Antibiotic Susceptibility Testing Methods Research Articles

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.

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.

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.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

Rapid Phenotypic Antibiotics Susceptibility Analysis by a 3D Printed Prototype.

One of the most important public health concerns is the increase in antibiotic-resistant pathogens and corresponding treatment of associated infections. Addressing this challenge requires more efficient use of antibiotics, achievable by the use of evidence-based, effective antibiotics identified by antibiotic susceptibility testing (AST). However, the current standard method of phenotypic AST used for this purpose requires 48 h or more from sample collection to result. Until results are available, broad-spectrum antibiotics are used to avoid delaying treatment. The turnaround time must therefore be shortened in order for the results to be available before the second administration of antibiotics. The phenotypic electrochemical AST method presented here identifies effective antibiotics within 5-10 h after sampling. Spiked serum samples, including polymicrobial samples, with clinically relevant pathogens and respective concentrations commonly found in bloodstream infections (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa) are used. Direct loading of the test with diluted serum eliminates the need for a pre-culture, as required by existing methods. Furthermore, by combining several electrochemical measurement procedures with computational analysis, allowing the method to be used both online and offline, the AST achieves a sensitivity of 94.44% and a specificity of 95.83% considering each replicate individually.

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.

Multipad agarose plate: a rapid and high-throughput approach for antibiotic susceptibility testing.

We describe a phenotypic antibiotic susceptibility testing (AST) method that can provide an eightfold speed-up in turnaround time compared with the current clinical standard by leveraging advances in microscopy and single-cell imaging. A newly developed growth plate containing 96 agarose pads, termed the multipad agarose plate (MAP), can be assembled at low cost. Pads can be prepared with dilution series of antibiotics. Bacteria are seeded on the pads and automatically imaged using brightfield microscopy, with a fully automated segmentation pipeline quantifying microcolony formation and growth rate. Using a test set of nine antibiotics with very different targets, we demonstrate that accurate minimum inhibitory concentration (MIC) measurements can be performed based on the growth rate of microcolonies within 3 h of incubation with the antibiotic when started from exponential phase. Faster, reliable and high-throughput methods for AST, such as MAP, could improve patient care by expediting treatment initiation and alleviating the burden of antimicrobial resistance.

Rapid Phenotypic Antibiotic Susceptibility Profiling of Clinical Escherichia coli and Klebsiella pneumoniae Blood Cultures.

Bloodstream infections (BSI) are defined by the presence of viable bacteria or fungi, accompanied by systemic signs of infection. Choosing empirical therapy based solely on patient risk factors and prior antibiotic susceptibility test (AST) may lead to either ineffective treatment or unnecessarily broad-spectrum antibiotic exposure. In general, Clinical & Laboratory Standards Institute guideline-approved ASTs have a turnaround time of 48-72 h from sample to answer, a period that may result in a critical delay in the appropriate selection of therapy. Therefore, reducing the time required for AST is highly advantageous. We have previously shown that our novel rapid AST method, MAPt (Micro-Agar-PCR-test), accurately identifies susceptibility profiles for spiked bioterrorism agents like Bacillus anthracis, Yersinia pestis and Francisella tularensis directly from whole-blood and blood culture samples, even at low bacterial levels (500 CFU/mL). This study evaluated the performance of MAPt on routine bloodstream infection (BSI), focusing on Escherichia coli and Klebsiella pneumoniae isolates from clinical cultures, including resistant strains to some of the six tested antibiotics. Notably, MAPt yielded results exceeding 95% agreement with the standard hospital method within a significantly shorter timeframe of 6 h. These findings suggest significant potential for MAPt as a rapid and reliable BSI management tool.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data

DNA sequencing could become an alternative to invitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan14,2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and invitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), andlinezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

Prevalence of piperacillin/tazobactam resistance in invasive Haemophilus influenzae in Germany.

Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi. To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms. According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller-Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing. Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates. Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations.

Comparative Evaluation of Broth Microdilution With Disc Diffusion and VITEK 2 for Susceptibility Testing of Colistin on Multidrug-Resistant Gram-Negative Bacteria.

Background The rise of antibiotic resistance, particularly in Gram-negative bacteria, poses a significant global health threat. Colistin, a last-resort antibiotic, has witnessed renewed use. However, accurate susceptibility testing for colistin is challenging, with various methods available, leading to potential discrepancies. Ensuring reliable testing is crucial for effective patient treatment and antimicrobial stewardship. This study addresses the need to compare different colistin susceptibility testing methods, providing insights into their accuracy and relevance in clinical settings. Methods In this one-year prospective observational cross-sectional study conducted atIndira Gandhi Institute of Medical Sciences (IGIMS), Bihar, India, atertiary care hospital from July 2021 to June 2022, we aimed to evaluate the concordance between two widely used methods, VITEK 2 and Disc Diffusion, for antibiotic susceptibility testing in clinical multidrug-resistant Gram-negative bacterial isolates. These isolates, including species like Klebsiella pneumoniae, Acinetobacter baumannii, Klebsiella oxytoca, Pseudomonas aeruginosa, Citrobacter freundii, and Escherichia coli, were isolated from various clinical specimens. After rigorous species-level identification and quality control measures, antibiotic susceptibility testing was performed using both methods, and their agreement was assessed through Percentage Agreement analysis. Results In our study, we isolated and identified bacterial isolates from 105 patients, with a mean age of 47.30 years, demonstrating a wide age range. Pus samples were the most common type (25.7%), and K. pneumoniae was the most prevalent organism (45.7%). Antibiotic resistance patterns revealed significant challenges in treating infections caused by K. pneumoniae and A. baumannii, with resistance rates exceeding 70% for certain antibiotics. Among the 48 isolates of K. pneumoniae, the agreement was 93.8%, with 89.6% being sensitive and 6.3% being resistant by Disc Diffusion, while VITEK 2 indicated 0% resistance. E. coli isolates (n=21) had an agreement of 90.5%, with 90.5% sensitivity and 9.5% resistance by Disc Diffusion, and no resistance by VITEK 2. Conclusion The comparative analysis of antibiotic susceptibility testing methods reveals the superior performance of the VITEK 2 system, particularly in sensitivity and negative predictive value, emphasizing its potential as a reliable tool for guiding antibiotic therapy decisions.

Cost-Efficient Micro-Well Array-Based Colorimetric Antibiotic Susceptibility Testing (MacAST) for Bacteria from Culture or Community.

Rapid and cost-efficient antibiotic susceptibility testing (AST) is key to timely prescription-oriented diagnosis and precision treatment. However, current AST methods have limitations in throughput or cost effectiveness, and are impractical for microbial communities. Here, we developed a high-throughput micro-well array-based colorimetric AST (macAST) system equipped with a self-developed smartphone application that could efficiently test sixteen combinations of bacteria strains and antibiotics, achieving comparable AST results based on resazurin metabolism assay. For community samples, we integrated immunomagnetic separation into the macAST (imacAST) system to specifically enrich the target cells before testing, which shortened bacterial isolation time from days to only 45 min and achieved AST of the target bacteria with a low concentration (~103 CFU/mL). This proof-of-concept study developed a high-throughput AST system with an at least ten-fold reduction in cost compared with a system equipped with a microscope or Raman spectrum. Based on colorimetric readout, the antimicrobial susceptibility of the bacteria from microbial communities can be delivered within 6 h, compared to days being required based on standard procedures, bypassing the need for precise instrumentation in therapy to combat bacterial antibiotic resistance in resource-limited settings.

Fifty years devoted to anaerobes: historical, lessons, and highlights.

Renew interest and enthusiasm for anaerobes stem from both technological improvements (culture media, production of an adequate anaerobic atmosphere, identification methods) and greater awareness on the part of clinicians. Anaerobic infections were historically treated empirically, targeting the species known to be involved in each type of infection. Prevotella, fusobacteria, and Gram-positive cocci (GPAC) were considered responsible for infections above the diaphragm whereas for intra-abdominal infections, Bacteroides of the fragilis group (BFG), GPAC and clostridia were predominantly implicated. The antibiotic susceptibility of anaerobes was only taken into consideration by the clinician in the event of treatment failure or when faced with infections by multidrug-resistant bacteria (MDR). The evolution of antibiotic resistance together with clinical failures due to the absence of detection of hetero-resistant clones has resulted in a greater need for accessible antibiotic susceptibility testing (AST) and disc diffusion method. Improved isolation and identification of anaerobes, along with the availability of accessible and robust methods for performing AST, will ensure that treatment, whether empirical or guided by an antibiogram, will lead to better outcomes for anaerobic infections.

Microwell-enhanced optical rapid antibiotic susceptibility testing of single bacteria

Bacteria that are resistant to antibiotics present an increasing burden on healthcare. To address this emerging crisis, novel rapid antibiotic susceptibility testing (AST) methods are eagerly needed. Here, we present an optical AST technique that can determine the bacterial viability within 1h down to a resolution of single bacteria. The method is based on measuring intensity fluctuations of a reflected laser focused on a bacterium in reflective microwells. Using numerical simulations, we show that both refraction and absorption of light by the bacterium contribute to the observed signal. By administering antibiotics that kill the bacteria, we show that the variance of the detected fluctuations vanishes within 1 h, indicating the potential of this technique for rapid sensing of bacterial antibiotic susceptibility. We envisage the use of this method for massively parallelizable AST tests and fast detection of drug-resistant pathogens.

B-236 Portable 2 Hour Sample-to-Answer Multi-pathogen Phenotypic Antibiotic Susceptibility Test (AST)

Abstract Background Antimicrobial Resistance is a rapidly growing issue worldwide, arising from the overuse and misuse of antibiotics. The strongest tool the medical community has available to combat AMR is rapid, correct application of antibiotics to identified bacterial infections. Clinics and physician offices seek on-site rapid solutions to speed the pathway to timely and appropriate antibiotic therapies. GeneCapture is prototyping a rapid, sample-to-answer product to provide antibiotic susceptibility results directly from infected urine or wound samples. The test provides a susceptible vs non-susceptible call in under two hours and can be applied to samples with single or mixed pathogens. With support from the Defense Health Agency, a portable table-top instrument and disposable cartridge are being engineered to run this test at the point of care, allowing same-day prescriptions. The process is currently operational on the lab bench. Methods Fluorescent labeling of bacterial species, followed by optical counting, determines population growth with/without a suite of antibiotics. An on-board algorithm instantly converts raw cell numbers into a differential call of Susceptible or Non-susceptible for each bug/drug pair. A disposable cartridge with on-board antibiotics has been designed and is being finalized for fabrication. The optical counting is operating in a desktop device. Results Testing to date includes 124 strains from 7 of the most common Urinary Tract Infection and wound pathogens against a panel of six antibiotics. A total of 541 antibiotic/bacterial strain pairs in single or multiple pathogen antibiotic susceptibility testing (AST), are producing an initial 88.7% agreement to gold standard culture results. These results are all generated in two hours or less of assay time and represent a strong preliminary data set. Conclusion This 2-hour portable, novel, phenotypic AST profiling will produce informed and effective treatment options for medics, clinics and physician offices long before existing AST methods could return results.

Drug-Resistant Helicobacter pylori: Diagnosis and Evidence-Based Approach.

Helicobacter pylori (H. pylori) is the most common chronic bacterial infection, affecting approximately half of the world's population. H. pylori is a Class I carcinogen according to the World Health Organization, and the International Agency for Research on Cancer (IARC) has linked it to 90% of stomach cancer cases worldwide. The overall pattern points to a yearly reduction in eradication rates of H. pylori with the likelihood of success further decreasing after each unsuccessful therapeutic effort. Antimicrobial resistance in Helicobacter pylori is a major public health concern and is a predominant cause attributed to eradication failure. As a result, determining H. pylori's antibiotic susceptibility prior to the administration of eradication regimens becomes increasingly critical. Detecting H. pylori and its antimicrobial resistance has traditionally been accomplished by time-consuming culture and phenotypic drug susceptibility testing. The resistance of H. pylori to different antibiotics is caused by various molecular mechanisms, and advances in sequencing technology have greatly facilitated the testing of antibiotic susceptibility to H. pylori. This review will summarize H. pylori antibiotic resistance patterns, mechanisms, and clinical implications. We will also review the pros and cons of current antibiotic susceptibility testing methods. Along with a comparison of tailored susceptibility-guided regimens and empirical therapy based on the latest evidence, an evidence-based approach to such situations will be explored.

Susceptibility‐guided sequential strategy versus empirical therapy for Helicobacter pylori infection: study protocol for a randomised controlled trial

BackgroundNew treatment strategies are required against infections caused by Helicobacter pylori, which grows increasingly resistant to antibiotics. Polymerase chain reaction-based methods for antibiotic susceptibility testing are available for detecting H. pylori-specific mutations that confer resistance to clarithromycin and levofloxacin. Several meta-analyses have compared eradication rates for susceptibility-guided versus empirical therapy for H. pylori treatment; however, all have significant limitations and high heterogeneity, and the results are contradictory. The main objective of this trial is to assess whether a sequential strategy based on molecular susceptibility testing-guided therapy for H. pylori has a better eradication rate than empirical therapy.MethodsThis trial is designed as a prospective, randomised, open-label, active-controlled and single-centre study. Men and women who are H. pylori-positive, naïve to treatment, and aged 18–65 years will be recruited. A total of 500 participants will be randomised to receive either empirical therapy or a susceptibility-guided sequential strategy. Bismuth quadruple therapy will be the empirical first-line therapy, and in case of failure, high-dose dual (proton-pump inhibitor + amoxicillin) treatment will be the rescue therapy. For the susceptibility-guided sequential strategy, regimen selection will be based on H. pylori susceptibility to clarithromycin (first-line) and levofloxacin (rescue). A first-line treatment of clarithromycin triple therapy will be selected for clarithromycin-sensitive strains. For clarithromycin resistance, a high-dose dual therapy will be selected. During the rescue treatment, a levofloxacin quadruple regimen will be selected for levofloxacin-sensitive strains, and a furazolidone quadruple regimen will be selected for others. The primary outcome is the first-line eradication rate in both groups, and the overall (including first and rescue therapies) H. pylori eradication rate in both groups is one of the secondary outcomes. The eradication rates of H. pylori will be analysed by intention-to-treat analysis, modified intention-to-treat analysis, and per-protocol analysis.DiscussionThis randomised controlled trial will provide objective and valid evidence about the value of polymerase chain reaction-based molecular methods for antibiotic susceptibility testing in guiding H. pylori eradication.Trial registrationClinicaltrials.gov NCT05549115. Released on 18 September 2022. First posted on 22 September 2022. Enrolment of the first participant on 20 September 2022. The study is retrospectively registered.

Rapid Antibiotic Susceptibility Testing of Gram-Negative Bacteria Directly from Urine Samples of UTI Patients Using MALDI-TOF MS.

Urinary tract infections (UTIs) are one of the most common human infections and are most often caused by Gram-negative bacteria such as Escherichia coli. In view of the increasing number of antibiotic-resistant isolates, rapidly initiating effective antibiotic therapy is essential. Therefore, a faster antibiotic susceptibility test (AST) is desirable. The MALDI-TOF MS-based phenotypic antibiotic susceptibility test (MALDI AST) has been used in blood culture diagnostics to rapidly detect antibiotic susceptibility. This study demonstrates for the first time that MALDI AST can be used to rapidly determine antibiotic susceptibility in UTIs directly from patients' urine samples. MALDI-TOF MS enables the rapid identification and AST of Gram-negative UTIs within 4.5 h of receiving urine samples. Six urinary tract infection antibiotics, including ciprofloxacin, cotrimoxazole, fosfomycin, meropenem, cefuroxime, and nitrofurantoin, were analyzed and compared with conventional culture-based AST methods. A total of 105 urine samples from UTI patients contained bacterial isolates for MALDI AST. The combination of ID and AST by MALDI-TOF allowed us to interpret the result according to EUCAST guidelines. An overall agreement of 94.7% was found between MALDI AST and conventional AST for the urinary tract pathogens tested.

Handyfuge Microfluidic for On-Site Antibiotic Susceptibility Testing.

Low-cost, rapid, and accurate acquisition of minimum inhibitory concentrations (MICs) is key to limiting the development of antimicrobial resistance (AMR). Until now, conventional antibiotic susceptibility testing (AST) methods are typically time-consuming, high-cost, and labor-intensive, making them difficult to accomplish this task. Herein, an electricity-free, portable, and robust handyfuge microfluidic chip was developed for on-site AST, termed handyfuge-AST. With simply handheld centrifugation, the bacterial-antibiotic mixtures with accurate antibiotic concentration gradients could be generated in less than 5 min. The accurate MIC values of single antibiotics (including ampicillin, kanamycin, and chloramphenicol) or their combinations against Escherichia coli could be obtained within 5 h. To further meet the growing demands of point-of-care testing, we upgraded our handyfuge-AST with a pH-based colorimetric strategy, enabling naked eye recognition or intelligent recognition with a homemade mobile app. Through a comparative study of 60 clinical data (10 clinical samples corresponding to six commonly used antibiotics), the accurate MICs by handyfuge-AST with 100% categorical agreements were achieved compared to clinical standard methods (area under curves, AUCs = 1.00). The handyfuge-AST could be used as a low-cost, portable, and robust point-of-care device to rapidly obtain accurate MIC values, which significantly limit the progress of AMR.

Teicoplanin-Resistant Coagulase-Negative Staphylococci: Do the Current Susceptibility Testing Methods Reliably Detect This Elusive Phenotype?

Coagulase-negative staphylococci (CoNS), members of the skin commensal microbiota, are increasingly associated with local or systemic infections due to a shift in patient populations in recent decades. Subsequently, more CoNS strains have been subjected to antibiotic susceptibility testing (AST), thus leading to the increased detection of teicoplanin resistance. However, data concerning teicoplanin resistance among CoNS strains remain limited, heterogeneous, and inconclusive. We collected 162 consecutive CoNS strains identified using Vitek-2 as teicoplanin-resistant and tested them with a range of AST methods. The results of standard and high inoculum broth microdilution (sBMD; hBMD), agar dilution (AD) after 24 h and 48 h incubation, standard and macrogradient diffusion strip (sGDT, MET), screening agar, and disc diffusion were compared to assess their robustness and to establish a diagnostic algorithm to detect teicoplanin resistance. sBMD was used as the reference method, and the lowest number of strains were teicoplanin-resistant using this method. sGDT and disc diffusion generated similar results to sBMD. Compared with sBMD, AD-24 h generated the lowest number of false teicoplanin-resistant strains, followed by hBMD, AD-48 h, and Vitek-2. sGDT, a fast, easy, affordable method in diagnostic settings, generated the highest rate of false teicoplanin-susceptible strains. Vitek-2 testing produced the highest number of teicoplanin-resistant strains. Only in two strains was the initial Vitek-2 teicoplanin resistance confirmed using five other AST methods. In conclusion, the different antibiotic susceptibility testing methods generated inconsistent, inconclusive, and discrepant results, thus making it difficult to establish a diagnostic algorithm for suspected teicoplanin resistance. Teicoplanin testing proved to be challenging and easily influenced by technical factors. This study aimed not only to raise awareness of teicoplanin resistance testing but also of the need for future studies focusing on the clinical efficacy of teicoplanin in relation to its susceptibility results.