Evaluation of an ultra-rapid antibiotic susceptibility testing method on positive blood cultures with Escherichia coli

Blood stream infection (BSI) is related to high mortality and morbidity. Early antimicrobial therapy is crucial in treating patients with BSI. The most common Gram-negative bacteria causing BSI is Escherichia coli. Targeted effective treatment of patients with BSI is only possible if it is based on antibiotic susceptibility testing (AST) data after blood culture positivity. However, there are very few methods available for rapid phenotypic AST and the fastest method takes 4 h. Here we analyzed the performance of a 30 min ultra-rapid method for AST of E. coli directly from positive blood cultures (BC). In total, 51 positive BC with E. coli were studied, and we evaluated the ultra-rapid method directly on positive BC as well as on E. coli colonies cultured on agar plates. The results obtained by the new method were compared with disk diffusion. The method provided accurate AST result in 30 min to Ciprofloxacin and Gentamicin for 92% and 84% of the positive BC samples, respectively. For E. coli isolates retrieved from agar plates, 86% and 96% of the AST results were accurate for Ciprofloxacin and Gentamicin, respectively, after 30 min of assay time. When time to result was modulated in-silico from 30 to 60 minutes for the agar plate samples, accuracy of AST results went up to 92% for Ciprofloxacin and to 100% for Gentamicin. The present study shows that the method is reliable and delivers ultra-rapid AST data in 30 minutes directly from positive BC and as well as from agar plates.

Phenotypical and Genotypical Comparison of Clostridium difficile Isolated from Clinical Samples: Homebrew DNA Fingerprinting versus Antibiotic Susceptibility Testing (AST) and Clostridial Toxin Genes

Background. Clostridium (Clostridioides) difficile is recognized as the major cause of healthcare antibiotic-associated diarrhea. We surveyed a molecular epidemiological correlation between the clinical isolates from two general hospitals in Iran through clustering toxigenic types and antibiotic susceptibility testing (AST) accuracy. Methods. Study population included 460 diarrhoeic specimens from inpatients with a history of antibiotic therapy. All samples underwent enriched anaerobic culture, confirmed by detection of gluD gene with PCR. Toxin status and AST were assessed by the disk diffusion method (DDM) and minimal inhibitory concentrations (MICs) of metronidazole, vancomycin, and rifampin. C. difficile outbreak was analyzed through conventional PCR by tracing toxin genes and Homebrew pulsed-field gel electrophoresis (PFGE) for characterizing isolates within our healthcare systems. Results. A total of 29 C. difficile strains were isolated by enriched anaerobic culture from the clinical samples. Among them, 22 (4.8%) toxigenic profiles yielded toxins A and B (tcdA, tcdB) and binary toxins (cdtA, cdtB). The minimum inhibitory concentration (MIC) was 18.1% and 9% for vancomycin and metronidazole, and all isolates were susceptible to rifampicin and its minimum inhibitory concentration was at <0.003 μg/mL. The most dominant toxigenic and antibiotic-resistant “pulsotype F” was detected through PFGE combined with multiple Clostridial toxigenic pattern and AST. Conclusions. DNA fingerprinting studies represent a powerful tool in surveying hypervirulent C. difficile strains in clinical settings. Resistance to vancomycin and metronidazole, as first-line antibiotics, necessitate accomplishment of proper control strategies and also prescription of tigecycline as a more appropriate option.

Staphylococcus aureus with inducible clindamycin resistance and methicillin resistance in a tertiary hospital in Nepal

Background Staphylococcus aureus is a global public health issue in both community and hospital settings. Management of methicillin-resistant S. aureus (MRSA) infections are tough owing to its resistance to many antibiotics. Macrolide-lincosamide-streptogramin B (MLSB) antibiotics are commonly used for the management of MRSA. This study was aimed to determine the occurrence of inducible clindamycin- and methicillin-resistant S. aureus at a tertiary care hospital in Kathmandu, Nepal. Methods A total of 1027 clinical samples were processed following standard laboratory procedures and antibiotic susceptibility testing of S. aureus was performed by disc diffusion method. MRSA isolates were detected phenotypically using cefoxitin disc, and inducible clindamycin resistance was detected phenotypically using the D-zone test. Results Of 1027 samples, 321 (31.2%) were culture positive, of which 38 (11.8%) were S. aureus. All S. aureus isolates were susceptible to vancomycin, and 25 (67%) of S. aureus isolates were multidrug-resistant. Similarly, 15 (39.5%) of S. aureus were MRSA and 14 (36.5%) were inducible clindamycin-resistant phenotypes. Conclusion Inducible clindamycin and methicillin resistance were common in S. aureus. This emphasizes that the methicillin resistance test and the D-zone test should be incorporated into the routine antibiotic susceptibility testing in hospital settings.

A portable and high-integrated 3D microfluidic chip for bacterial quantification and antibiotic susceptibility testing

On-site single-cell antibiotic susceptibility testing (sc-AST) provides unprecedented technical potential to improve the treatment of bacterial infections and study heterogeneous resistance to antibiotics. Herein, we developed a portable and high-integrated 3D polydimethylsiloxane (PDMS) chip to perform fast and on-site bacteria quantification and sc-AST. The 3D arrangement of the chambers significantly improved the integration of reaction units (~10000/cm2) and widened the dynamic range to 5 orders of magnitude. A capillary valve-based flow distributor was adopted for flow equidistribution in 64 parallel channels and uniform sample loading in as short as 2 s. The degassed PDMS enabled this device to independently dispense the sample into 3D chamber array with almost 100% efficiency. The quantification of Escherichia coli (E. coli) strains with various activity was accomplished in 0.5-2 h, shortened by 20 h in comparison to the traditional plate counting. The functionality of our platform was demonstrated with several effective antibiotics by determining minimum inhibitory concentrations at single-cell level. Furthermore, we utilized the lyophilization of test reagents and needle-mediated reagents rehydration to realize one-step on-site sc-AST. The results indicate that the proposed sc-AST platform is portable, highly sensitive, fast, accurate and user-friendly, thus it has the potential to facilitate precise therapy in time and monitor the treatment. Meanwhile, it could serve as an approach for investigating the mechanisms of heteroresistance at single-cell resolution.