Conventional Antibiotic Susceptibility Tests Research Articles

P10 Impact of miniaturization and early readout on antimicrobial susceptibility tests

Abstract Background New technologies offer the promise of faster, more accessible antibiotic susceptibility testing. UK policy includes the development and introduction of innovative diagnostics to tackle antimicrobial resistance, and whilst many new technologies are emerging, we still have limited evidence of their performance. Many of these emerging technologies are changing two core aspects of antimicrobial susceptibility tests (AST). Firstly, using microfluidic platforms to miniaturize standard antibiotic susceptibility testing and secondly measuring susceptibility at earlier timepoints, here, we report a comprehensive assessment of 1-mL volume broth microdilution with an early endpoint readout, proving that miniaturization and earlier timepoints of conventional antibiotic susceptibility testing is feasible and accurate. Methods The test measures bacterial growth in 1-mL capillaries in the presence of the metabolic dye resazurin. All other aspects of the test remain standard. Images of the capillaries are taken every 5 minutes over 18 h to determine accuracy at different timepoints during incubation at 37°C. Over a total of 3 sites in the UK, Spain and Thailand, we have tested 367 isolates from clinical samples for 8–17 antibiotics. Results Preliminary data shows that for 99 Gram-negative isolates tested in Thailand, after 6-h incubation, accuracy for antibiotics is as follows: cefotaxime 96%, ceftazidime 92%, meropenem 93%, amikacin 94%, amoxicillin–clavulanic acid 79%, cefuroxime 97%, cefoxitin 80%, ciprofloxacin 84%, gentamicin 93% and trimethoprim 97%. Further testing is ongoing, and isolates from urine samples are currently being collected from Basingstoke and North Hampshire hospital to expand the type of organism tested. Conclusions Unlike many microfluidic systems, the dip and test microcapillary–based broth microdilution test is manufactured in bulk, allowing rapid and testing of large sample sets with minimal investment. An understanding of the effect of miniaturization is vital to developers of microfluidic phenotypic antibiotic susceptibility tests and can provide confidence in emerging systems.

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.

Precise digital bacteria enumeration and antibiotic susceptibility testing via a portable vibrating capillary-based droplet platform

Antibiotic resistance can lead to multidrug-resistant superbugs and threaten global health, putting forward a high demand for accurate and fast antibiotic susceptibility testing (AST). Conventional AST methods are labor-intensive and time-consuming, far from meeting the requirement for rapid clinical decision-making. Here, we develop a portable vibrating capillary-based droplet platform for precise digital bacteria enumeration and rapid AST. Multi-volume droplets encapsulating bacteria with or without antibiotics are generated on demand, and the elevated fluorescence from resazurin reduction is measured, which corresponds to the growing bacteria. Automated data analysis is achieved by a self-developed image processing program. Multi-volume droplet digital counting allows precise bacteria enumeration over a wide dynamic range spanning more than 4 orders of magnitude. Droplet digital AST of ampicillin against E. coli demonstrates that it is feasible for accurately assessing the minimum inhibitory concentration and investigating the inoculum effect, with the assay time reduced to 5 h. In sum, this method is easy to operate, cost-effective, with high accuracy and short time-to-result, which is highly promising for rapid and precise assessment of antimicrobial susceptibility in resource-limited settings.

Accelerated antibiotic susceptibility testing of pseudomonas aeruginosa by monitoring extracellular electron transfer on a 3-D paper-based cell culture platform

Pseudomonas aeruginosa is the most important opportunistic pathogen leading to serious and life-threatening infections, especially in immunocompromised patients. Because of its remarkable capacity to resist antibiotics, the selection of the right antibiotics with the exact dose for the appropriate duration is critical to effectively treat the infections and prevent antibiotic resistance. Although conventional genotypic and phenotypic antibiotic susceptibility testing (AST) methods have been dramatically advanced, they have suffered from many technical and operational issues as a generalized antibiotic stewardship program. Furthermore, given that most microbial infections are caused by their biofilms, the existing AST methods do not provide evidence-based antibiotic prescribing guidance for biofilm-based infections because the results are based on individual bacteria traditionally grown in their planktonic form. In this work, we create an innovative susceptibility testing technique for P. aeruginosa that offers clinically relevant guidelines and widely adaptable stewardship to effectively treat the infections and minimize antibiotic resistance. Our approach evaluates the antibiotic efficacy by continuously monitoring the accumulated electrical outputs from the bacterial extracellular electron transfer (EET) process in the presence of antibiotics. Our innovative paper-based culturing 3-D scaffold promotes surface-associated growth of bacterial colonies and biofilms. The platform replicates a natural habitat for P. aeruginosa where it can grow similarly to sites it infects. Our technique enables an all-electrical, real-time, easy-to-use, portable AST that can be easily translatable to clinical settings. The entire procedure takes 96 min to provide evidence-based antimicrobial prescribing guidance for biofilm-based infections.

Performance Evaluation of the Quantamatrix QMAC-dRAST System for Rapid Antibiotic Susceptibility Testing Directly from Blood Cultures.

Objectives: Rapid antibiotic susceptibility testing (AST) for positive blood cultures can improve patient clinical outcomes if the time to an effective antimicrobial therapy is shortened. In this study, we tested the Quantamatrix dRAST system (QMAC-dRAST), a rapid AST system based on time-lapse microscopic imagery of bacterial colony formation in agarose. Methods: Evaluation of the QMAC-dRAST was performed from 250 monobacterial blood cultures including 130 Enterobacterales, 20 non-fermentative Gram-negative bacteria, 69 staphylococci and 31 enterococci. Blood cultures were recovered from anonymous patients or from spiking experiments to enrich our study with bacterial species and resistant strains. Categorical agreement (CA), minor errors (me), major errors (ME) and very major errors (VME) were calculated in comparison to the results obtained from the BD Phoenix™ M50. Discrepancies between the Phoenix™ M50 and QMAC-dRAST results were investigated using the gradient strip method. The repeatability and reproducibility performance of the QMAC-dRAST was assessed for 16 strains, each strain being tested five times from a spiked blood culture. Results: The overall CAs for Enterobacterales, non-fermentative Gram-negative bacteria, staphylococci and enterococci were 95.1%, 91.2%, 93.4% and 94.5%, respectively. The VME percentage was below 4% for all the groups except for staphylococci, which showed a VME rate of 7%. The median time to result was 6.7 h (range: 4.7–7.9). Repeatability and reproducibility assays showed a high reliability of AST results with best and worst ratios of 98.8% and 99.6% and 95.0% and 98.3%, respectively. Conclusions: The QMAC-dRAST is a fast and reliable system to determine AST directly from monobacterial blood cultures with a major TAT reduction compared to conventional AST testing.

Effectiveness of antimicrobial stewardship programmes based on rapid antibiotic susceptibility testing of haematological patients having high-risk factors for bacteraemia-related mortality: a post-hoc analysis of a randomised controlled trial

A randomised controlled trial showed that rapid phenotypic antibiotic susceptibility testing (AST) with antimicrobial stewardship programme (ASP) increases the proportion of haematological patients with bacteraemia receiving optimal targeted therapy within 72 h of blood culture collection. This post-hoc analysis aimed to evaluate the effects of rapid phenotypic AST intervention in haematological patients at high risk of a poor outcome from bacteraemia. Haematological patients with bacteraemia (n=116) were assigned randomly to a conventional AST group or a rapid AST group. The two outcome measures were the proportion of patients receiving optimal targeted therapy at 72 h post blood culture collection and the time to optimal targeted therapy; subgroup analysis was conducted based on baseline demographics (age, sex) and prognostic (Charlson comorbidity index, haematological treatment intensity, Pitt bacteraemia score, appropriateness of empirical antibiotic therapy and multidrug-resistant organism) indicators. The interaction effect between the intervention and subgroup factors was examined using regression model analysis. Age, sex, Charlson comorbidity index, haematological treatment intensity, Pitt bacteraemia score and appropriateness of empirical antibiotic therapy had no significant interaction effects on the proportion of patients receiving optimal targeted therapy (P=0.129-0.826). However, infection by a multidrug-resistant organism did have a significant interaction effect (P=0.042). Regarding time to optimal targeted therapy, there were no significant interaction effects between the intervention and subgroup factors (P=0.156-0.848). In conclusion, rapid phenotypic AST with ASP intervention may accelerate early optimal targeted antimicrobial treatment of haematological patients, even those in high-risk subgroups with bacteraemia.

Hand-powered vacuum-driven microfluidic gradient generator for high-throughput antimicrobial susceptibility testing

The growth of bacterial resistance to antimicrobials is a serious problem attracting much attention nowadays. To prevent the misuse and abuse of antimicrobials, it is important to carry out antibiotic susceptibility testing (AST) before clinical use. However, conventional AST methods are relatively laborious and time-consuming (18–24 h). Here, we present a hand-powered vacuum-driven microfluidic (HVM) device, in which a syringe is used as the only vacuum source for rapid generating concentration gradient of antibiotics in different chambers. The HVM device can be preassembled with various amounts of antibiotics, lyophilized, and stored for ready-to-use. Bacterial samples can be loaded into the HVM device through a simple suction step. With the assistance of Alamar Blue, the AST assay and the minimum inhibitory concentration (MIC) of different antibiotics can be investigated by comparing the growth results of bacteria in different culture chambers. In addition, a parallel HVM device was proposed, in which eight AST assays can be performed simultaneously. The results of MIC of three commonly used antibiotics against E. coli K-12 in our HVM device were consistent with those obtained by traditional method while the detection time was shortened to less than 8 h. We believe that our platform is high-throughput, cost-efficient, easy to use, and suitable for POCT applications.

P10 Rapid capture of uropathogenic bacteria and on-chip determination of antimicrobial resistance

BackgroundUrinary tract infection (UTI) is one of the most common bacterial infections responsible for increased annual incidence of antimicrobial resistance (AMR) cases. Clinical diagnosis of UTI AMR relies heavily on conventional urine culture and antibiotic susceptibility testing (AST) which has a turnaround time of ∼3 days. Often, irrespective of the infection status, antibiotics are prescribed to patients even before the test results are available, leading to non-judicious use of antibiotics. Over the years, several technologies have been developed for the rapid detection and diagnosis of UTI AMR, however, most of them are limited to traditional microbiological techniques and large laboratory equipment that are not readily available in low-to-middle income countries (LMICs). To address these diagnostic limitations, we are developing a rapid and affordable UTI-AMR diagnostic microfluidic device that is clinical friendly aimed at improving UTI management and AMR stewardship.ResultsOur device enables the flow of a large volume of urine specimens for the capture/enrichment of uropathogenic bacteria and determination of AST via a porous membrane that is augmented with a multifunctional polymer-based material. Important objectives for the development of UTI AMR diagnostic microfluidic device are: (i) development of a multifunctional polymer-based material; and (ii) validation of UTI AMR diagnostic device. We have successfully developed a polysaccharide-based platform to (i) selectively capture uropathogenic bacteria from urine specimen by immobilizing concanavalin A (con A) lectin as bacterial capture agent on the polymer surface via chemical modification; (ii) encapsulate and release bacterial nutrient media and antibiotics for AST; and (iii) detect AST via encapsulation of bacterial growth indicator. In addition, we have also determined the development of methacrylate-based and acrylamide-based synthetic polymer-based material for our application. Further, we have demonstrated the uniform augmentation of the polysaccharide-based polymer onto porous membrane via dip-coating technique for on-chip bacterial capture/enrichment and AST in fluid (urine) flow conditions. The porous membrane is a conducting material which enables us to perform electrochemical measurements such as impedance spectroscopy that accelerates the detection process of antibiotic susceptibility. As a proof-of-concept, we have determined the capture of biosafety level I Escherichia coli expressing kanamycin resistance gene on chemically surface modified polysaccharide-based polymer containing con A and the antibiotic susceptibility of captured bacteria against different antibiotics with and without the porous membrane. We have quantitatively determined the limit of detection of E. coli on multifunctional polysaccharide-based polymer material.ConclusionsThe utility of the UTI AMR microfluidic device in clinical settings enables clinicians to make informed decisions on the most appropriate antibiotic for treatment in less than a day. Integration of impedance spectroscopy will further accelerate the detection by significantly reducing the time of detection. Further, the device allows for off-chip analysis by retrieving the captured uropathogenic bacteria to perform high throughput sequencing for identifying AMR genetic determinants. Therefore, with the ability to selectively capture uropathogenic bacteria and determine AST in a short time, our technology has the potential to overcome some of the current limitations in UTI AMR diagnostics.

Speciation and Antibiotic Susceptibility Pattern of Shigella isolates in a Rural Tertiary Care Hospital, Kolar

Shigellosis is the most important cause of bacillary dysentery. It’s a major public health problem. It is endemic in many developing countries resulting in a significant amount of mortality and morbidity. The incidence is 90 million with 6,00,000 deaths world over. In India, there is an emergence of resistance to Shigella resulting in limitation of treatment. To study the prevalence of Shigella species and their antibiogram in stool samples obtained from patients treated for dysentery at Rural Tertiary Care Hospital. A retrospective analysis of the Shigella isolates obtained from 400 stool samples from patients with dysentery, treated at Rural Tertiary Care Hospital from 2015 -2019 was conducted. The isolates were identified by conventional methods and antibiotic susceptibility testing was performed by Kirbybauers disk diffusion method and analysed. The percentage of isolation of Shigella species at our centre was 5% with Shigella flexneri being the predominant species isolated. A progressive pattern of resistance was observed. 100% strains of Shigella flexneri and Shigella sonnei were resistant to cotrimoxazole and nalidixic acid. 75% of Shigella flexneri were resistant to fluoroquinolones and 25% resistant to third-generation cephalosporins. 100% isolates were sensitive to azithromycin. In view of rampant resistance observed among Shigella species, it is a need to test these isolates for antibiotic susceptibility and treat the patients based on the antibiogram.

Mini-review: Recent advances in imaging-based rapid antibiotic susceptibility testing

The rapid emergence and spread of antibiotic-resistant bacteria have become a major threat to public health. In clinics, antibiotic susceptibility testing (AST) is employed to guide appropriate prescription to suppress the aggravation of antibiotic resistance. However, the conventional culture-based AST methods are time-consuming and labor-intensive. Therefore, rapid AST approaches are urgently needed. In this review, conventional, commercial, and recent progress in novel imaging-based rapid AST modalities are reviewed with the merits and limitations of each recent imaging-based method highlighted. Moreover, we also discuss the future directions of applying imaging-based rapid AST to point-of-care diagnosis.

High resolution melting assay as a reliable method for diagnosing drug-resistant TB cases: a systematic review and meta-analysis

BackgroundTuberculosis (TB) is one of the most contagious infectious diseases worldwide. Currently, drug-resistant Mycobacterium tuberculosis (Mtb) isolates are considered as one of the main challenges in the global TB control strategy. Rapid detection of resistant strains effectively reduces morbidity and mortality of world’s population. Although both culture and conventional antibiotic susceptibility testing are time-consuming, recent studies have shown that high resolution melting (HRM) assay can be used to determine the types of antibiotic resistance. In the present meta-analysis, we evaluated the discriminative power of HRM in detecting all drug-resistance cases of TB.MethodsA systematic search was performed using databases such as Cochrane Library, Scopus, PubMed, Web of Science, and Google Scholar. Related studies on the effect of HRM in the diagnosis of drug-resistant (DR) TB cases were retrieved by April 2021. We used Meta-Disc software to evaluate the pooled diagnostic sensitivity and specificity of HRM for the detection of each type of drug-resistant cases. Finally, diagnostic value of HRM was characterized by summary receiver operating characteristic (SROC) curve and the area under the curve (AUC) method.ResultsOverall 47 studies (4,732 Mtb isolates) met our criteria and were included in the present meta-analysis. Sensitivity, specificity, and AUC of HRM were measured for antibiotics such as isoniazid (93%, 98%, 0.987), rifampin (94%, 97%, 0963), ethambutol (82%, 87%, 0.728), streptomycin (82%, 95%, 0.957), pyrazinamide (72%, 84%, 0.845), fluoroquinolones (86%, 99%, 0.997), MDR-TB (90%, 98%, 0.989), and pan-drug-resistant TB (89%, 95%, 0.973).ConclusionsThe HRM assay has high accuracy for the identification of drug-resistant TB, particularly firs-line anti-TB drugs. Therefore, this method is considered as an alternative option for the rapid diagnosis of DR-TB cases. However, due to heterogeneity of included studies, the results of HRM assays should be interpreted based on conventional drug susceptibility testing.

Advances in Antimicrobial Resistance Monitoring Using Sensors and Biosensors: A Review

The indiscriminate use and mismanagement of antibiotics over the last eight decades have led to one of the main challenges humanity will have to face in the next twenty years in terms of public health and economy, i.e., antimicrobial resistance. One of the key approaches to tackling antimicrobial resistance is clinical, livestock, and environmental surveillance applying methods capable of effectively identifying antimicrobial non-susceptibility as well as genes that promote resistance. Current clinical laboratory practices involve conventional culture-based antibiotic susceptibility testing (AST) methods, taking over 24 h to find out which medication should be prescribed to treat the infection. Although there are techniques that provide rapid resistance detection, it is necessary to have new tools that are easy to operate, are robust, sensitive, specific, and inexpensive. Chemical sensors and biosensors are devices that could have the necessary characteristics for the rapid diagnosis of resistant microorganisms and could provide crucial information on the choice of antibiotic (or other antimicrobial medicines) to be administered. This review provides an overview on novel biosensing strategies for the phenotypic and genotypic determination of antimicrobial resistance and a perspective on the use of these tools in modern health-care and environmental surveillance.

Rapid Determination of Antibiotic Resistance in Klebsiella pneumoniae by a Novel Antibiotic Susceptibility Testing Method Using SYBR Green I and Propidium Iodide Double Staining.

Due to the broad-spectrum antibiotic usage and empirical treatments, the pathogenic bacterium, Klebsiella pneumoniae, has shown extremely high detection rates at hospitals with an increasing antibiotic resistance. Therefore, rapid detection of the antibiotic resistance is urgently required and essential for effective treatments. In this study, we evaluated the performance of a newly developed method for ultra-rapid detection of antibiotic resistance in 30–60 min in K. pneumoniae by using the SYBR Green I and propidium iodide (PI) staining. A total of 100 clinical isolates were tested for antibiotic resistance using four different antibiotics (ceftriaxone, cefepime, meropenem, and ciprofloxacin). The results showed that the SYBR Green I/PI rapid antibiotic susceptibility test (AST) could reliably detect antibiotic resistance to the four drugs in 60 min, and the results were highly concordant with the conventional AST (i.e., Kirby-Bauer method and broth microdilution method) for detection of ceftriaxone, cefepime, meropenem, and ciprofloxacin resistance with a high accuracy of 99, 96, 96, and 93%, respectively. Therefore, the rapid AST established in our study helps to enable targeted therapy to save lives and reduce the empirical use of antibiotics and ultimately the health and economic burdens of antibiotic resistance.

Prevalence, serotype and antibiotic susceptibility of Group B Streptococcus isolated from pregnant women in Jakarta, Indonesia.

Group B Streptococcus (GBS) is a bacterial pathogen which is a leading cause of neonatal infection. Currently, there are limited GBS data available from the Indonesian population. In this study, GBS colonization, serotype distribution and antimicrobial susceptibility profile of isolates were investigated among pregnant women in Jakarta, Indonesia. Demographics data, clinical characteristics and vaginal swabs were collected from 177 pregnant women (mean aged: 28.7 years old) at 29-40 weeks of gestation. Bacterial culture identification tests and latex agglutination were performed for GBS. Serotyping was done by conventional multiplex PCR and antibiotic susceptibility testing by broth microdilution. GBS colonization was found in 53 (30%) pregnant women. Serotype II was the most common serotype (30%) followed by serotype III (23%), Ia and IV (13% each), VI (8%), Ib and V (6% each), and one non-typeable strain. All isolates were susceptible to vancomycin, penicillin, ampicillin, cefotaxime, daptomycin and linezolid. The majority of GBS were resistant to tetracycline (89%) followed by clindamycin (21%), erythromycin (19%), and levofloxacin (6%). The serotype III was more resistant to erythromycin, clindamycin, and levofloxacin and these isolates were more likely to be multidrug resistant (6 out of 10) compared to other serotypes. This report provides demographics of GBS colonization and isolate characterization in pregnant women in Indonesia. The results may facilitate preventive strategies to reduce neonatal GBS infection and improve its treatment.

Antimicrobial Susceptibility Testing in a Rapid Single Test via an Egg-like Multivolume Microchamber-Based Microfluidic Platform.

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-MVM2) platform, which is used to produce a wide range of gradient-based antibiotic concentrations quickly (∼10 min). The EL-MVM2 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-MVM2 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-MVM2 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-MVM2 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.

Diffusion-Convection Hybrid Microfluidic Platform for Rapid Antibiotic Susceptibility Testing.

Conventional antibiotic susceptibility testing (AST) assays such as broth microdilution and Kirby-Bauer disk diffusion are time-consuming (e.g., 24-72 h) and labor-intensive. Here, we present a microfluidic platform to perform AST assays with a broad range of antibiotic concentrations and controls. A culture medium stream was serially enriched with antibiotics along the length of the platform via diffusion and flow-directing mass convection mechanisms, generating a concentration gradient captured in a series of microchamber duplicates. We observed an agreement between the simulated and experimental concentration gradients and applicability to a variety of different molecules by changing the loading time according to a simple linear equation. The AST assay in our platform is based on bacterial metabolism, indicated by resazurin fluorescence. The small reaction volume enabled a minimum inhibitory concentration (MIC) to be determined in 4-5 h. Proof-of-concept functionality testing, using human isolates and clinically important antibiotics from different classes, indicated a high rate of agreement (94%: MIC within ±1 two-fold dilution of the reference method) of on-chip MICs and conventional broth microdilution. Overall, our results showed that this microfluidic platform is capable of determining antibiotic susceptibility in a rapid and reliable manner.

Microscopic Analysis of Bacterial Inoculum Effect Using Micropatterned Biochip

Antimicrobial resistance has become a major problem in public health and clinical environments. Against this background, antibiotic susceptibility testing (AST) has become necessary to cure diseases in an appropriate and timely manner as it indicates the necessary concentration of antibiotics. Recently, microfluidic based rapid AST methods using microscopic analysis have been shown to reduce the time needed for the determination of the proper antibiotics. However, owing to the inoculum effect, the accurate measurement of the minimal inhibitory concentration (MIC) is difficult. We tested four standard bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, against five different antibiotics: piperacillin, cefotaxime, amikacin, levofloxacin, and ampicillin. The results showed that overall, the microfluidic system has a similar inoculum effect compared to the conventional AST method. However, due to the different testing conditions and determination protocols of the growth of the microfluidic based rapid AST, a few results are not identical to the conventional methods using optical density. This result suggests that microfluidic based rapid AST methods require further research on the inoculum effect for practical use in hospitals and can then be used for effective antibiotic prescriptions.

Isolation and Molecular Characterization of Enterotoxigenic Escherichia coli (ETEC) Strains From Industrial Dairy Farms of Hamedan, Iran

Background: Enterotoxigenic Escherichia coli (ETEC) is considered as one of the most common causes of infectious diarrhea in calves, infecting animals during the first week of age. The secretory diarrhea is attributed to the virulence factors of ETEC strains mainly including heat stable toxin (STa), as well as F5 (K99) and F41 fimbriae. Objectives: The present study was undertaken to investigate ETEC infection in neonatal calves of industrial dairy farms of Hamedan, Iran. Additionally, it was undertaken to investigate the genotypic screening of virulence genes in enterotoxigenic E. coli isolated from from dairy farms calves of Hamedan county. Materials and Methods: A total of 120 rectal swab samples were collected from healthy and diarrheic calves at one week of age belonging to eight farms. Conventional bacteriological methods, multiplex PCR, and antibiotic susceptibility test of the ETEC isolates were performed. Results: Nine E. coli isolates were found to be ETEC strains, carrying STa enterotoxin along with F5 and/or F41 fimbriae as the indicators of ETEC cells. Additionally, antibiotic susceptibility test of the ETEC isolates revealed that all of them were sensitive to trimethoprim-sulfamethoxazole, ciprofloxacin, and enrofloxacin, whereas complete resistance was observed against amoxicillin-clavulanic acid (100%) and polymyxin B (100%). The present study, conducted for the first time in Hamedan, indicated a prevalence of 7.5% for Enterotoxigenic Escherichia coli in the examined animals. Conclusion: Regarding economic losses of the infection in claves as well as the zoonotic nature of ETEC cells, it is recommended that measures should be taken, such as immunization of pregnant cows prior to the delivery, feeding of adequate colostrum to newborn calves at the right time, and adherence to hygiene practices on the farms to prevent and/or reduce the incidence of diarrhea cases caused by infection with these bacteria.

Real-time monitoring of extracellular ATP in bacterial cultures using thermostable luciferase.

Adenosine triphosphate (ATP) is one of the most important indicators of cell viability. Extracellular ATP (eATP) is commonly detected in cultures of both eukaryotic and prokaryotic cells but is not the focus of current scientific research. Although ATP release has traditionally been considered to mainly occur as a consequence of cell destruction, current evidence indicates that ATP leakage also occurs during the growth phase of diverse bacterial species and may play an important role in bacterial physiology. ATP can be conveniently measured with high sensitivity in luciferase-based bioluminescence assays. However, wild-type luciferases suffer from low stability, which limit their use. Here we demonstrate that an engineered, thermostable luciferase is suitable for real-time monitoring of ATP release by bacteria, both in broth culture and on agar surfaces. Different bacterial species show distinct patterns of eATP accumulation and decline. Real-time monitoring of eATP allows for the estimation of viable cell number by relating luminescence onset time to initial cell concentration. Furthermore, the method is able to rapidly detect the effect of antibiotics on bacterial cultures as Ampicillin sensitive strains challenged with beta lactam antibiotics showed strongly increased accumulation of eATP even in the absence of growth, as determined by optical density. Patterns of eATP determined by real-time luminescence measurement could be used to infer the minimal inhibitory concentration of Ampicillin. Compared to conventional antibiotic susceptibility testing, the method presented here is faster and more sensitive, which is essential for better treatment outcomes and reducing the risk of inducing antibiotic resistance. Real-time eATP bioluminescence assays are suitable for different cell types, either prokaryotic or eukaryotic, thus, permitting their application in diverse fields of research. It can be used for example in the study of the role of eATP in physiology and pathophysiology, for monitoring microbial contamination or for antimicrobial susceptibility testing in clinical diagnostics.