resistant and the susceptible genotype.The association of clarithromycin resistance with gender was not statistically significant, but metronidazole resistant strains were isolated more frequently in females (67.8%) than in males (32.2%) and the difference was significant. As for gastroduodenal diseases, the difference between strains isolated from patients with peptic ulceration and those with non peptic ulceration was not statistically significant. When about the distribution of resistant strains to clarithromycin and metronidazole between the three Tunisian cities (Tunis, Menzel Bourguiba and Mahdia), the difference was not statistically significant.ConclusionLocal data regarding the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and the main genetic mutation involved in clarithromycin resistance in vivo (A2143G) are necessary to prove a clear need for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Tunisia in order to avoid the emergence of resistant strains.

Laboratory Evaluation of Iranian Commercially Provided Antibiotic Disks With Conventional E-Test Method for Susceptibility Testing in Three Most Isolated Multi-drug Resistant Organisms

Antimicrobial susceptibility testing is one of the most important tasks of clinical microbiology laboratories. This study was performed to compare accuracy of susceptibility testing carried out by disk diffusion method and E-test minimal inhibitory concentration (MIC) for detection antimicrobial resistance among three prevalent gram-negative organism isolated in an Iranian hospital . These organisms included Pseudomonas aeroginosa, Acenitobacter bumannii, and Klebsiella pneumoniaew which isolated from different Baquiatalah hospital wards .Disk diffusion method were carried out initially in Baqyatalah hospital microbiology laboratory. All isolates were sent to Iranian reference microbiology laboratory for detection of MIC by E-test method. Of 100 isolates, 52 isolates were Acinetobacter bumannii, 32 Pseudomonas aeroginosa and 16 Klebsiella pneumonia. All isolated strains were resistant to ciprofloxacin, imipenem, and amikacin by disk diffusion method. However there were significant differences compared with E-test method. Many strains were susceptible to above mentioned antibiotics by E-test method .Our study revealed that there was a controversy results between disk diffusion method and MIC determination technique by E-test. It may be due to lack of suitable internal quality control as well as poor quality of commercially provided disks in our country.

Prevalence of beta-lactamase-producing strains among 149 anaerobic gram-negative rods isolated from periodontal pockets.

In a prospective study, 47 adults presenting a rapidly progressive periodontitis were selected in order to evaluate the prevalence of beta-lactamase-producing strains among oral anaerobic gram-negative rods. Predominant anaerobes were identified from two of the deepest periodontal pockets. beta-Lactamase-positive strains fulfilled to at least two of three criteria: positive nitrocefin test, penicillin Etest minimal inhibitory concentration > 1 microgram/ml, and disk diffusion synergy between amoxycillin and clavulanic acid > 10 mm. At least one beta-lactamase-producing strain was found in 53.2% of patients and 39.4% of the periodontal pockets investigated. Prominent beta-lactamase-positive species were Prevotella buccae and Prevotella intermedia (respectively 16 of 38: 42% and 18 of 52: 35% positive strains), followed by Prevotella bivia, Prevotella disiens, Prevotella denticola and Fusobacterium nucleatum (respectively 1 of 6: 17%, 1 of 10: 10%, 1 of 10: 10%, and 1 of 13: 8% positive strains). No beta-lactamase producer could be evidenced in Porphyromonas gingivalis (10 strains tested). All the beta-lactamase-positive strains with the nitrocefin test had penicillin minimal inhibitory concentrations > 1 microgram/ml with the Etest, and a strong synergy between amoxicillin and clavulanic acid was always observed.

In vitro activity of antituberculous agents against Mycobacterium tuberculosis isolates from Bogota, DC (Colombia) evaluated by the Etest

Tuberculosis tests for antimicrobial susceptibility takes weeks. However, delayed therapy, can compromise the patient, as well as lead to an increase in disease incidence. Among infectious diseases, tuberculosis continues to be a leading cause of death in the world. The E-test is a new concept for Minimal Inhibitory Concentrations (MIC) determinations for antimicrobial agents that is based on a predefined antibiotic gradient on a plastic strip calibrated with a continuous logarithmic MIC scale covering 15 two-fold dilutions. MICs of rifampin, isoniazid, and ethambutol were determined by using the E-test (AB BIODISK, Solna, Sweden) for 30 clinical strains of Mycobacterium tuberculosis isolated from four hospitals, and were compared with the Bactec method. To make the inoculum with a turbidity equivalent to a McFarland 3.0 standard, we obtained a sample from an agar surface and the Bactec 460, as described by the manufacturer. Excellent agreement (100% for rifampin, 96.8% for ethambutol, and 90% for isoniazid) was demonstrated between the E-test MIC distributions and the Bactec interpretive criteria for all clinical isolates of M. tuberculosis tested. The E-test appears to be a good alternative method for testing the susceptibility of M. tuberculosis isolates to the three, most-commonly-used therapeutic agents.

Evaluation of the E-Test for susceptibility testing of pneumococci

The E-Test (AB Biodisk, Sweden) is an antibiotic gradient strip that is applied to an inoculated agar plate and results in an elliptical zone of inhibition that intercepts the graded strip, producing a quantitative (microgram per milliliter) result. Pneumococci (100) were used in this study, 38 penicillin-susceptible [minimal inhibitory concentrations (MICs), ≤0.12 μg/ml], 42 intermediately resistant (MICs, 0.12–1.0 μg/ml), and 20 resistant (MICs, >1 μg/ml). E-Test strips were evaluated on Mueller-Hinton agar plates with 5% sheep blood. Agar dilution MICs were determined by the National Committee for Clinical Laboratory Standards (NCCLS) method. Penicillin MICs for the E-Test tended to be slightly lower (one log 2 dilution) than reference MICs due to the continuous scale from which E-Test MICs were read. All but two penicillin-susceptible isolates were correctly categorized by the E-Test method. Of the 62 penicillin-resistant strains, 59 had E-Test MICs of ≥0.12 μg/ml, with 88% of these strains having E-Test MICs within one doubling dilution of the reference MICs. However, using the current NCCLS breakpoint MICs, many of the penicillin-resistant strains with reference MICs of 2 μg/ml were categorized as intermediate by the E-Test, with MICs of 0.38–1 μg/ml. For chloramphenicol, erythromycin, and tetracycline, correlation of the two methods was excellent. E-Test chloramphenicol MICs provided clearer separation of susceptible and resistant strains than did the reference method. We conclude that the E-Test is a reliable method for determination of MICs of the antibiotics evaluated for pneumococci.