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Introduction. Sepsis rates are increasing, with Gram-negative organisms representing a large proportion of bloodstream infections. Rapid antibiotic administration, alongside diagnostic investigations, is required for the effective management of these patients. Gap statement. Current diagnostics take ~48 h for a final report; therefore, rapid diagnostics are required. Aim. This study investigated a novel antibiotic sensitivity method, the scattered light integrating collector (SLIC), combined with a rapid identification method using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) technology to determine if an accurate identification and susceptibility result can be provided within 4 h of a positive blood culture report. Methodology. A total of 47 blood cultures containing Gram-negative bacteria from 46 patients were processed using the MALDI-TOF Biotyper Sepsityper for identification directly from the blood and the SLIC instrument for susceptibility testing. All organisms were also tested using the current standard workflow used in the host laboratory. Categorical agreement (CA), major errors (MaEs) and very major errors (VMEs) were determined. Results. SLIC produced susceptibility results with a 71.9% CA, 30.6% MaE and 17.5% VME. The median difference in time to the final result was 44.14 (43 : 05-45 : 15) h earlier compared to the current method. Conclusion. We conclude that SLIC was unable to consistently provide sufficiently accurate antibiotic susceptibility results compared to the current standard method.

The reduction of antimicrobial susceptibility testing (AST) time-to-result is a central need, especially in sepsis treatment. The current automated rapid ASTs are still too expensive for many laboratories. We aimed to evaluate three pre-treatment methods for a same-day inoculation on both automated AST platforms available in our laboratory. We tested 100 Enterobacterales or staphylococci positive bottles. We obtained good results with the different methods and instruments. In particular, Vitek-2 showed good performances with Enterobacterales AST when inoculated with bacterial pellet (96.6% categorical agreement - CA-, 93.3% essential agreement - EA). Also short-term incubation colonies for staphylococci AST had acceptable CA (94.2%), even if with 77.5% EA. MicroScan system for staphylococci AST with both short-term incubation and direct blood inoculation reached >95% CA, but 92.5% and 83.6% EA, respectively. On the other hand, Enterobacterales AST showed optimal performances only with bacterial pellet inoculation (97.6% CA). In fact, direct blood inoculation showed not acceptable parameters for several molecules. Both systems allow a 24-h reduction in time-to-result, by using the same instruments of routine activity after rapid and cheap pre-treatments., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier B.V.)

Urinary tract infections (UTIs) represent the most prevalent type of outpatient infection, with significant adverse health and economic burdens. Current culture-based antibiotic susceptibility testing can take up to 72 h resulting in ineffective prescription of broad-spectrum antibiotics, poor clinical outcomes and development of further antibiotic resistance. We report an electrochemical lab-on-a-chip (LOC) for testing samples against seven clinically-relevant antibiotics. The LOC contained eight chambers, each housing an antibiotic-loaded hydrogel (cephalexin, ceftriaxone, colistin, gentamicin, piperacillin, trimethoprim, vancomycin) or antibiotic-free control, alongside a resazurin bulk-modified screen-printed electrode for electrochemical detection of metabolically active bacteria using differential pulse voltammetry. Antibiotic susceptibility in simulated UTI samples or donated human urine with either Escherichia coli or Klebsiella pneumoniae could be established within 85 min. Incorporating electrochemical detection onto a LOC provides an inexpensive, simple method for the sensitive determination of antibiotic susceptibility that is significantly faster than using a culture-based approach., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: This study compared the results of the new Sysmex PA-100 AST System, a point-of-care analyser, with routine microbiology for the detection of urinary tract infections (UTI) and performance of antimicrobial susceptibility tests (AST) directly from urine., Methods: Native urine samples from 278 female patients with suspected uncomplicated UTI were tested in the Sysmex PA-100 and with reference methods of routine microbiology: urine culture for bacteriuria and disc diffusion for AST., Results: The analyser delivered bacteriuria results in 15 min and AST results within 45 min. Sensitivity and specificity for detection of microbiologically confirmed bacteriuria were 84.0% (89/106; 95% CI: 75.6-90.4%) and 99.4% (155/156; 95% CI: 96.5-100%), respectively, for bacterial species within the analyser specifications. These are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus, which are common species causing uncomplicated UTI. Overall categorical agreement (OCA) for AST results for the five antimicrobials tested in the Sysmex PA-100 (amoxicillin/clavulanic acid, ciprofloxacin, fosfomycin, nitrofurantoin and trimethoprim) ranged from 85.4% (70/82; 95%CI: 75.9-92.2%) for ciprofloxacin to 96.4% (81/84; 95% CI: 89.9-99.3%) for trimethoprim. The Sysmex PA-100 provided an optimal treatment recommendation in 218/278 cases (78.4%), against 162/278 (58.3%) of clinical decisions., Conclusion: This first clinical evaluation of the Sysmex PA-100 in a near-patient setting demonstrated that the analyser delivers phenotypic AST results within 45 min, which could enable rapid initiation of the correct targeted treatment with no further adjustment needed. The Sysmex PA-100 has the potential to significantly reduce ineffective or unnecessary antibiotic prescription in patients with UTI symptoms., (© 2024. The Author(s).)

Treatment assessment and patient outcome for sepsis depend predominantly on the timely administration of appropriate antibiotics 1-3 . However, the clinical protocols used to stratify and select patient-specific optimal therapy are extremely slow 4 . In particular, the major hurdle in performing rapid antimicrobial susceptibility testing (AST) remains in the lengthy blood culture procedure, which has long been considered unavoidable due to the limited number of pathogens present in the patient's blood. Here we describe an ultra-rapid AST method that bypasses the need for traditional blood culture, thereby demonstrating potential to reduce the turnaround time of reporting drug susceptibility profiles by more than 40-60 h compared with hospital AST workflows. Introducing a synthetic beta-2-glycoprotein I peptide, a broad range of microbial pathogens are selectively recovered from whole blood, subjected to species identification or instantly proliferated and phenotypically evaluated for various drug conditions using a low-inoculum AST chip. The platform was clinically evaluated by the enrolment of 190 hospitalized patients suspected of having infection, achieving 100% match in species identification. Among the eight positive cases, six clinical isolates were retrospectively tested for AST showing an overall categorical agreement of 94.90% with an average theoretical turnaround time of 13 ± 2.53 h starting from initial blood processing., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The escalation of antimicrobial resistance (AMR) due to the excessive and inappropriate use of antimicrobials has prompted the urgent need for more rapid and effective antimicrobial susceptibility testing (AST) methods. Conventional AST techniques often take 16-24 h, leading to empirical prescription practices and the potential emergence of AMR. The study aimed to develop a rapid disk diffusion (RDD) method utilizing laser speckle formation (LSF) technology to expedite AST results. The study aimed to evaluate the performance of LSF technology in determining antimicrobial susceptibility. In this study, preclinical and clinical settings were established to compare the LSF technology with conventional disk diffusion (DD) methods to measure the inhibition zones. Preclinical experiments with different bacterial strains demonstrated more than 70% categorical agreement (CA) against most antimicrobials. Further, clinical experiments with multiple strains and antibiotics revealed CA ranging from 40 to 79%, while major and minor discrepancies were observed around 30% and 11%, respectively. These observations revealed high concordance between RDD and DD for multiple antimicrobials in multiple species. The results underscore the potential of RDD-based LSF technology for hastening AST procedures. The current study is marked by a unique equipment setup and analysis approach. Collectively, the suggested laser-based RDD showed greater potential than previously developed comparable methods. The proposed method and design have a higher application potential than formerly developed similar technologies. Together, the study contributes to the ongoing development of rapid AST methods., (© 2024. The Author(s).)

Background: Tuberculosis (TB) is caused due to the infection of Mycobacterium tuberculosis (MTB) and it can infect the various parts of the human body. The disease is highly prevalent and is the second most common cause of death worldwide after COVID-19. Apart from sputum specimen, it is exceedingly difficult to diagnose due to its paucibacillary nature. The current study was intended to evaluate the accuracy of Smart Sure™ MTB and multidrug-resistant-TB (MDR-TB) kits (Genetix Biotech Asia Pvt. Ltd., India) with Xpert ultra and Mycobacterium growth indicator tube (MGIT) culture on nonsputum specimens from TB suspects., Methods: A total of 205 nonsputum specimens were received between October 2023 and May 2024 at Intermediate Reference Laboratory, Department of Medicine, All India Institute of Medical Sciences, New Delhi, India. Xpert ultra and Smart Sure™ MTB and MDR-TB tests were done directly on samples. However, processed specimens were used for MGIT culture and drug-susceptibility testing (DST). Invalid and MGIT contaminated specimens were excluded from the final calculation., Results: Overall, sensitivity and specificity of Smart Sure™ MTB screening kit was 71.59% and 98.28%, respectively, with Xpert ultra and 68.35% and 90.83%, respectively, with MGIT culture. While comparing with both Xpert ultra and MGIT-DST to detect rifampicin (RIF) resistant, Smart Sure™ MDR-TB kits showed sensitivity of 75.0% and 100% of specificity. However, for isoniazid (INH) resistance, Smart Sure™ MDR-TB kits showed 100% of sensitivity and specificity with MGIT-DST., Conclusion: For the detection of MTB and its drug-resistance patterns (RIF and INH) in the specimens other than sputum, Smart Sure™ MTB and MDR-TB kits could play a vital role in TB endemic countries. While comparing the set-ups and skilled staffs, it required almost same as compared with previously approved WHO diagnostics used in resource-limited countries., (Copyright © 2024 Copyright: © 2024 International Journal of Mycobacteriology.)

Background: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA., Results: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively., Conclusion: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship., (© 2024. The Author(s).)

Antibiotic susceptibility testing (AST) is a routine procedure in diagnostic laboratories to determine pathogen resistance profiles toward antibiotics. The need for fast and accurate resistance results is rapidly increasing with a global rise in pathogen antibiotic resistance over the past years. Microfluidic technologies can enable AST with lower volumes, lower cell numbers, and a reduction in the sample-to-result time compared to state-of-the-art systems. We present a protocol to perform AST on a miniaturized nanoliter chamber array platform. The chambers are filled with antibiotic compounds and oxygen-sensing nanoprobes that serve as a viability indicator. The growth of bacterial cells in the presence of different concentrations of antibiotics is monitored; living cells consume oxygen, which can be observed as an increase of a luminesce signal within the growth chambers. Here, we demonstrate the technique using a quality control Escherichia coli strain, ATCC 35218. The AST requires 20 μL of a diluted bacterial suspension (OD 600  = 0.02) and provides resistance profiles about 2-3 h after the inoculation. The microfluidic method can be adapted to other aerobic pathogens and is of particular interest for slow-growing strains., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Success in the global fight against antimicrobial resistance (AMR) is likely to improve if surveillance can be performed on an epidemiological scale. An approach based on agars with incorporated antimicrobials has enormous potential to achieve this. However, there is a need to identify the combinations of selective agars and key antimicrobials yielding the most accurate counts of susceptible and resistant organisms. A series of experiments involving 1,202 plates identified the best candidate combinations from six commercially available agars and five antimicrobials, using 18 Escherichia coli strains as either pure cultures or inocula-spiked feces. The effects of various design factors on colony counts were analyzed in generalized linear models. Without antimicrobials, Brilliance E. coli and CHROMagar ECC agars yielded 28.9% and 23.5% more colonies, respectively, than MacConkey agar. The order of superiority of agars remained unchanged when fecal samples with or without spiking of resistant E. coli strains were inoculated onto agars with or without specific antimicrobials. When antimicrobials were incorporated at various concentrations, it was revealed that ampicillin, tetracycline, and ciprofloxacin were suitable for incorporation into Brilliance and CHROMagar agars at all defined concentrations. Gentamicin was suitable for incorporation only at 8 and 16 μg/ml, while ceftiofur was suitable only at 1 μg/ml. CHROMagar extended-spectrum β-lactamase (ESBL) agar supported growth of a wider diversity of extended-spectrum-cephalosporin-resistant E. coli strains. The findings demonstrate the potential for agars with incorporated antimicrobials to be combined with laboratory-based robotics to deliver AMR surveillance on a vast scale with greater sensitivity of detection and strategic relevance. IMPORTANCE Established models of surveillance for AMR in livestock typically have a low sampling intensity, which creates a tremendous barrier to understanding the variation of resistance among animal and food enterprises. However, developments in laboratory robotics now make it possible to rapidly and affordably process large volumes of samples. Combined with modern selective agars incorporating antimicrobials, this forms the basis of a novel surveillance process for identifying resistant bacteria by chromogenic reactions, including accurately detecting and quantifying the presence of bacteria even when they are present at low concentrations. Because Escherichia coli is a widely preferred indicator bacterium for AMR surveillance, this study identifies the optimal selective agar for quantifying resistant E. coli strains by assessing the growth performance on agars with antimicrobials. The findings are the first step toward exploiting laboratory robotics in an up-scaled approach to AMR surveillance in livestock, with wider adaptations in food, clinical microbiology, and public health.

Antimicrobial resistance surveillance data is lacking from many resource-limited settings mainly due to limited laboratory testing. Novel culture systems may address some of the limitations of conventional culture media and expand the availability of microbiology services. The aims of this study were to evaluate the performance of InTray COLOREX Screen/ESBL and Compact Dry for the detection of uropathogens and of extended-spectrum beta-lactamase (ESBL)-producing organisms from urine samples. Urines samples were collected from patients presenting with symptoms of urinary tract infection to primary care clinics in Harare. Performance of the InTray COLOREX Screen, ESBL and Compact Dry chromogenic media were compared to the reference of culture using Brilliance UTI agar and conventional antimicrobial susceptibility testing. A total of 414 samples were included in the analysis. Of the included samples, 98 were positive on Brilliance UTI agar and 83 grew Enterobacterales. The sensitivities and specificities for Enterobacterales were 89.2% (95% CI 80.4-94.9) and 98.2% (95% CI 96.1-99.3) for InTray Screen and 95.2% (95% CI 88.1-98.7) and 99.7% (95% CI 98.3-100) for Compact Dry. Extended-spectrum beta-lactamases were present in 22 isolates from the Brilliance UTI agar. The sensitivity of the InTray COLOREX ESBL culture plates for the detection of ESBL-producing organisms was 95.5% (95% CI 77.2-99.9) and specificity was 99.5% (95% CI 98.2-99.9%). Our findings show good performance of the novel culture systems for the detection of uropathogens and ESBL-producing organisms. Both systems have several advantages over conventional media and have the potential to expand and decentralize laboratory testing., (© 2021. The Author(s).)

The industrial version of SuperCAZ/AVI® medium developed for screening CAZ/AVI resistant Gram-negative isolates has been evaluated here using a collection of 87 well-characterized clinical isolates of worldwide origin. In addition, testing was performed by spiking stools with a series of resistant and susceptible isolates. In those conditions, the SuperCAZ/AVI® medium exhibited a sensitivity and specificity of 100 %, down to the lower limit of detection of 10 1 to 10 2 CFU/ml. The SuperCAZ/AVI® medium is a sensitive and specific screening medium for detection of CZA-resistant bacteria regardless of their resistance mechanisms., Competing Interests: Declaration of Competing Interest All authors have nothing to declare., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gepotidacin is a triazaacenaphthylene antibiotic with activity against Neisseria gonorrhoeae including strains resistant to current agents. We tested 145 N. gonorrhoeae isolates by agar dilution according to Gonococcal Isolate Surveillance Program and Clinical and Laboratory Standards Institute methodologies. Gepotidacin demonstrated a minimum inhibitory concentration (MIC) 50 of 0.25 μg/mL and a MIC 90 of 0.5 μg/mL (highest gepotidacin MIC was 1 μg/mL) against the 145 N. gonorrhoeae isolates tested. We also assessed the impact of test variables on antimicrobial susceptibility test results for gepotidacin, ciprofloxacin, and ceftriaxone against 10 N. gonorrhoeae isolates. Media type had the biggest effect but wasn't specific to gepotidacin. Gepotidacin MICs were also affected by inoculum, pH, and 10% CO 2 . These in vitro data indicate that further study of gepotidacin is warranted for potential use in treating gonorrhea infections and highlight the importance of controlling for media type, inoculum, CO 2 , and pH when performing MIC testing with gepotidacin., (Copyright © 2021 GlaxoSmithKline plc. Published by Elsevier Inc. All rights reserved.)

Severe, invasive Streptococcus pyogenes (Strep A) infections result in greater than 500,000 deaths annually. First line treatment for such infections is benzylpenicillin, often with the addition of clindamycin, but treatment failure can occur with this regimen. This failure has been partially attributed to the inoculum effect, which presents as reduced antibiotic susceptibility during high bacterial density and plateau-phase growth. Hollow fibre infection models (HFIM) have been proposed as an in vitro alternative to in vivo research to study these effects. To re-evaluate the inoculum effect for benzylpenicillin, clindamycin, linezolid, and trimethoprim-sulfamethoxazole using a Strep A HFIM. Differential antibiotic susceptibility of Strep A was measured in a HFIM starting from low- and high-density inocula with an average difference in bacterial concentration of 56-fold. Dynamic antibiotic concentrations were delivered over 48 h to simulate in vivo human pharmacokinetics in an in vitro model. Differences in antibiotic susceptibility were measured by plate count of colony-forming units over time. Inoculum effects were seen in benzylpenicillin and linezolid at 24 h, and benzylpenicillin, linezolid, and clindamycin at 48 h. The effect size was greatest for continuously infused benzylpenicillin at 48 h with a log 10 -fold difference of 4.02 between groups. No inoculum effect was seen in trimethoprim-sulfamethoxazole, with a maximal log 10 -fold difference of 0.40. Inoculum effects were seen using benzylpenicillin, linezolid, and clindamycin, which may predict reduced clinical efficacy following treatment delay. The model has proven robust and largely in agreeance with published data, recommending it for further Strep A study., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Delaying effective antibiotic therapy is a major cause of sepsis-associated mortality. The EUCAST rapid antibiotic susceptibility test (RAST) is performed from positive blood cultures to provide rapid results. Disc diffusion tests inoculated with positive blood culture broth are read at 4, 6, and 8 h and interpreted against species and time-specific criteria. Potential problems are the possibility of missing specific reading times for tests and slower growth in incubators that are frequently opened. The current study aimed to assess if digital visualization by the BD Kiestra™ total laboratory automation system is suitable for reading RASTs by capturing images at the correct times and retaining them for review. Utilizing the Kiestra™ InoqulA, 100 μl of positive blood culture broth was lawn-inoculated onto Mueller-Hinton agar and incubated at 35 °C for automated digital zone measurement at 4, 6, and 8 h. Aliquots from 135 positive blood cultures were tested against EUCAST-recommended and other drugs and assessed for readability of digital images. Microdilution MICs were determined in parallel to RASTs. All isolates except 7/10 enterococci yielded images of suitable quality for zone measurement. Of the 641 digitally read tests for other organisms, 207 (32.3%) were readable in 4 h, 555 (86.6%) in 6 h, and 641 (100%) in 8 h. For tests included in EUCAST criteria, 92.1% provided categorical agreement with microdilution MICs. Digital image reading of RASTs is a potentially viable, inexpensive tool for providing rapid susceptibility results which can help reduce sepsis-associated mortality., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To compare the performance of agar dilution and broth microdilution by commercial and in-house prepared plates for the Bacteroides fragilis group. The cost analysis was performed to demonstrate that in-house prepared BMD plates were a suitable alternative to agar dilution given the high cost and low feasibility of incorporating commercial BMD plates in routine, particularly in the tertiary care institutes of many low- and middle-income countries., Methods: Thirty B. fragilis group isolates were tested against six antibiotics, frequently used as empirical therapy for anaerobic infections including metronidazole, clindamycin, imipenem, piperacillin-tazobactam, cefoxitin, and chloramphenicol. The running consumable expenditure for all methodologies was calculated., Results: The results demonstrated essential and categorical agreement of >90% for all antibiotics except cefoxitin, which showed <90% categorical agreement. No major or very major errors were observed. We observed a high agreement and strong concordance for MIC values between both methods and inter-rate reliability of >0.9 by Cohen's kappa analysis, indicating almost perfect agreement between both methods using either of the plates. In contrast to agar dilution, a 20.5 fold cost reduction was seen in BMD using in-house plates and a 5.8 fold reduction using commercial plates to test a single isolate. However, when testing 30 isolates concurrently the cost significantly increased for commercial BMD plates by 8.4 folds, and only 1.03 fold cost reduction was seen with in-house BMD plates., Conclusion: BMD gives comparable results to agar dilution and can be considered a method of choice to test a small number of samples. The technique is an economical option when plates are standardized in-house and could be employed for susceptibility testing of the B. fragilis group., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

We sought to evaluate the MICRONAUT MIC-Strip colistin (MMS) assay, a commercial broth microdilution (BMD) panel, using 273 carbapenem-resistant Acinetobacter baumannii and Enterobacterales isolates. BMD was used as gold standard. MMS had 98.5% sensitivity and 99.5% specificity. All 37 isolates with MICs close to the breakpoint exhibited complete agreement., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses., (© 2021. The Author(s).)

Slow growing, mucoid isolates of Pseudomonas aeruginosa require adaptation of the protocol used for automated antimicrobial susceptibility testing (AST). In the present study we used a water soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) in combination with menadione for possibly improving AST of slow growing and biofilm-forming P. aeruginosa isolates from cystic fibrosis (CF) patients. WST-1 and menadione addition ensures sensitive detection of microbial growth increase in the presence of antibiotics that may remain undetected with the automated VITEK® 2 method. We observed that 32.8% of P. aeruginosa isolates from CF and bronchiectasis patients produced an elevated absorbance signal intensity thereby increasing the sensitivity while maintaining the accuracy of VITEK 2. Our study merits future investigation with other slow growing pathogenic bacterial species., (Copyright © 2021 bioMérieux. Published by Elsevier B.V. All rights reserved.)

Rapid detection of carbapenemases as a cause of resistance is beneficial for infection control and antimicrobial therapy. The BD Phoenix NMIC-502 panel and CPO detect test identifies presence of carbapenemases in Enterobacterales such as Klebsiella pneumoniae and assigns them to Ambler classes. To evaluate the performance of the CPO detect panel, we employed a European collection of 1222 K. pneumoniae including carbapenem non-susceptible and susceptible clinical isolates from 26 countries, for which draft genomes were available after Illumina sequencing and the presence of carbapenemase genes had been identified by ARIBA gene calling. The CPO panel detected 488 out of 494 carbapenemase-encoding isolates as positive and six as negative. One-hundred and two isolates were tested positive for carbapenemase in the absence of any carbapenemase gene. The CPO panel identified 229 out of 230 KPC-positive isolates as carbapenemase producing and classified 62 of these as class A enzyme. Similarly, the CPO panel correctly specified 167 of 182 as class D. Regarding metallo-beta-lactamases, the CPO panel assigned 78 of 90 MBL positive isolates to class B enzymes. The sensitivity of the CPO panel in detecting carbapenemase activity was 99.5%, 97.7% and 98.3% for class A, B and D enzymes, respectively. The sensitivity in assignation to Ambler class A, B and D was 27%, 86% and 91%, respectively. An overall sensitivity of 98.8% and specificity of 86% in unclassified detection of carbapenemases was observed, with frequent false positive detection of carbapenemase producing organisms, thus rendering further confirmatory tests necessary.

Otitis media (OM), known as a middle ear infection, is the leading cause of antibiotic prescriptions for children. With wide-spread use of antibiotics in OM, resistance to antibiotics continues to decrease the efficacy of the treatment. Furthermore, as the presence of a middle ear biofilm has contributed to this reduced susceptibility to antimicrobials, effective interventions are necessary. A miniaturized 3D-printed microplasma jet array has been developed to inactivate Pseudomonas aeruginosa, a common bacterial strain associated with OM. The experiments demonstrate the disruption of planktonic and biofilm P. aeruginosa by long-lived molecular species generated by microplasma, as well as the synergy of combining microplasma treatment with antibiotic therapy. In addition, a middle ear phantom model was developed with an excised rat eardrum to investigate the antimicrobial effects of microplasma on bacteria located behind the eardrum, as in a patient-relevant setup. These results suggest the potential for microplasma as a new treatment paradigm for OM.

Optimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST's RAST on primarily sterile body fluids in blood culture bottles with total lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197 Staphylococcus aureus, 91 Enterococcus spp., 38 Escherichia coli, 11 Klebsiella pneumoniae and 8 Pseudomonas aeruginosa were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for P. aeruginosa and meropenem and ciprofloxacin, 1/9 (11.1%) for K. pneumoniae and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for Enterococcus spp. and vancomycin and ampicillin, respectively. Minor errors for P. aeruginosa and meropenem (1/8; 12.8%) and for E. coli and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.

Background: Rapid and accurate microbial identification and antimicrobial susceptibility testing (AST) are essential for timely use of appropriate antimicrobial agents for bloodstream infection. To shorten the time for isolating colonies from the positive blood culture, various preparation methods for direct identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system were developed. Here, we evaluated the SepsiPrep kit (ASTA Corp.) for direct identification of microorganisms and AST from positive blood cultures using MicroIDSys Elite MALDI-TOF MS system (ASTA Corp.) and VITEK-2 system (bioMérieux)., Methods: For direct identification, a total of 124 prospective monomicrobial positive blood culture bottles were included. For direct identification, the pellet was prepared by centrifugation and washing twice. For direct AST, the pellet was suspended in 0.45% saline and adjusted to McFarland 0.5. The results from the direct identification and AST using MicroIDSys Elite and VITEK-2 system were compared to those from the conventional method performed with pure colony subcultured on agar plate., Results: Compared to the conventional method using pure colony, correct direct identification rate was 96.5% and 98.5% for 57 gram-positive isolates and 67 gram-negative isolates, respectively. For direct AST, among the 55 gram-positive isolates, the categorical agreement (CA) for staphylococci, streptococci, and enterococci was 96.7%, 98.4%, and 94.1%, respectively. For 66 gram-negative isolates, the CA for Enterobacterales and non-fermentative gram-negative rods was 99.0% and 96.6%, respectively., Conclusions: The SepsiPrep kit was easy to use combined with MicroIDSys Elite and VITEK-2 system and also, the correct identification and AST rate were very high., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Fast determination of antimicrobial agents' effectiveness (susceptibility/resistance pattern) is an essential diagnostic step for treating bacterial infections and stopping world-wide outbreaks. Here, we report an egg-like multivolume microchamber-based microfluidic (EL-MVM 2 ) platform, which is used to produce a wide range of gradient-based antibiotic concentrations quickly (∼10 min). The EL-MVM 2 platform works based upon testing a bacterial suspension in multivolume microchambers (microchamber sizes that range from a volume of 12.56 to 153.86 nL). Antibiotic molecules from a stock solution diffuse into the microchambers of various volumes at the same loading rate, leading to different concentrations among the microchambers. Therefore, we can quickly and easily produce a robust antibiotic gradient-based concentration profile. The EL-MVM 2 platform's diffusion (loading) pattern was investigated for different antibiotic drugs using both computational fluid dynamics simulations and experimental approaches. With an easy-to-follow protocol for sample loading and operation, the EL-MVM 2 platform was also found to be of high precision with respect to predicting the susceptibility/resistance outcome (>97%; surpassing the FDA-approval criterion for technology-based antimicrobial susceptibility testing instruments). These features indicate that the EL-MVM 2 is an effective, time-saving, and precise alternative to conventional antibiotic susceptibility testing platforms currently being used in clinical diagnostics and point-of-care settings.

Antimicrobial susceptibility testing (AST) is essential for detecting resistance in Pseudomonas aeruginosa and other bacterial pathogens. Here we evaluated the performance of broth microdilution (BMD) panels created using a semi-automated liquid handler, the D300e Digital Dispenser (Tecan Group Ltd., CH) that relies on inkjet printing technology. Microtitre panels (96-well) containing nine twofold dilutions of 12 antimicrobials from five classes (β-lactams, β-lactam/β-lactamase inhibitors, aminoglycosides, fluoroquinolones, polymyxins) were prepared in parallel using the D300e Digital Dispenser and standard methods described by CLSI/ISO. To assess performance, panels were challenged with three well characterized quality control organisms and 100 clinical P. aeruginosa isolates. Traditional agreement and error measures were used for evaluation. Essential (EA) and categorical (CA) agreements were 92.7% and 98.0% respectively for P. aeruginosa isolates with evaluable on-scale results. The majority of minor errors that fell outside acceptable EA parameters (≥ ± 1 dilution, 1.9%) were seen with aztreonam (5%) and ceftazidime (4%), however all antimicrobials displayed acceptable performance in this situation. Differences in MIC were often log 2 dilution lower for D300e dispensed panels. Major and very major errors were noted for aztreonam (2.6%) and cefepime (1.7%) respectively. The variable performance of D300e panels suggests that further testing is required to confirm their diagnostic utility for P. aeruginosa.

Introduction: This study aimed to evaluate the etiology of lower respiratory tract infections (LRTIs) and their antibiotic resistance., Methodology: Bacterial culture results of LRT samples from 17 hospitals between 2016-2019 were included in the study. All isolates were identified and AST were performed by automated microbiology systems. AST was performed according to EUCAST., Results: Non-duplicate 30,051 (26,890 HA and 3156 CA) isolates detected as causative pathogen. LRTIs are caused by 85.1% Gram-negative bacterial pathogens and 14.9% Gram-positive. The most common isolates among HA pathogens were Acinetobacter spp. (27.4%), P.aeruginosa (22.2%), K.pneumoniae (17.9%); among CA pathogen S.pneumoniae (19.9%), P. aeruginosa (18.9%), H.influenzae (14.6%). ESBL rate was 62.5% in K.penumoniae; 53.1% in E.coli; 19.1% in Klebsiella spp; 13.9% in Enterobacter spp.; 8.6% in Proteus spp.; 6.3% in Citrobacter spp.; and 4.3% in Serratia spp. Resistance rates to carbapenems and colistin were 92.8% and 12.8% in A baumannii, 39.8% and 7.5% in P.aeruginosa, 47.3% and 18.5% in K.penumoniae. Among staphylococci, 27.3% of S. aureus and 82.4% of CoNS were methicillin resistant. 7.6% of E.faecium and 0.9% of E.faecalis were vancomycin resistant.  Linezolid resistant S. aureus, CoNS, E.faecalis and E.faecium rates were 0.3%, 2.9%, 0.0% and 4.6%. Inducible clindamycin resistant rate was 17.2% in S. aureus 38.2% in CoNS. Non-susceptible S.pneumoniae isolate rate to penicillin was 37.0%. 6.5% of S.maltophilia and 4.4% of B.cepacia isolates were resistant to trimethoprim/sulfamethoxazole., Conclusions: Antibiotic resistance was mainly observed among A.baumannii and K.pneumoniae and continuous surveillance of antimicrobial resistance patterns in the management of LRTIs is important., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Aylin Uskudar Guclu, Aylin Altay Kocak, Mehtap Akcil Ok, Bulent Tutluoglu, Ahmet Celal Basustaoglu.)

Antibiotic resistance has become a serious threat to food safety and public health globally. Therefore, the development of a sensitive, quick, and simple method for antibiotic susceptibility testing is an urgent and crucial need. A novel concentration gradient microfluidic chip was designed in this work to generate antibiotic concentration gradient, culture bacteria, and produce fluorescence emission. An in-house-assembled fluorescence detection platform was constructed, and experiments were conducted to verify the linearity of the generated concentration gradient, explore the appropriate incubation time and flow rate for the microfluidic chip, and study the effect of long-term acid-based food processing on antibiotic susceptibility testing. Experimental results show that the concentration gradient generated by the microfluidic chip exhibited good linearity, stability, and controllability. The appropriate flow rate and incubation time for the microfluidic chip were 2 μL/min and 5 h, respectively. The use of this microfluidic chip for testing antibiotic resistance of Salmonella to ofloxacin and ampicillin generated results that were completely consistent with test results obtained using the gold-standard method. Furthermore, Salmonella showed greater sensitivity to antibiotics under strong acid conditions, confirming the potential influence of acid-based food processing on antibiotic susceptibility testing of real samples. The designed microfluidic chip provides a high-throughput, sensitive, and rapid antibiotic susceptibility testing method that combines the microfluidic chip and the fluorescence detection platform. The application of this method would facilitate determination of antibiotic-resistant bacterial strains for clinicians and researchers, and enable monitoring of changes in bacterial resistance during food processing.

The problem of antimicrobial resistance (AMR) is becoming increasingly serious. Bacteria producing extended spectrum beta-lactamase (ESBL), which can hydrolyze beta-lactam antibiotics, are among the most important drug resistant bacteria. Rapid AMR analysis methods are essential for identifying antibiotic resistant bacteria, which is of significant positive value to the clinical therapy of infectious disease. We developed a platform which integrates a sandwich microfluidic filter device with electrospray ionization mass spectrometry (ESI-MS). Bacterial cells were loaded in the sandwich microfluidic chip and antibiotic drugs were injected to pass through the blocked bacterial cells. By online ESI-MS analysis of the antibiotic drugs and their hydrolysis products, the AMR of the bacteria can be assessed within 30 minutes. Four Escherichia coli strains, namely two ESBL-positive and two ESBL-negative, were successfully discriminated using ampicillin and the third generation cephalosporin ceftriaxone. Considering the simplicity and high efficiency of the assay, the microfluidic chip integrated online ESI-MS system is promising in the rapid clinical diagnosis of ESBL-producing bacteria.

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca 2+ ]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 10 5 cells are analysed one-by-one with microfluidic impedance cytometry for 2-3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species.

The first objective of this study was to determine the GenoType NTM-DR assay performance for subspecies identification in Mycobacterium abscessus complex isolates. The second objective was to evaluate the GenoType NTM-DR assay ability to detect clarithromycin and amikacin resistance in M. abscessus complex isolates compared with drug susceptibility testing (DST) and PCR sequencing of the erm(41), rrl and rrs genes. The concordance between the GenoType NTM-DR and MLST results concerning subspecies identification was 100%. The wild type and mutated alleles of the rrl and rrs genes were detected by the GenoType NTM-DR assay and PCR sequencing with 100% (115/115) agreement. Similarly, 100% concordance between GenoType NTM-DR and DST was observed for clarithromycin and amikacin testing. Sensitivity for the detection of clarithromycin and amikacin resistance was 100%. The GenoType NTM-DR assay provides a robust and complementary tool to the gold standard methods (MLST and broth microdilution) for subspecies identification and drug resistance detection., Competing Interests: The authors have declared that no competing interests exist.

Antibiotic combination therapies are important for the efficient treatment of many types of infections, including those caused by antibiotic-resistant pathogens. Combination treatment strategies are typically used under the assumption that synergies are conserved across species and strains, even though recent results show that the combined treatment effect is determined by specific drug-strain interactions that can vary extensively and unpredictably, both between and within bacterial species. To address this problem, we present a new method in which antibiotic synergy is rapidly quantified on a case-by-case basis, allowing for improved combination therapy. The novel CombiANT methodology consists of a 3D-printed agar plate insert that produces defined diffusion landscapes of 3 antibiotics, permitting synergy quantification between all 3 antibiotic pairs with a single test. Automated image analysis yields fractional inhibitory concentration indices (FICis) with high accuracy and precision. A technical validation with 3 major pathogens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showed equivalent performance to checkerboard methodology, with the advantage of strongly reduced assay complexity and costs for CombiANT. A synergy screening of 10 antibiotic combinations for 12 E. coli urinary tract infection (UTI) clinical isolates illustrates the need for refined combination treatment strategies. For example, combinations of trimethoprim (TMP) + nitrofurantoin (NIT) and TMP + mecillinam (MEC) showed synergy, but only for certain individual isolates, whereas MEC + NIT combinations showed antagonistic interactions across all tested strains. These data suggest that the CombiANT methodology could allow personalized clinical synergy testing and large-scale screening. We anticipate that CombiANT will greatly facilitate clinical and basic research of antibiotic synergy., Competing Interests: The authors of this manuscript have the following competing interests: A patent comprising the described method has been filed by the authors and is pending under number 640 SE 2050304-1.

Broth microdilution (BMD), the reference method to determine bacterial susceptibility to polymyxins, is a laborious and time-consuming technique. Policimbac® is a commercial test panel which uses lyophilized polymyxin B to determine the minimum inhibitory concentration for Gram-negative isolates. This study evaluated the performance of Policimbac® in comparison with BMD for 110 isolates. Although the Policimbac® presented a very low essential agreement, the categorical agreement with BMD was optimal. Policimbac® is an alternative approach to BMD for evaluating the susceptibility to polymyxin B.

Background: The spread of multi-drug resistant tuberculosis (MDR-TB) is a leading global public-health challenge. Because not all biological mechanisms of resistance are known, culture-based (phenotypic) drug-susceptibility testing (DST) provides important information that influences clinical decision-making. Current phenotypic tests typically require pre-culture to ensure bacterial loads are at a testable level (taking 2-4 weeks) followed by 10-14 days to confirm growth or lack thereof., Methods and Findings: We present a 2-step method to obtain DST results within 3 days of sample collection. The first involves selectively concentrating live mycobacterial cells present in relatively large volumes of sputum (~2-10mL) using commercially available magnetic-nanoparticles (MNPs) into smaller volumes, thereby bypassing the need for pre-culture. The second involves using microchannel Electrical Impedance Spectroscopy (m-EIS) to monitor multiple aliquots of small volumes (~10μL) of suspension containing mycobacterial cells, MNPs, and candidate-drugs to determine whether cells grow, die, or remain static under the conditions tested. m-EIS yields an estimate for the solution "bulk capacitance" (Cb), a parameter that is proportional to the number of live bacteria in suspension. We are thus able to detect cell death (bactericidal action of the drug) in addition to cell-growth. We demonstrate proof-of-principle using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of ~ 2000-10,000 CFU of mycobacteria were extracted from ~5mL of artificial sputum during the decontamination process with efficiencies of 84% -100%. Subsequently, suspensions containing ~105 CFU/mL of mycobacteria with 10 mg/mL of MNPs were monitored in the presence of bacteriostatic and bactericidal drugs at concentrations below, at, and above known MIC (Minimum Inhibitory Concentration) values. m-EIS data (ΔCb) showed data consistent with growth, death or stasis as expected and/or recorded using plate counts. Electrical signals of death were visible as early as 3 hours, and growth was seen in < 3 days for all samples, allowing us to perform DST in < 3 days., Conclusion: We demonstrated "proof of principle" that (a) live mycobacteria can be isolated from sputum using MNPs with high efficiency (almost all the bacteria that survive decontamination) and (b) that the efficacy of candidate drugs on the mycobacteria thus isolated (in suspensions containing MNPs) could be tested in real-time using m-EIS., Competing Interests: Shramik Sengupta owns equity interest in ImpeDx Diagnostics. ImpeDx has licensed technology from the University of Missouri. Shramik Sengupta, Sachidevi Puttaswamy and Roli Kargupta are co-inventors of this technology. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The point-of-care detection of pathogens in biological samples in resource-limited settings should be inexpensive, rapid, portable, simple and accurate. Here, we describe a custom-made fidget spinner that rapidly concentrates pathogens in 1-ml samples of undiluted urine by more than 100-fold for the on-device colorimetric detection of bacterial load and pathogen identification. In Tiruchirappalli, India, the device enabled the on-site detection of infection with the naked eye within 50 min in urine samples from 39 patients suspected of having a urinary tract infection. We also show that, in 30 clinical samples of urinary tract infection, the device can be used to perform an antimicrobial susceptibility test for the antimicrobial drugs ciprofloxacin and cefazolin within 120 min. The fidget spinner could be used in low-resource settings as an inexpensive handheld point-of-care device for the rapid concentration and detection of pathogens in urine samples.

Proper treatment of bloodstream infections requires rapid, early determination of appropriate antibiotic agents, emphasizing the need for more rapid drug susceptibility testing. The Drug Susceptibility Testing Microfluidic (DSTM) device represents a novel method in which a small amount of bacterial suspension is injected into the microchip-like device and cultured for 3 h. However, it remains unknown whether the DSTM method can directly determine antibiotic susceptibilities from positive blood cultures. Here, we developed a new approach to directly assess drug susceptibility, using the DSTM method for positive blood cultures. We compare the utility and accuracy of DSTM with those of conventional susceptibility testing methods. Fifty positive blood cultures identified as gram-negative bacilli were used herein. The outcomes of drug susceptibility and resistance assays for positive blood cultures were compared to those of conventional susceptibility testing methods to evaluate their utility and accuracy. Method agreement rates between DSTM and standard methods often exceed 90%, suggesting a high positive correlation with conventional methods. Furthermore, our results show that a combination of multiple drugs in the DSTM device helps identify extended-spectrum β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC-)-producing microorganisms. In conclusion, DSTM method enables effective drug susceptibility and resistance screening within 3 h from positive blood cultures and is suitable for the rapid and personalized determination of the antimicrobial regimen., Competing Interests: Declaration of Competing Interest This work was supported by Fluidware technologies Inc., Kasukabe Japan, and Fukoku Co., Ltd., Ageo, Japan., (Copyright © 2020. Published by Elsevier Ltd.)

The lack of rapid antibiotic susceptibility tests adversely affects the treatment of bacterial infections and contributes to increased prevalence of multidrug-resistant bacteria. Here, we describe an all-electrical approach that allows for ultrasensitive measurement of growth signals from only tens of bacteria in a microfluidic device. Our device is essentially a set of microfluidic channels, each with a nanoconstriction at one end and cross-sectional dimensions close to that of a single bacterium. Flowing a liquid bacteria sample (e.g., urine) through the microchannels rapidly traps the bacteria in the device, allowing for subsequent incubation in drugs. We measure the electrical resistance of the microchannels, which increases (or decreases) in proportion to the number of bacteria in the microchannels. The method and device allow for rapid antibiotic susceptibility tests in about 2 h. Further, the short-time fluctuations in the electrical resistance during an antibiotic susceptibility test are correlated with the morphological changes of bacteria caused by the antibiotic. In contrast to other electrical approaches, the underlying geometric blockage effect provides a robust and sensitive signal, which is straightforward to interpret without electrical models. The approach also obviates the need for a high-resolution microscope and other complex equipment, making it potentially usable in resource-limited settings., Competing Interests: Competing interest statement: K.L.E. discloses a potential of conflict of interest, as he is the cofounder of a company, which aims to commercialize this technology. No potential conflicts of interest exist for Y.Y. and K.G.

This study aimed to understand the efficacy and mechanisms of action of an aerosolized glycol-ethanol formulations against bacteria. We validated a small-scale in-house test chamber to determine the microbicidal efficacy of four aerosolized formulations combining dipropylene glycol and ethanol against Staphylococcus aureus and Escherichia coli embedded in alginate. The aerosolized glycol/ethanol formulation decreased bacterial viability by 3 log 10 and was more efficacious than an ethanol only control formulation. Electron microscopic examination indicated extensive structural damage in both bacteria, and membrane damage was confirmed with potassium release in S. aureus and DNA release in E. coli. The development of a small test chamber facilitated the measurement of the microbicidal efficacy and experiments to understand the mechanism of action of an aerosolized microbicidal formulation. SIGNIFICANCE AND IMPACT OF THE STUDY: There is an increased interest in developing effective microbicidal-aerosolized formulations. The development of a small in-house test chamber allowed the measurement of the microbicidal efficacy of an aerosolized glycol/ethanol formulation at a low cost. We showed that a glycol/ethanol aerosolized formulation caused extensive structural damage in Gram-negative and -positive bacteria resulting in a 3 log 10 reduction in viability., (© 2020 The Society for Applied Microbiology.)

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

Background: Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide. Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis. We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization (RDBH) assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M. tuberculosis isolated in China., Methods: In this study, we applied a RDBH assay to simultaneously detect the resistance of rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) in 320 clinical M. tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing (DST) and sequencing. The RDBH assay was designed to test up to 42 samples at a time. Pearson's chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method, and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing., Results: The results showed that the concordances between phenotypic DST and RDBH assay were 95% for RIF, 92.8% for INH, 84.7% for SM, 77.2% for EMB and the concordances between sequencing and RDBH assay were 97.8% for RIF, 98.8% for INH, 99.1% for SM, 93.4% for EMB. Compared to the phenotypic DST results, the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5% for RIF, 90.3 and 97.3% for INH, 77.4 and 91.5% for SM, 61.4 and 85.7% for EMB, respectively; compared to sequencing, the sensitivity and specificity of the RDBH assay were 97.7 and 97.9% for RIF, 97.9 and 100.0% for INH, 97.8 and 100.0% for SM, 82.6 and 99.1% for EMB, respectively. The turnaround time of the RDBH assay was 7 h for testing 42 samples., Conclusions: Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF, INH, SM and EMB, enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

Colistin has become a critical antibiotic for fatal Gram-negative infections owing to the proliferation of multidrug-resistant carbapenemase-producing bacteria. Thus, cheaper, faster, efficient and easier-to-use colistin diagnostics are required for clinical surveillance, diagnoses and therapeutics. The sensitivity, specificity, major error (ME), very major error (VME), categorial agreement, essential agreement, turnaround time (TAT), average cost, and required skill for four colistin resistance diagnostics viz., CHROMagar COL-APSE, ComASP Colistin, MicroScan, and Colistin MAC Test (CMT) were evaluated against broth microdilution (BMD) using 84 Gram-negative bacterial isolates. A multiplex PCR (M-PCR) was used to screen all isolates to detect the presence of the mcr-1 to mcr-5 genes. A 15-point grading scale was used to grade the tests under skill, ease, processing time etc. mcr-1 was detected by both M-PCR and CMT in a single E. coli isolate, with other PCR amplicons suggestive of mcr-2, -3 and -4 genes being also observed on the gel. The sensitivity and specificity of CHROMagar COL-APSE, MicroScan, and ComASP Colistin, were 82.05% and 66.67%, 92.31% and 76.92%, and 100% and 88.89% respectively. The MicroScan was the most expensive at a cost (per sampe tested) of R221.6 ($15.0), followed by CHROMagar COL-APSE (R118.3; $8.0), M-PCR (R75.1; $5.1), CMT (R20.1; $1.4) and ComASP Colistin (R2.64; $0.2). CHROMagar was the easiest to perform, followed by ComASP Colistin, M-PCR, MicroScan, CMT and BMD whilst M-PCR and MicroScan required higher skill. The ComASP Colistin was the best performing diagnostic test, with low VME and ME, making it recommendable for routine colistin sensitivity testing in clinical laboratories; particularly, in poorer settings. It is however limited by a TAT of 18-24 hours.

Antimicrobial resistance (AMR) has been identified by the World Health Organisation as a global threat that currently claims at least 25,000 deaths each year in Europe and 700,000 globally; the number is projected to reach 10 million per year between 2015 and 2050. Therefore, there is an urgent need for low-cost but reliable point-of-care diagnostics for early screening of infections especially in developing countries lacking in basic infrastructure and trained personnel. This work is aimed at developing such a device, a paper-based microfluidic device for infection testing by an unskilled user in a low resource setting. Here, we present our work relating to the use of our laser-patterned paper-based devices for detection and susceptibility testing of Escherichia coli, via a simple visually observable colour change. The results indicate the suitability of our integrated paper devices for timely identification of bacterial infections at the point-of-care and their usefulness in providing a hugely beneficial pathway for accurate antibiotic prescribing and thus a novel route to tackling the global challenge of AMR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Many patients with severe infections receive inappropriate empirical treatment, and rapid detection of bacterial antibiotic susceptibility can improve clinical outcome and reduce mortality. To this end, we have developed a multiplex fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli , Klebsiella pneumoniae , and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime, and meropenem (Gram-negative bacteria) or gentamicin, ofloxacin, and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in an array of growth chambers in the chip where bacterial microcolony growth was monitored over time using automated image analysis. MIC values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2 to 4 h, and the results showed categorical agreement with reference MIC values as determined by broth microdilution in 86% of the cases. IMPORTANCE Prompt and effective antimicrobial therapy is crucial for the management of patients with severe bacterial infections but is becoming increasingly difficult to provide due to emerging antibiotic resistance. The traditional methods for antibiotic susceptibility testing (AST) used in most clinical laboratories are reliable but slow with turnaround times of 2 to 3 days, which necessitates the use of empirical therapy with broad-spectrum antibiotics. There is a great need for fast and reliable AST methods that enable starting targeted treatment within a few hours to improve patient outcome and reduce the overuse of broad-spectrum antibiotics. The multiplex fluidic chip for phenotypic AST described in the present study may enable data on antimicrobial resistance within 2 to 4 h, allowing for an early initiation of appropriate antibiotic therapy., (Copyright © 2020 Wistrand-Yuen et al.)

Xpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra's predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on  CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥10 3 CFU/ml (C Tmin <25, >"low semi-quantitation") detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10 -3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.

For the first time, a rapid method was proposed to determine the susceptibility of Escherichia coli cells to antibiotics by the example of ampicillin by using a biological sensor based on a slot mode in an acoustic delay line. It has been established that an indicator of the antibiotic activity to microbial cells is the difference between the recorded sensor's signal before and after exposure cells with antibiotic. The depth and frequency of the peaks of resonant absorption in the frequency dependence of the insertion loss of sensor varied after adding an antibiotic with different concentrations to the microbial cells. By using the acoustic sensor based on slot-mode a criterion of E. coli sensitivity to ampicillin was established. The advantages of this method are the ability to carry out the analysis directly in the liquid, the short analysis time (within 10-15 min), and the possibility to reusable sensor.

Rapid diagnosis of tuberculosis (TB) and antibiotic resistances are imperative to initiate effective treatment and to stop transmission of the disease. A new generation of more sensitive, automated molecular TB diagnostic tests has been recently launched giving microbiologists more choice between several assays with the potential to detect resistance markers for rifampicin and isoniazid. In this study, we determined analytical sensitivities as 95% limits of detection (LoD95) for Xpert MTB/Rif Ultra (XP-Ultra) and BD-MAX MDR-TB (BD-MAX) as two representatives of the new test generation, in comparison to the conventional FluoroType MTB (FT-MTB). Test matrices used were physiological saline solution, human and a mucin-based artificial sputum (MUCAS) each spiked with Mycobacterium tuberculosis in declining culture- and qPCR-controlled concentrations. With BD-MAX, XP-Ultra, and FT-MTB, we measured LoD95TB values of 2.1 cfu/ml (CI95%: 0.9-23.3), 3.1 cfu/ml (CI95%: 1.2-88.9), and 52.1 cfu/ml (CI95%: 16.7-664.4) in human sputum; of 6.3 cfu/ml (CI95%: 2.9-31.8), 1.5 cfu/ml (CI95%: 0.7-5.0), and 30.4 cfu/ml (CI95%: 17.4-60.7) in MUCAS; and of 2.3 cfu/ml (CI95%: 1.1-12.0), 11.5 cfu/ml (CI95%: 5.6-47.3), and 129.1 cfu/ml (CI95%: 82.8-273.8) in saline solution, respectively. LoD95 of resistance markers were 9 to 48 times higher compared to LoD95TB. BD-MAX and XP-Ultra have an equal and significantly increased analytical sensitivity compared to conventional tests. MUCAS resembled human sputum, while both yielded significantly different results than normal saline. MUCAS proved to be suitable for quality control of PCR assays for TB diagnostics., Competing Interests: Xpert cartridges, FluoroType reagents and diarella MTB/NTM/MAC qPCR kits were provided free of charge by Cepheid, Hain Lifescience GmbH and gerbion GmbH & Co. KG, respectively. The BD-MAX machine was provided by Becton Dickinson free of charge. MB received a salary from BD for giving a talk at a conference (REMMDI, 11-April-2019). Gerbion GmbH & Co. KG and IML red GmbH are partners in further research projects (TB-Tube and TB-SeqDisK). A patent named “ARTIFICIAL SPUTUM, METHOD OF PRODUCING AN ARTIFICIAL SPUTUM” is submitted in the name of IML red GmbH to the European Patent Office and currently pending (application number EP19165015.9). The patent will not be published before the 25th of September 2020. Commercial affiliations and the pending patent do not alter our adherence to PLOS ONE policies on sharing data and materials.

Detecting carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly difficult due to the emergence of diverse enzymes. The aim of the study was to evaluate an agar plate-based modified carbapenem inactivation method (p-mCIM) for detection of CPE. Stock strains and clinical isolates of CPE were used to evaluate the p-mCIM. The p-mCIM was performed as described for the mCIM, except that meropenem disks were placed on the lawn of test organisms on Mueller-Hinton agar (MHA) plates. Among 17 stock strains of CPE, six of eight KPC-2-like- and all six NDM-1-like carbapenemase-producing strains were positive by the p-mCIM without incubation in the carbapenem inactivation (CI) step. Among 380 CPE clinical isolates detected, 308 and 38 were KPC-2-like and NDM-1-like enzyme producers, respectively. The required incubation time in the CI step to show all isolates were positive by p-mCIM was 3 h for isolates with KPC-2-like enzyme and 1 h for isolates with metallo-β-lactamases. Twenty-eight of 30 isolates with OXA-48-like enzymes were p-mCIM positive. Sensitivities of both the p-mCIM and the mCIM (based on inhibition zone of ≤15 mm) for detection of CPE were 100%. All 70 ertapenem-nonsusceptible, but carbapenemase gene-negative isolates tested were both p-mCIM (based on inhibition zone of ≥21 mm) and mCIM negative. In conclusion, performance of the p-mCIM, which uses a lawn of bacterial colonies on MHA plate instead of a bacteria-suspended Tryptic soy broth tube in the CI step, is essentially identical to that of the CLSI-recommended mCIM in the detection of clinical isolates of Enterobacteriaceae producing carbapenemases including difficult to detect bla OXA-48 -like enzymes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) of bacteria-causing bloodstream infections can improve patients' outcome. In this study, we evaluated the performance of Alfred60 AST (Alifax) which provides AST directly on positive blood culture (BC) bottles by light scattering. In a selected group of patients with a clinical suspicion of severe sepsis or at risk for infections with multiresistant organisms, we compared Alfred60 AST AST results with traditional AST results (Vitek2 (bioMérieux) or disk diffusion). Discrepancy analysis was performed by Etest (bioMérieux) or broth microdilution. In total, 222 samples were evaluated. On 595 susceptibility determinations, 93.4% showed categorical agreement (CA) with the standard method. Eighty-one percent of isolates showed a 100% categorical agreement (CA) which increased to 84.3% after discrepancy analysis. There were 8 very major discrepancies (VMD), 18 major discrepancies (MD), and 13 minor discrepancies (MiD). Most discrepant results were observed for piperacillin-tazobactam (15.6%) and clindamycin (18.9%). Analysis time was 6-6.5 h for a complete Alfred60 AST AST result. In addition, we evaluated the behavior of clinicians in adjusting antibiotic therapy according to the routine AST results. In 37% of all patients, antibiotic therapy was altered after reporting of AST result and adjustment was more frequent for Gram-negative than for Gram-positive isolates. With some improvements, Alfred60 AST provides accurate and rapid preliminary AST results for organisms causing bloodstream infections and may have at least a potential clinical benefit in about one-third of patients with severe sepsis, by delivering faster results compared with conventional methods.