Susceptibility Testing Methods Research Articles

A Road Less Traveled: E-test Method for Antifungal Susceptibility Testing in Trichophyton mentagrophyte Isolates Among Patients Presenting With Dermatophytosis at a Tertiary Healthcare Center in North India

A Road Less Traveled: E-test Method for Antifungal Susceptibility Testing in Trichophyton mentagrophyte Isolates Among Patients Presenting With Dermatophytosis at a Tertiary Healthcare Center in North India

Sedimentation field-flow fractionation for rapid phenotypic antimicrobial susceptibility testing: a pilot study.

The increase in antibiotic resistance is a major public health issue. The development of rapid antimicrobial susceptibility testing (AST) methods is becoming a priority to ensure early and appropriate antibiotic therapy. To evaluate sedimentation field-flow fractionation (SdFFF) as a method for performing AST in less than 3 h. SdFFF is based on the detection of early biophysical changes in bacteria, using a chromatographic-type technology. One hundred clinical Escherichia coli strains were studied. A calibrated bacterial suspension was incubated for 2 h at 37°C in the absence (untreated) or presence (treated) of five antibiotics used at EUCAST breakpoint concentrations. Bacterial suspensions were then injected into the SdFFF machine. For each E. coli isolate, retention times and elution profiles of antibiotic-treated bacteria were compared with retention times and elution profiles of untreated bacteria. Algorithms comparing retention times and elution profiles were used to determine if the strain was susceptible or resistant. Performance evaluation was done according to CLSI and the ISO standard 20776-2:2021 with broth microdilution used as the reference method. AST results from SdFFF were obtained in less than 3 h. SdFFF showed high categorical agreement (99.8%), sensitivity (99.5%) and specificity (100.0%) with broth microdilution. Results for each antimicrobial were also in agreement with the ISO 20776-2 recommendations, with sensitivity and specificity of ≥95.0%. This study showed that SdFFF can be used as a rapid, accurate and reliable phenotypic AST method with a turnaround time of less than 3 h.

Atmospheric Pressure Enhanced Self-Sealing Rotation-SlipChip with Programmable Concentration Gradient Generation for Microbiological Applications.

In microbiological research, traditional methods for bacterial screening and antibiotic susceptibility testing are resource-intensive. Microfluidics offers an efficient alternative with rapid results and minimal sample consumption, but the demand for cost-effective, user-friendly platforms persists in communities and hospitals. Inspired by the Magdeburg hemispheres, the strategy adapts to local conditions, leveraging omnipresent atmospheric pressure for self-sealing of Rotation-SlipChip (RSC) equipped with a 3D circular Christmas tree-like microfluidic concentration gradient generator. This innovative approach provides an accessible and adaptable platform for microbiological research and testing in diverse settings. The RSC can avoid leakage concerns during multiple concentration gradient generation, chip-rotating, and final long-term incubation reaction (≥24h). Furtherly, RSC subtypes adapted to different reactions can be fabricated in less than 15min with cost less than $1, the result can be read through designated observational windows by naked-eye. Moreover, the RSC demonstrates its capability for evaluating bacterial biomarker activity, enabling the rapid assessment of β-galactosidase concentration and enzyme activity within 30min, and the limit of detection can be reduced by 10-fold. It also rapidly determines the minimum antibiotic inhibitory concentration and antibiotic combined medications results within 4 h. Overall, these low-cost and user-friendly RSC make them invaluable tools in determinations at previously impractical environment.

Uji Kerentanan Aedes aegypti terhadap Malation, Permetrin, dan Bendiokarb di Kelurahan Tangkerang Timur Kecamatan Tenayan Raya, Pekanbaru

Insecticide resistant of Aedes aegypti populations is one of the main problems in controlling dengue hemorrhagic fever vectors because it will produce resistant offspring. This study aimed to determine the insecticide susceptibilty status of Ae. aegypti to 5% malathion, 0,75% permethrin, and 0,1% bendiocarb in Tangkerang Timur Village, Tenayan Raya District, Pekanbaru. This study is a descriptive observational study with a cross-sectional design. The resistance test method in this study was the World Health Organization (WHO) susceptibility test method with impregnated paper (5% malation, 0,75% permethrin, and 0,1% bendiocarb). The larval that were reared into adult mosquitoes were collected from 100 houses in Tangkerang Timur Village. Total adult mosquitoes aged 3-5 days tested in this study were 100 mosquitoes. The test mosquitoes were exposed to impregnated paper for 1 hour and the number of knock down mosquitoes was counted. Mosquito mortality was counted after 24 hours. The results of the susceptibility test in this study showed the mortality of Ae. aegypti to 5% malathion, 0,75% permethrin, and 0,1% bendiocarb were 100%, 85%, and 3.75%. The results of this study indicated that Ae. Aegypti has been resistant to 0,75% permethrin and 0,1% bendiocarb and susceptible to 5% malathion.

Outcomes of 23 patients diagnosed with New Delhi metallo-beta-lactamase (NDM)-producing Klebsiella pneumoniae infection treated with ceftazidime/avibactam and aztreonam at a single center in Poland.

Amongst all etiologic hospital-acquired infection factors, K. pneumoniae strains producing New Delhi metallo-β-lactamase (KP-NDM) belong to pathogens with the most effective antibiotic resistance mechanisms. Clinical guidelines recommend using ceftazidime/avibactam with aztreonam (CZA + AT) as the preferred option for NDM-producing Enterobacterales. However, the number of observations on such treatment regimen is limited. This retrospective study reports the clinical and microbiological outcomes of 23 patients with KP-NDM hospital-acquired infection treated with CZA + AT at a single center in Poland. The isolates were derived from the urine, lungs, blood, peritoneal cavity, wounds, and peritonsillar abscess. In microbiological analysis, mass spectrometry for pathogen identification, polymerase chain reaction, or an immunochromatographic assay for detection of carbapenemase, as well as VITEK-2 system, broth microdilution, and microdilution in agar method for antimicrobial susceptibility tests were used, depending of the pathogens' nature. CZA was administered intravenously (IV) at 2.5g every eight hours in patients with normal kidney function, and aztreonam was administered at 2g every eight hours IV. Such dosage was modified when renal function was reduced. KP-NDM was eradicated in all cases. Four patients (17.4%) died: three of them had a neoplastic disease, and one - a COVID-19 infection. The combination of CZA + AT is a safe and effective therapy for infections caused by KP-NDM, both at the clinical and microbiological levels. The synergistic action of all compounds resulted in a good agreement between the clinical efficacy of CZA + AT and the results of in vitro susceptibility testing.

Colistin, the last resort antibiotic: challenges in the implementation of its routine susceptibility testing

Background: colistin has become a critical antibiotic for lifethreatening multidrug resistance Gram-negative infections, particularly carbapenemase-producing bacteria. Detecting colistin resistance in routine microbiology laboratories is crucial for combating these fatal infections poses a challenge. Especially in developing countries, there is a need for a cost-effective, rapid, and user-friendly diagnostic method. Objective: implementing the various available methods for colistin testing is a significant challenge in resource-limited settings due to logistic difficulties and the need for technical expertise. Materials and Methods: this study shares experiences and insights gained while implementing in-vitro colistin susceptibility testing in a high-load bacteriology laboratory of a tertiary care center in Delhi, India. The following test methods for colistin susceptibility testing were incorporated in the routine antimicrobial susceptibility testing of our laboratory: Colistin Agar Test, Colistin Broth Disk Elution Test, Broth Microdilution susceptibility testing. Results: inconsistent growth patterns were observed in the colistin agar dilution Minimum Inhibitory Concentration (MIC) method, which could be resolved only after the preparation of fresh plates containing that specific concentration of colistin. The contamination issue of plates on use over a few days was addressed by pouring agar containing various concentrations of colistin in cottonplugged glass tubes. In the colistin broth disk elution test, due to the non-availability of screw-capped 10 mL glass tubes, MacCornety bottles (30 mL) were used. Subcultures were performed from the turbid wells to rule out the growth of contaminants when encountering discordant MIC values or skipped wells on the colistin broth microdilution test. Conclusions: despite several technical issues in in-vitro colistin susceptibility testing, we have successfully implemented it in our laboratory. Our experiences can offer guidance to laboratories that are still in the process of implementing it.

Comparison of testing methods assessing the in vitro efficacy of the combination of aztreonam with avibactam on multidrug-resistant Gram-negative bacilli

BackgroundAztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches.Methods96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher).ResultsAccording to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%).ConclusionsThe ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

MultiRapid ATB NP test for detecting concomitant susceptibility and resistance of last-resort novel antibiotics available to treat multidrug-resistant Enterobacterales infections

BackgroundRecently developed therapeutics against Gram-negative bacteria include the β-lactam-β-lactamase inhibitor combinations ceftazidime–avibactam (CZA), meropenem–vaborbactam (MEV), and imipenem–relebatam (IPR), and the siderophore cephalosporin cefiderocol (FDC). The aim of this study was to develop a test for rapid identification of susceptibility/resistance to CZA, MEV, IPR, and FDC for Enterobacterales in a single test for rapid clinical decision making. MethodsThe MultiRapid ATB NP test is based on the detection of glucose metabolism occurring after bacterial growth in the presence of defined concentrations of CZA, MEV, IPR, and FDC, followed by visual detection of colour change of the pH indicator red phenol (red to yellow) generated by the acidification of the medium upon bacterial growth. This test is performed in 96-well microplates. The MultiRapid ATB NP test was evaluated using 78 Enterobacterales isolates and compared to the reference method broth microdilution. ResultsThe MultiRapid ATB NP test displayed 97.0% (confidence interval [CI] 92.6–98.8) sensitivity, 97.7% (CI 94.3–99.1) specificity, and 97.4% (CI 95.0–98.7) accuracy. The results were obtained after 3 h of incubation at 35 °C ± 2 °C, representing at least a 15-h gain-of-time compared with currently used antimicrobial susceptibility testing methods. ConclusionThe MultiRapid ATB NP test provided accurate results for the concomitant detection of susceptibility/resistance to CZA, MEV, IPR, and FDC in Enterobacterales, independent of the resistance mechanism. This test may be suitable for implementation in any microbiology routine laboratory.

Evaluation of a real-time PCR assay performance to detect Mycobacterium tuberculosis, rifampicin, and isoniazid resistance in sputum specimens: a multicenter study in two major cities of Indonesia.

Tuberculosis (TB) is one of the major global health issues due to its high mortality rate, especially in low- and middle-income countries. One of the key success points of the TB eradication program is early TB diagnosis, which requires rapid and accurate diagnostic testing. This study aimed to evaluate the performance of a newly developed RT-PCR kit (Indigen MTB/DR-TB RT-PCR) in a routine TB clinical setting. A multi-fluorescence RT-PCR assay was designed and developed to detect regions within IS6110, rpoB, katG, and inhA of the Mycobacterium tuberculosis (MTB) genes. Sputum specimens were obtained from suspected TB patients who visited TB healthcare facilities in two major cities of Indonesia from September 2022 to May 2023. Specimens were assessed using Indigen MTB/DR-TB RT-PCR, acid-fast bacillus (AFB) smear microscopy, MTB culture, and drug susceptibility testing (DST) methods. Fisher's exact test (χ2) was used to analyze the Indigen performance relative to culture methods. The performance of Indigen MTB/DR-TB RT-PCR to detect MTB was assessed using 610 sputum specimens obtained from suspected patients. The overall sensitivity and specificity were 94.12% (95% CI: 90.86-96.48%) and 98.32% (95% CI: 96.20-99.46%), respectively. When the analysis was performed on AFB smear-negative TB subjects (386 subjects), a lower sensitivity level was found at 78.57% (95% CI: 68.26-86.78%), while the specificity level remained similar at 98.34% (95% CI: 96.18-99.46%). The overall performance of Indigen MTB/DR-TB RT-PCR to detect MTB showed substantial agreement with the MTB culture method (kappa value 0.93). In comparison to DST, the sensitivity and specificity levels of Indigen to detect RIF resistance or INH resistance were 78.2% (95% CI: 61.8-90.2%) and 82.8% (95% CI: 64.2-94.2%), respectively, while the specificity level for both groups was at 100% (95% CI, 87.7-100%). Indigen MTB/DR-TB RT-PCR demonstrated reliable performance for TB molecular diagnostic testing and can be implemented in routine TB diagnostic settings.

Microbroth dilution method for antibiotic susceptibility testing of fastidious and anaerobic bacteria of the urinary microbiome.

Antibiotic susceptibilities of fastidious and anaerobic bacteria of the human urinary microbiome are largely underreported due to difficulty in growing them in the lab environment. The current standard medium, Muller-Hinton broth, has difficulty supporting the growth of many of these species, leaving microbiologists without a standardized method. To address this need, this study offers a methodology to survey susceptibilities in a high-throughput manner of these understudied microbes with a proposed harmonized medium, NYCIII, which is capable of supporting the growth of both fastidious and non-fastidious urinary microbes. Broader standardization of this method can allow for the development of antibiotic-resistant breakpoints of the many uncharacterized urinary microbes.

Chemical composition and biological evaluation of tea tree (Melaleuca alternifolia L.) leaves essential oils

Melaleuca alternifolia L. essential oils (MaEO), extracted through steam, distillation of its leaves, offer a multitude of benefits. The aim of the study was to determine the physicochemical and biological properties, such as relative density, absolute density, acid value, saponification value, ester value, freezing point, fragrance retention, antioxidant and antibacterial activity of MaEO. Gas chromatography mass spectrometry (GC-MS) analysis was used to analyze the chemical composition of essential oils (EO), and the obtained results displayed those 45 compounds were identified and quantified; among them, the main component was Terpinen-4-ol (44.55%). In addition, the antioxidant capacity (AC) was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with the half maximal inhibitory concentration (IC50) of 360 mg mL-1. Moreover, MaEO also had excellent antibacterial activity (AA) against certain types of bacteria, using the paper disc diffusion method for antibiotic susceptibility testing. The diameter of the inhibitory zones were 12.33, 14, 15.67, and 24.33 mm for Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 25923), Salmonella enterica (ATCC 13076), and Escherichia coli (ATCC 25922), respectively.

Investigation of JGS frost susceptibility testing method

Frost heave is a type of frost action that occurs when the soil temperature is below freezing temperature. Construction in cold regions is challenging and can cause severe damage to structures due to frost heave. The segregation potential (SP) is widely accepted in engineering design and for frost heave predictions as an indicator for frost susceptibility classification. The purpose of this study is to investigate the frost heave experiments in the JGS method and to evaluate the JGS frost susceptibility testing results for different soil mixtures with different SP through numerical simulation with a Thermal-Hydraulic coupled model. The results show good agreement between numerical simulation and test results for different degrees of frostsusceptible soils. This model is expected to provide accurate predictions of the frost heave behavior of different soils.

Comparison of Rapid Antibiotic Susceptibility Test Method Directly from Blood Culture Bottle with Standard Disc Diffusion Method

Aim: Early determination of antimicrobial susceptibility of sepsis pathogens is important. In this study, we aimed to compare the standard disc diffusion method with the rapid antimicrobial susceptibility testing (RAST) method performed directly from blood culture bottles. Material and Methods: Bacteria isolated from samples that gave a positive signal on the blood culture device between April 2019 and September 2019 were included in the study, and antimicrobial susceptibilities were determined by the standard disc diffusion method and the RAST method. Categorical agreement, small error, large error, very large error, and area of technical uncertainty ratios were recorded. Results: A total of 103 bacteria including 19 S. aureus, 10 Enterococcus spp. and 24 E. coli, 24 K. pneumoniae, 13 P. aeruginosa, and 13 A. baumannii were included in the study. When the RAST method was compared with the standard disc diffusion method, 100% agreement was found between the methods against imipenem, meropenem, gentamicin, and trimethoprim-sulfamethoxazole in E. coli isolates at all hours evaluated, and against meropenem in K. pneumoniae isolates at the 6th and 8th hour. For S. aureus and P. aeruginosa isolates, very major errors were found in the RAST results. For A. baumannii isolates, 100% agreement between methods was observed for many antibiotics. Conclusion: It was concluded that the RAST method is a simple and inexpensive test for life-threatening infections such as sepsis. It was also felt that similar studies should be carried out with a large number of isolates, as compliance rates vary depending on the bacteria tested.

Practice of standardization of CLSI M45 A3 antimicrobial susceptibility testing of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in Guangdong Province 2017-2021.

The concentration of antimicrobial agents in environments like water and food has increased rapidly, which led to a rapid increase in antimicrobial resistance levels in the environment. Monitoring of bacterial resistance levels is considered as a necessary means to control the bacterial resistance. Reference standards are critical for antimicrobial susceptibility testing. CLSI M45 A3 standard defines pathogenic microorganisms that cause infections less frequently than those covered by CLSI M02, M07, and M100 as Infrequently Isolated or Fastidious Bacteria and specifies antimicrobial susceptibility testing methods. Our study investigated the epidemiology and antimicrobial susceptibility testing data of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in 70 hospitals in Guangdong Province between 2017 and 2021. We defined testing methods other than those specified in CLSI M45 A3 as "Non-Standardized." The proportion of standardized antimicrobial susceptibility testing for penicillin increased significantly (Corynebacterium spp. 17.4% vs. 50.0% p < 0.05; Micrococcus spp. 50.0% vs. 77.8% p < 0.05; Abiotrophia spp. and Granulicatella spp. 21.4% vs. 90.9% p < 0.001), while for cefotaxime (Corynebacterium spp. 0.0% vs. 45.2% p < 0.05; Abiotrophia spp. and Granulicatella spp. 0.0% vs. 14.3% p = 0.515) and vancomycin increased finitely. Non-standardized methods were used for all other antimicrobials. Due to limitations in the economic and medical environment, some clinical laboratories are unable to fully comply with CLSI M45 A3 standard. We recommend that CLSI should add breakpoints for disk diffusion method to improve the standardization of antimicrobial susceptibility testing.

Comparative performance of biofire pneumonia panel and standard culture-based methods for diagnosing pneumonia in critically ill patients: Impact on antibiotic stewardship

IntroductionLower respiratory tract infections (LRTIs) are a common cause of morbidity and mortality worldwide. Accurate identification of the pathogens causing LRTIs is crucial for ensuring of diagnostic and antibiotic stewardship. The Biofire Pneumonia Panel (BFPP) is a molecular diagnostic test that allows rapid detection of various bacterial and viral pathogens. In this study, we compared the performance of BFPP with standard culture methods for the detection of pathogens. Materials and methodsRespiratory samples from 70 patient with suspected LRTIs were tested using both BFPP and standard culture methods. The distribution of isolated bacterial pathogens was analyzed, and the sensitivity and specificity of BFPP were calculated. Additionally, the performance of BFPP in detecting antimicrobial resistance genes was evaluated. The results were compared with those obtained from VITEK-2 antimicrobial susceptibility testing and culture-based methods. ResultsAmong the suspected LRTI cases, BFPP identified a single pathogen in 32.8% of cases and multiple pathogens in 40% of cases. The standard culture method detected a single pathogen in 47.1% of cases. BFPP showed a sensitivity of 93.9% and a specificity of 45.9% for the total sample. The performance of BFPP in detecting antimicrobial resistance genes varied for different pathogens with overall sensitivity of 40.1% and specificity of 95.9%. ConclusionThe Biofire Pneumonia Panel (BFPP) demonstrated high sensitivity for several bacterial pathogens, indicating its potential as a rapid diagnostic tool. However, its performance varied for different microorganisms, and it had limitations in detecting certain pathogens and antimicrobial resistance genes for which still required more further studies to explore different resistance gene mechanism that can be incorporated in this panel in future. The BFPP can complement standard culture methods as a rapid tool in the diagnosis of LRTIs.

A Day Saved is a Life Saved: Direct Antimicrobial Susceptibility Testing from Positively Flagged Blood Culture Bottles and their Concordance with the Routine Method.

Sepsis is a major health problem worldwide and is associated with high morbidity and mortality with every hour delay in initiation of therapy. A conventional method of blood culture and Antimicrobial Susceptibility Testing (AST) takes around 48-72 hours. Empirical antibiotics need to be administered until the sensitivity report is made available. It has been estimated that 20-50% of the empirical antibiotics are inappropriate, resulting in prolonged hospital stays, adverse effects, and emergence of drug resistance. Additionally, this also puts an extra financial burden on both the patients and healthcare settings. Performing direct Antimicrobial Sensitivity Testing (dAST) is an important tool to reduce turn-around time (TAT) by at least 18-24 hours, thus reducing morbidity and mortality among critically ill patients. Direct AST (dAST) was performed from the positively flagged blood culture bottles received between December, 2021 to May, 2022 from Intensive Care Units (ICUs) on MuellerHinton Agar (MHA) using four drops of withdrawn blood. dAST was performed for six drugs: Ceftriaxone-30 µg (CTR), Piperacillin/Tazobactam-100/10 µg (PIT), Meropenem-10 µg (MRP), Ciprofloxacin-5 µg (CIP), Aztreonam-30 µg (AT), and Colistin (CL). The zone of inhibition was interpreted as per CLSI M100 ed32, 2022 guidelines. A parallel conventional method was also performed to examine for categorical agreement and disagreement. Identification was carried out using MALDI-TOF MS from the colonies that appeared on the dAST plate on the subsequent day. A total of 162 positively flagged blood culture bottles were included in the study. The majority of the Gram-negative organisms were from Enterobacterales (n=109), followed by Acinetobacter spp. (n=28) and Pseudomonas aeruginosa (n=25). Out of the 972 isolate-antimicrobial combinations, overall Categorical Agreement (CA) was seen in 936 (96.3%), whereas disagreement was observed in 36 with minor error (mE) in 21 (2.2%), major error (ME) in 7 (0.7%), and very major error (VME) in 8 (0.8%) when compared to the routine method. Categorical agreement (CA) of > 99% was seen in ceftriaxone (CTR) and ciprofloxacin (CIP). In comparison, the lowest CA was observed with meropenem (MRP) at 92%. Colistin dAST was performed using the E-strip method, and the result obtained was highly convincing, with an overall disagreement of only 1.2%. Rapid dAST from positively flagged blood culture bottles proved to significantly reduce the TAT from the time of sample collection to the first availability of antimicrobial susceptibility report with excellent categorical agreement of > 95% using the conventional disc diffusion method. Results obtained were within the acceptance criteria set by U. S. Food and Drug Administration (FDA) guidelines of > 90% categorical agreement for a new method. We were able to obtain excellent concordance for colistin using the E-strip method. Performing dAST not only saves a "day", but its proper implementation would save a "life".

Revisiting the Checkerboard to Inform Development of β-Lactam/β-Lactamase Inhibitor Combinations.

A two-dimensional "checkerboard" array employing systematic titration (e.g., serial two-fold dilutions) is a well-established in vitro method for exploring the antibacterial effects of novel drug combinations. Minimum inhibitory concentrations (MICs) on the checkerboard are isoeffective points at which the antibiotic potency is the same. Representations of checkerboard MIC curves for a β-lactam and β-lactamase inhibitor combination are used in hypothetical "thought experiments" and reveal the ways in which current practices can be improved. Because different types of response (i.e., independence vs. additivity vs. one effective agent; interaction vs. noninteraction) produce different MIC curves, data from different strains/isolates should not be pooled indiscriminately, as the composition of a pooled dataset will influence any derived pharmacokinetic/pharmacodynamic (PK/PD) index. Because the β-lactamase inhibitor threshold concentration (CT) parameter is a function of the β-lactam partner dosing regimen, it is not possible to derive a universal PK/PD index target based on CT. Alternative susceptibility testing methods represent different planes through the checkerboard; a fixed ratio method is less prone to bias for all β-lactam and β-lactamase inhibitor combinations. Susceptibility test MICs will often not reflect the sensitivity of the strain/isolate to the β-lactamase inhibitor, so the use of these MICs to normalize PK/PD indices is inappropriate.

Identification of Mycobacterium tuberculosis Resistance to Common Antibiotics: An Overview of Current Methods and Techniques.

Multidrug-resistant tuberculosis (MDR-TB) is an essential cause of tuberculosis treatment failure and death of tuberculosis patients. The rapid and reliable profiling of Mycobacterium tuberculosis (MTB) drug resistance in the early stage is a critical research area for public health. Then, most traditional approaches for detecting MTB are time-consuming and costly, leading to the inappropriate therapeutic schedule resting on the ambiguous information of MTB drug resistance, increasing patient economic burden, morbidity, and mortality. Therefore, novel diagnosis methods are frequently required to meet the emerging challenges of MTB drug resistance distinguish. Considering the difficulty in treating MDR-TB, it is urgently required for the development of rapid and accurate methods in the identification of drug resistance profiles of MTB in clinical diagnosis. This review discussed recent advances in MTB drug resistance detection, focusing on developing emerging approaches and their applications in tangled clinical situations. In particular, a brief overview of antibiotic resistance to MTB was present, referred to as intrinsic bacterial resistance, consisting of cell wall barriers and efflux pumping action and acquired resistance caused by genetic mutations. Then, different drug susceptibility test (DST) methods were described, including phenotype DST,genotype DST and novel DST methods. The phenotype DST includes nitrate reductase assay, RocheTM solid ratio method, and liquid culture method and genotype DST includes fluorescent PCR, GeneXpert, PCR reverse dot hybridization, ddPCR, next-generation sequencing and gene chips. Then, novel DST methods were described, including metabolism testing, cell-free DNA probe, CRISPR assay, and spectral analysis technique. The limitations, challenges, and perspectives of different techniques for drug resistance are also discussed. These methods significantly improve the detection sensitivity and accuracy of multidrug-resistant tuberculosis (MRT) and can effectively curb the incidence of drug-resistant tuberculosis and accelerate the process of tuberculosis eradication.

Comparison of Colistin Susceptibility Tests

Introduction: Polymyxins are important antimicrobial agents for the treatment of infections caused by Gram-negative bacteria. The susceptibility testing for polymyxins is a challenge for clinical laboratories due to the difficulty of performance, reproducibility, and accuracy of available methods. Aim: To compare the performance of the colistin susceptibility test of an automated system and a gradient test with the gold standard broth microdilution method (BMD). Materials and Methods: Multidrug-resistant isolates of Acinetobacter baumannii (n=102), Klebsiella pneumoniae (n=40), and Pseudomonas aeruginosa(n=11) were included. The VITEK 2 systems and gradient test were studied according to the manufacturer’s instructions. Broth microdilution tests were performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Commercial susceptibility testing methods were compared to BMD. Results: Rates of essential agreement of colistin test results between BMD, VITEK 2, and gradient test were 96.1% and 79.7%, respectively. The VITEK 2 and gradient test showed 95.4% and 94.8% of categorial agreement. The very major error rate of VITEK 2 was 3.2%, and the gradient test was 5.2%. The major error rate of VITEK 2 was 1.3%, and there was no major error for the gradient test. Conclusion and Suggestions: The very major error rate was higher in the gradient test (5.2%) than VITEK 2 (3.2%). Even if the very major error rate of VITEK 2 was lower, both resistance and susceptility results of VITEK 2 should be confirmed with the BMD test. Further studies for susceptibility testing are needed with a focus on the correlation of MIC’s results of different tests.

Fosfomycin susceptibility testing and resistance mechanisms in Enterobacterales in South Africa.

Fosfomycin treatment of urinary tract infections is increasingly attractive due to escalating antibiotic resistance rates among urinary pathogens. Standard antibiotic susceptibility testing methods perform poorly for fosfomycin as there is poor correlation between susceptibility results and clinical outcomes in urinary pathogens other than Escherichia coli. We evaluated the performance of fosfomycin susceptibility testing in E. coli and Klebsiella pneumoniae to determine whether fosfomycin susceptibility is associated with molecular resistance mechanisms. Forty-six each of E. coli and K. pneumoniae clinical isolates were obtained from a tertiary hospital in South Africa, from 01 June 2017 to 31 January 2018. Agar dilution, disk diffusion, and gradient diffusion were performed and interpreted using the Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines. Molecular resistance mechanisms were identified by whole genome sequence analysis. Disk diffusion and gradient diffusion were accurate alternatives for fosfomycin susceptibility testing in E. coli (98% categorical agreement), but not in K. pneumoniae (47% categorical agreement). All E. coli isolates contained at least one resistance mechanism, but only one isolate with a fosA gene was resistant. In K. pneumoniae, 63% (29/46) and 70% (32/46) of isolates were susceptible to fosfomycin, using Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively, despite all isolates containing a fosA gene and a uhpT mutation. A better understanding of fosfomycin susceptibility and improved antibiotic susceptibility testing tools could improve diagnostic capability and clinical guidelines for fosfomycin treatment of urinary tract infections. This study highlights the importance of adhering to interpretive guidelines when performing antimicrobial susceptibility testing and the need for simplified, accurate and standardised susceptibility testing methodology and interpretation for fosfomycin in Enterobacterales organisms.