Platinum nanoparticles-based electrochemical H2O2 sensor for rapid antibiotic susceptibility testing.

With the increase of antimicrobial resistance, rapid antibiotic susceptibility testing (AST) to guide precise antibiotic administration has become increasingly important. However, current gold standard AST approaches tend to take up to 24-48 h. In this work, based on the nature of catalase-positive bacteria decomposing H2O2, we developed a rapid, portable, straightforward, and cost-effective phenotypic AST approach by detecting residual H2O2 using a Pt nanoparticles-based electrochemical sensor. The pulse current of the sensor exhibited a linear increase with rising H2O2 concentration, demonstrating a high sensitivity of ∼382.2 µA cm-2 mM-1. This approach showed superb diagnostic performance, with an area under the curve of 1 for 24 clinical samples of Escherichia coli and Staphylococcus aureus, with a total detection time of 60 and 45 min, respectively. Furthermore, the performance of the sensor showed no degradation even after 100 detections, promising a substantial reduction in AST costs. Overall, the proposed approach exhibited immense potential for diagnosing bacterial antibiotic resistance.

Detection of mecA-mediated methicillin resistance and evaluation of disk-diffusion antimicrobial susceptibility characteristics of Staphylococcus saprophyticus isolates from geographically diverse locations: Staphylococcus saprophyticus antibiotic susceptibility testing.

Staphylococcus saprophyticus, a common uropathogen, is usually susceptible to urine-concentrating antimicrobials, so routine AST is not recommended by CLSI. Our study evaluated the antimicrobial resistance profiles of 277 S. saprophyticus isolates from North America and a globally diverse cohort. Notably, 24% (67/277) of our isolates come from non-urinary sources. AST was performed against 12 antimicrobials using standard disk diffusion, PCR for mecA and mecC, PBP2a production assays, and cefinase. 5% (13/277) of isolates were mecA positive and cefinase positive, 63% (176/277) were mecA negative but cefinase positive, 4% (11/277) were mecA positive but cefinase negative, and 28% (77/277) were mecA and cefinase negative. All (277/277) isolates were susceptible to delafloxacin, ciprofloxacin, rifampin, linezolid, and nitrofurantoin and 95% (262/277) were susceptible to trimethoprim-sulfamethoxazole. Our results showed that regardless of using CLSI or EUCAST breakpoints oxacillin had low categorical agreement for mecA presence, making it unsuitable for surrogate testing, while cefoxitin disk diffusion had high very major error rate. If possible, PBP2a or mecA testing is recommended for guiding therapy for non-urinary infections. Our work supports CLSI guidelines on routine susceptibility to urinary tract antibiotics.

Precision medicine in practice: unravelling the prevalence and antibiograms of urine cultures for informed decision making in federal tertiary care- a guide to empirical antibiotics therapy.

Background and Objectives: Urinary tract infections (UTIs), one of the most prevalent bacterial infections, are facing limited treatment options due to escalating concern of antibiotic resistance. Urine cultures significantly help in identification of etiological agents responsible for these infections. Assessment of antibiotic susceptibility patterns of these bacteria aids in tackling the emerging concern of antibiotic resistance and establishment of empirical therapy guidelines. Our aim was to determine various agents responsible for urinary tract infections and to assess their antibiotic susceptibility patterns. Materials and Methods: This cross-sectional study was performed over a period of six months from January 2023 to July 2023 in Department of Microbiology of Pakistan Institute of Medical Sciences (PIMS). Results: Out of 2957 positive samples, Gram negative bacteria were the most prevalent in 1939 (65.6%) samples followed by Gram positive bacteria in 418 (14.1%) and Candida spp. in 269 (9.1%) samples. In gram negative bacteria, Escherichia coli (E. coli) was the most prevalent bacteria isolated from 1070 samples (55.2%) followed by Klebsiella pneumoniae in 397 samples (20.5%). In Gram positive bacteria, Enterococcus spp. was the most common bacteria in 213 samples (51%) followed by Staphylococcus aureus in 120 samples (28.7%). Amikacin was the most sensitive drug (91%) for Gram negative bacteria. Gram positive bacteria were most susceptible to linezolid (97%-100%). Conclusion: The generation of a hospital tailored antibiogram is essential for the effective management of infections and countering antibiotic resistance. By adopting antimicrobial stewardship strategies by deeper understanding of sensitivity patterns, we can effectively combat antibiotic resistance.

Evaluation of EUCAST rapid antimicrobial susceptibility test directly from positive blood culture for Pseudomonas aeruginosa.

In this study, we evaluated the performance of the EUCAST RAST method on a collection of 154 clinical strains of P. aeruginosa, including strains resistant to ceftazidime and carbapenems. While the test is convenient for routine laboratories, we observed significant rates of VME (ranging from 0.0 to 15.0%) and ME (ranging from 1.3 to 16.3%) after 6 h, particularly for key antibiotics such as ceftazidime, piperacillin/tazobactam, and meropenem. Extending the incubation time to 8 h may improve results (CA ranging from 87.2 to 99%), but caution is required in interpretation due to persistence of VME (ranging from 0.0 to 15.6%) and ME (ranging from 0.0 to 11.7%).

Rapid determination of antibiotic susceptibility of clinical isolates of Escherichia coli by SYBR green I/Propidium iodide assay.

Infections caused by pathogenic Escherichia coli are a serious threat to human health, while conventional antibiotic susceptibility tests (AST) have a long turn-around time, and rapid antibiotic susceptibility methods are urgently needed to save lives in the clinic, reduce antibiotic misuse and prevent emergence of antibiotic-resistant bacteria. We optimized and validated the feasibility of a novel rapid AST based on SYBR Green I and Propidium Iodide (SGPI-AST) for E. coli drug susceptibility test. A total of 112 clinical isolates of E. coli were collected and four antibiotics (ceftriaxone, cefoxitin, imipenem, meropenem) were selected for testing. Bacterial survival rate of E. coli was remarkably linearly correlated with S value at different OD600 values. After optimizing the antibiotic concentrations, the sensitivity and specificity of SGPI-AST reached 100%/100%, 97.8%/100%, 100%/100% and 98.4%/99% for ceftriaxone, cefoxitin, imipenem and meropenem, respectively, and the corresponding concordances of the SGPI-AST with conventional AST were 1.000, 0.980, 1.000 and 0.979, respectively. The SGPI-AST can rapidly and accurately determine the susceptibility of E. coli clinical isolates to multiple antibiotics in 60 min, and has the potential to be applied to guide the precise selection of antibiotics for clinical management of infections caused by pathogenic E. coli.

Multiplex antibiotic susceptibility testing of urinary tract infections using an electrochemical lab-on-a-chip.

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.

Vibrio alginolyticus: A Rare Cause of Otitis Externa off the Coast of Florida.

Vibrio alginolyticus, a gram-negative marine bacterium, poses significant health risks through various infections transmitted via contaminated seawater or seafood consumption. This case report details a 42-year-old male presenting with chronic seropurulent discharge from his ear, ultimately diagnosed with otitis externa caused by V. alginolyticus. Examination findings and antibiotic sensitivity testing informed the treatment strategy, leading to a successful resolution. The increasing incidence of V. alginolyticus infections, particularly in warm coastal water, necessitated heightened clinical awareness and appropriate management. As global temperatures rise, proactive measures including patient education and accurate diagnosis become crucial in preventing disease progression and complications associated with V. alginolyticus infections.

CS/PVP Hydrogel-Based Nanocapillary for Monitoring Bacterial Growth and Rapid Antibiotic Susceptibility Testing.

Infection with drug-resistant bacteria poses a significant threat to human health. Judicious use of antibiotics could reduce the likelihood of bacterial resistance, which can be evaluated through antibiotic susceptibility testing (AST). This paper focuses on the application of a needle-like nanocapillary tip filled with chitosan (CS)/polyethylene pyrrolidone (PVP) hydrogel based on its specific pH-sensitive properties. The gel-filled nanocapillary has the potential to be used for electrical pH detection with a sensitivity of 3.06 nA/pH and a linear range from 7.3 to 4.3. Such sensitivity for pH measurement could be extended for monitoring of bacterial (such as Escherichia coli and Streptococcus salivarius) growth because of the relationship between pH and bacterial growth. Bacterial growth curves obtained using the hydrogel-filled nanocapillary showed good agreement with the OD600 method. Moreover, this device could be applied for rapid AST for tetracycline and norfloxacin on E. coli with minimum inhibitory concentrations of 2 and 0.125 µg/mL, respectively. This study expands the application of the hydrogel-based nanocapillary for bacterial research by monitoring changes in pH values.

Spontaneous resolution or antibiotic effectiveness? Reflection on a case of pediatric urinary tract infections caused by Enterococcus raffinosus.

Enterococcus raffinosus, named by Collins et al. in 1989, is a cocci-shaped bacterium that typically appears in pairs or short chains. As a Gram-positive and non-motile bacterium, it grows at 10°C-45°C, exhibiting negative peroxidase activity [1]. It is a normal flora in the oropharynx and gastrointestinal tract of domestic cats [2] and can also be isolated from human rectal swabs [3], it belongs to the same genus Enterococcus as Enterococcus faecalis and Enterococcus faecium. Enterococcus faecalis and Enterococcus faecium constitute 90% of clinically isolated strains. However, the incidence of other enterococci, excluding E. faecalis and E. faecium, is on the rise [4]. In this case report, a patient with pediatric urinary tract infections caused by E. raffinosus was presented, and a summary of relevant literature was provided.

A Thermoplastic Microsystem to Perform Antibiotic Susceptibility Testing by Monitoring Oxygen Consumption.

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 (OD600 = 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.

Strengthening clinical bacteriology laboratory diagnostics to combat sepsis and antimicrobial resistance in Benin: a train-the-trainer approach.

Introduction: Improved laboratory diagnostics is needed to support sepsis diagnosis and combat increasing antibiotic resistance in Benin. We trained clinical laboratory experts and technicians to improve their skills in accurate and up-to-date diagnostics. Methods: A Train-the-Trainer (TtT) approach was used to design the course that combines theoretical and practical laboratory skills, specifically addressing the knowledge gaps we had previously identified in our national survey. Pedagogical methods were student-centered, including peer learning, use of online materials, practical laboratory work and pre-and post-course tests. Results: We first trained 10 trainers who in turn trained 40 laboratory technicians from across the country, from both public and private clinical and veterinary laboratories. The trainers also prepared standard operation procedures for blood culture and antibiotic susceptibility testing based on international standards. Three months after the training, follow-up visits were made to the laboratories where the implementation of the new skills was evaluated. The progress of the participants observed during the course and the implementation of the new skills afterwards proved the training to be effective. Discussion: The professional networks created during the training, the empowerment that utilizes local knowledge resources, and the government support for our initiative can be expected to bring sustainability to the initiative and support the participation of Beninese laboratories in international surveillance programs in the future.

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

BACKGROUND: Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. METHODS: 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). RESULTS: According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). CONCLUSIONS: The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

ß-Galactosidase-triggered in situ synthesis of yellow emitting silicon nanoparticle and its application in visual detection of E. coli O157:H7 and drug susceptibility test.

The sensitive detection of foodborne pathogenic and rapid antibiotic susceptibility testing (AST) is of great significance. This paper reports the enzyme-triggered in situ synthesis of yellow emitting silicon nanoparticles (SiNPs) and the detection of Escherichia coli (E. coli) O157:H7 in food samples and the rapid AST. The rapid counting of E. coli O157:H7 has been achieved through direct visual observation, equipment detection, and smartphone digitalization. A simple detection platform based on smartphone senses and cotton swabs has been established. Meanwhile, rapid AST based on enzyme-catalyzed SiNPs can intuitively obtain colorimetric samples. This paper established a system for bacterial enzyme-triggered in situ synthesis of SiNPs, with high responsiveness, luminescence ratio, and specificity. The detection limit for E. coli O157:H7 can reach 100 CFU/mL during 5 h, and the recovery efficiency ranges from 90.14% to 110.16%, which makes it a promising strategy for the rapid detection of E. coli O157:H7 and AST.

Molecular rapid diagnostic testing for bloodstream infections: Nanopore targeted sequencing with pathogen-specific primers.

BACKGROUND: Nanopore sequencing, known for real-time analysis, shows promise for rapid clinical infection diagnosis but lacks effective assays for bloodstream infections (BSIs). METHODS: We prospectively assessed the performance of a novel nanopore targeted sequencing (NTS) assay in identifying pathogens and predicting antibiotic resistance in BSIs, analyzing 387 blood samples from December 2021 to April 2023. RESULTS: The positivity rate for NTS (69.5 %, 269/387) nearly matches that of metagenomic next-generation sequencing (mNGS) (74.7 %, 289/387; p = 0.128) and surpasses the positivity rate of conventional blood culture (BC) (33.9 %, 131/387; p < 0.01). Frequent pathogens detected by NTS included Klebsiella pneumoniae (n = 54), Pseudomonas aeruginosa (n = 36), Escherichia coli (n = 36), Enterococcus faecium(n = 30), Acinetobacter baumannii(n = 26), Staphylococcus aureus(n = 23), and Human cytomegalovirus (n = 37). Against a composite BSI diagnostic standard, NTS demonstrated a sensitivity and specificity of 84.0 % (95 % CI 79.5 %-87.7 %) and 90.1 % (95 % CI 81.7 %-88.5 %), respectively. The concordance between NTS and mNGS results (the percentage of total cases where both either detected BSI-related pathogens or were both negative) was 90.2 % (359/387), whereas the consistency between NTS and BC was only 60.2 % (233/387). In 80.6 % (50/62) of the samples with identical pathogens identified by both NTS tests and BCs, the genotypic resistance identified by NTS correlated with culture-confirmed phenotypic resistance. Using NTS, 95 % of samples can be tested and analyzed in approximately 7 h, allowing for early patient diagnosis. CONCLUSIONS: NTS is rapid, sensitive, and efficient for detecting BSIs and drug-resistant genes, making it a potential preferred diagnostic tool for early infection identification in critically ill patients.

The antibiogram of pus cultures in federal tertiary care hospital, Islamabad and its utility in antimicrobial stewardship.

Background and Objectives: Antimicrobial resistance has emerged as a significant global health threat. Infections caused by Multi Drug-Resistant (MDR) bacteria pose formidable challenges in terms of treatment options and patient outcomes. Pus cultures serve as crucial diagnostic tools in identifying the agents responsible for various infections, and their antimicrobial susceptibility patterns which help in establishment of empirical therapy guidelines. This study was conducted to determine the pathogen and its susceptibility pattern from pus cultures and to generate antibiogram in our tertiary care setting. Materials and Methods: It was a cross-sectional study, conducted for a period of six months, from July 2022 to December 2022, in the Pathology Department of Pakistan Institute of Medical Sciences (PIMS). Results: Out of total 2507 samples received, 1242 (49.5%) showed positive culture. Among the 1242 positive samples, 364 were Gram positive cocci (GPCs) and 878 were Gram negative rods (GNRs). Methicillin resistant Staphylococcus aureus (MRSA) was the most common isolate (23%) followed by Klebsiella pneumoniae (22.6%), Pseudomonas aeruginosa (16.9%), Enterobacter spp. (15.5%) and Escherichia coli (14.2%). Vancomycin was found to be highly effective (100%) against MRSA. GPCs were highly susceptible to linezolid (98%) while GNRs showed high level of sensitivity to colistin (96%) and tigecycline (92%). Conclusion: The generation of a local antibiogram specific to the hospital setting is essential to effectively manage infections empirically and preserve the efficacy of existing antibiotics. By implementing antimicrobial stewardship practices based on a better understanding of antibiotic susceptibility patterns, we can contribute to the mitigation of antibiotic resistance and improve patient outcomes.

Rapid antibiotic susceptibility testing (RAST) of blood cultures in enterobacteria with inducible chromosomal AmpC-type ß-lactamase.

INTRODUCTION: Early and adequate treatment of bloodstream infections decreases patient morbidity and mortality. The objective is to develop a preliminary method for rapid antibiotic susceptibility testing (RAST) in enterobacteria with inducible chromosomal AmpC. METHODS: RAST was performed directly on spiked blood cultures of 49 enterobacteria with inducible chromosomal AmpC. Results were read at 4, 6 and 8h of incubation. Commercial broth microdilution was considered the reference method. Disks of 10 antibiotics were evaluated. RESULTS: The proportion of readable tests at 4h was 85%. All RAST could be read at 6 and 8h. For most antibiotics, the S or R result at 4, 6 and 8h was greater than 80% after tentative breakpoints were established and Area of Technical Uncertainty was defined. CONCLUSIONS: This preliminary method seems to be of practical use, although it should be extended to adjust the breakpoints and differentiate them by species.

The microenvironment in antibiotic susceptibility testing.

Antibiotic susceptibility testing (AST) by agar diffusion has been repeatedly standardized and, in most cases, gives results which predict clinical success when antibiotic treatment is based on such results. The formation of the inhibition zone is due to a transition from planktonic to biofilm mode of growth. The kinetics of the interaction of antibiotics with bacteria is similar during AST by agar diffusion and during administration of antibiotics to the patients. However, the Mueller-Hinton agar (MHA) recommended for AST agar diffusion test is fundamentally different from the composition of the interstitial fluid in the human body where the infections take place and human cells do not thrive in MH media. Use of RPMI 1640 medium designed for growth of eucaryotic cells for AST of Pseudomonas aeruginosa against azithromycin results in lower minimal inhibitory concentration, compared to results obtained by MHA. The reason is that the RPMI 1640 medium increases uptake and reduces efflux of azithromycin compared to MHA. During treatment of cystic fibrosis patients with azithromycin, mutational resistance occur which is not detected by AST with MHA. Whether this is the case with other antibiotics and bacteria is not known but it is of clinical importance to be studied.

Infant Salmonella enterica Meningitis: A Rare Case Report and Review of Literature.

Meningitis caused by Salmonella enterica can be a fatal condition that is more common in low- and middle-income countries and uncommon in infants. This case of a 2-month-old male infant reported Salmonella meningitis symptoms, such as fever, irritability, altered sensorium, and diarrhoea. Clinical examination revealed bulging anterior fontanelles, dehydration, and sunken eyes. Screening for normal hearing, cranial ultrasound, and magnetic resonance imaging (MRI) revealed no brain abnormalities. A cerebrospinal fluid (CSF) culture revealed gram-negative Salmonella enterica bacilli. Treatment with meropenem and ampicillin was initiated after antibiotic susceptibility testing showed sensitivity. The patient's cerebrospinal fluid parameters and bacterial growth improved after antibiotic therapy. Two weeks later, the baby was neurologically healthy and discharged. Paediatricians should be aware that Salmonella enterica can cause meningitis in children with non-specific symptoms.

Mycobacterium porcinum Disseminated Infection in Non-severely Immunocompromised Host.

Mycobacterium porcinum is a nontuberculous mycobacteria (NTM) recently identified to cause human infection. Correct speciation of NTMs can be difficult and result in misdiagnosis and delayed treatment. Because of the paucity of the literature, there is a lack of awareness of the possibility of serious infections caused by M. porcinum. Although severe infections tend to occur in individuals with certain risk factors, the primary being an immunocompromised state, our case illustrates that it can also be possible in non-severely immunocompromised individuals. A 65-year-old male with a medical history of diabetes mellitus (DM), end-stage renal disease (ESRD) on hemodialysis (HD), congestive heart failure (CHF), and chronic obstructive pulmonary disease (COPD) was admitted to the emergency room due to a laceration on his right lower leg following a fall. He reported shortness of breath but denied other respiratory symptoms. On examination, he showed signs of infection and increased oxygen requirement compared to baseline. Blood culture was positive for acid-fast bacilli (AFB), initially reported as M. avium complex (MAC) and later confirmed as M. porcinum through gene sequencing and morphology analysis. Interval blood cultures taken a week later confirmed true M. porcinum bacteremia. Treatment initially involved intravenous antibiotics- imipenem and ciprofloxacin before transitioning to oral linezolid and ciprofloxacin based on sensitivities. Following 10 days of antibiotic therapy, subsequent blood cultures returned negative, and treatment with oral antibiotics was advised, with continued outpatient follow-up with infectious disease in two weeks. M. porcinum, typically considered a contaminant in healthy individuals, was identified as the causative agent of a disseminated infection in a non-severely immunocompromised patient. This case underscores the importance of accurately identifying the specific mycobacterial species, confirming true infection, and conducting antibiotic susceptibility testing due to the distinct antibiotic susceptibility profile of M. porcinum compared to other NTM like MAC.

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

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