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Background The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines are the most popular breakpoint guidelines used in antimicrobial susceptibility testing worldwide. The EUCAST guidelines are freely available to users while CLSI is available for non-members as a package of three documents for US $500 annually. This is prohibitive for clinical microbiology laboratories in resource poor settings. We set out to compare antibiotic susceptibility determined by the two guidelines to determine whether adoption of EUCAST guidelines would significantly affect our susceptibility patterns. Methods We reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa ) isolates from an automated microbiology identification system (VITEK-2) at the Aga Khan University Hospital Nairobi’s Pathology department. These MICs were then analyzed using both CLSI 2015 and EUCAST 2015 guidelines and classified as resistant, intermediate or susceptible. We compared the susceptibility and agreement between the CLSI and EUCAST categorizations. Results Susceptibility data from a total of 5165 E. coli , 1103 S. aureus and 532 P. aeruginosa isolates were included. The concordance rates of the two guidelines for E. coli, S. aureus and P. aeruginosa ranged from 78.2 to 100 %, 94.6 to 100 % and 89.1 to 95.5 % respectively. The kappa statistics for E. coli MICs revealed perfect agreement between CLSI and EUCAST for cefotaxime, ceftriaxone and trimethoprim–sulfamethoxazole, almost perfect agreement for ampicillin, ciprofloxacin, cefuroxime, gentamicin and ceftazidime, substantial agreement for meropenem, moderate agreement for cefepime and amoxicillin-clavulanate, fair agreement for nitrofurantoin and poor agreement for amikacin. For S. aureus the kappa statistics revealed perfect agreement for penicillin, trimethoprim–sulfamethoxazole, levofloxacin, oxacillin, linezolid and vancomycin, almost perfect agreement for clindamycin, erythromycin and tetracycline and moderate agreement for gentamicin. For P. aeruginosa the kappa analysis revealed moderate to almost perfect agreement for all the anti-pseudomonal antibiotics. Conclusion The results show comparable antibiotic susceptibility patterns between CLSI and EUCAST breakpoints. Given that EUCAST guidelines are freely available, it makes it easier for laboratories in resource poor settings to have an updated and readily available reference for interpreting antibiotic susceptibilities.

Background Blood stream infections (BSIs) are a major cause of morbidity and mortality. The time from taking blood cultures to obtain results of antibiotic sensitivity can be up to five days which impacts patient care. The Alfred 60 AST™ can reduce laboratory time from positive culture bottle to susceptibility results from 16 to 25 h to 5–6 h, transforming patient care. To evaluate the diagnostic accuracy of a rapid antimicrobial susceptibility system, the Alfred 60 AST™, in clinical isolates from patients with BSIs and confirm time to results. 301 Gram-negative and 86 Gram-positive isolates were analysed directly from positive blood culture bottles following Gram staining. Antimicrobial susceptibility results and time-to-results obtained by rapid Alfred 60 AST system and BD Phoenix were compared . Results A total of 2196 antimicrobial susceptibility test results (AST) were performed: 1863 Gram-negative and 333 Gram-positive. AST categorical agreement (CA) for Alfred 60 AST™ was 95% (1772/1863) for Gram-negative and 89% (295/333) for Gram-positive isolates. Gram-negative CA: ampicillin 96% (290/301); ciprofloxacin 95% (283/297); ceftriaxone 96% (75/78); meropenem 97% (288/297); piperacillin-tazobactam 95% (280/295); gentamicin 94% (279/297) and amikacin 93% (277/298). The median time to susceptibility results from blood culture flagging positive was 6.3 h vs 20 h ( p < 0.01 ) for Alfred system vs BD Phoenix™. Conclusion Alfred 60 AST system greatly reduced time to antimicrobial susceptibility results in Gram-negative and Gram-positive BSIs with good performance and cost, particularly for Gram-negative bacteraemia.

Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies.

Background: Bacteria produce volatile organic compounds (VOCs) during growth, which can be detected by colorimetric sensor arrays (CSAs). The SpecifAST® system (Specific Diagnostics) employs this technique to enable antibiotic susceptibility testing (AST) directly from blood cultures without prior subculture of isolates. The aim of this study was to compare the SpecifAST® AST results and analysis time to the VITEK®2 (bioMérieux) system. Methods: In a 12-month single site prospective study, remnants of clinical positive monomicrobial blood cultures were combined with a series of antibiotic concentrations. Volatile emission was monitored at 37 °C via CSAs. Minimal Inhibitory Concentrations (MICs) of seven antimicrobial agents for Enterobacterales, Staphylococcus, and Enterococcus spp. were compared to VITEK®2 AST results. MICs were interpreted according to EUCAST clinical breakpoints. Performance was assessed by calculating agreement and discrepancy rates. Results: In total, 96 positive blood cultures containing Enterobacterales, Staphylococcus, and Enterococcus spp. were tested (269 bug–drug combinations). The categorical agreement of the SpecifAST® system compared to the VITEK®2 system was 100% and 91% for Gram-negatives and Gram-positives, respectively. Errors among Gram-positives were from coagulase-negative staphylococci. Overall results were available in 3.1 h (±0.9 h) after growth detection without the need for subculture steps. Conclusion: The AST results based on VOC detection are promising and warrant further evaluation in studies with a larger sample of bacterial species and antimicrobials.

Antimicrobial resistance (AMR) has emerged as a major threat to public health globally. Accurate and rapid detection of resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment, combined with antimicrobial stewardship, are essential for controlling the emergence and spread of AMR. This article reviews common antimicrobial susceptibility testing (AST) methods and relevant issues concerning the advantages and disadvantages of each method. Although accurate, classic technologies used in clinical microbiology to profile antimicrobial susceptibility are time-consuming and relatively expensive. As a result, physicians often prescribe empirical antimicrobial therapies and broad-spectrum antibiotics. Although recently developed AST systems have shown advantages over traditional methods in terms of testing speed and the potential for providing a deeper insight into resistance mechanisms, extensive validation is required to translate these methodologies to clinical practice. With a continuous increase in antimicrobial resistance, additional efforts are needed to develop innovative, rapid, accurate, and portable diagnostic tools for AST. The wide implementation of novel devices would enable the identification of the optimal treatment approaches and the surveillance of antibiotic resistance in health, agriculture, and the environment, allowing monitoring and better tackling the emergence of AMR.

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

The aim of this study is to investigate the antimicrobial susceptibility of strains isolated from the major hospitals in China. A total of 44 teaching hospitals were involved. Antimicrobial susceptibility testing was conducted by Kirby-Bauer automated systems, and results were interpreted using CLSI criteria. Totally 244,843 strains were isolated in 2018, of which gram-negative bacilli and gram-positive cocci were accounting for 71.8% and 28.2%, respectively. 39.7% of isolates were cultured from lower respiratory tract, 18.8% from urine, 14.8% from blood, 1.3% from cerebrospinal fluid, respectively. Of those, the five major species were most often isolated (65.5%, 63%, 52.3%, and 30.3%). The resistance rate of MRSA to most antimicrobial agents was significantly higher than that of MSSA strains, except for to trimethoprim-sulfamethoxazole in urine specimen. E.coli was still highly susceptible to carbapenem antibiotics, and the resistance rate was less than 5%. Carbapenem resistance among Klebsiella pneumoniae, especially cultured from cerebrospinal fluid, increased significance from 18.6 to 64.1%. The resistance rates of Pseudomonas aeruginosa to carbapenems were nearly 30% in the blood, in urine, and in the lower respiratory tract, but about 60% of that in cerebrospinal fluid. About 80% of Acinetobacter baumannii strains was resistant to imipenem and meropenem, respectively. Bacterial resistance of five major clinical isolates from cerebrospinal fluid to common antibiotics (in particular Carbapenem-resistant Klebsiella pneumoniae) currently shows an increasing trend. It is worth to emphasize the importance of serious control of hospital infection and better management of clinical use of antimicrobial agents.

Infectious diseases caused by bacterial pathogens remain one of the most common causes of morbidity and mortality worldwide. Rapid microbiological analysis is required for prompt treatment of bacterial infections and to facilitate antibiotic stewardship. This study reports an adaptable microfluidic system for rapid pathogen classification and antimicrobial susceptibility testing (AST) at the single-cell level. By incorporating tunable microfluidic valves along with real-time optical detection, bacteria can be trapped and classified according to their physical shape and size for pathogen classification. By monitoring their growth in the presence of antibiotics at the single-cell level, antimicrobial susceptibility of the bacteria can be determined in as little as 30 minutes compared with days required for standard procedures. The microfluidic system is able to detect bacterial pathogens in urine, blood cultures, and whole blood and can analyze polymicrobial samples. We pilot a study of 25 clinical urine samples to demonstrate the clinical applicability of the microfluidic system. The platform demonstrated a sensitivity of 100% and specificity of 83.33% for pathogen classification and achieved 100% concordance for AST.

Several MALDI-TOF MS-based methods have been proposed for rapid detection of antimicrobial resistance. The most widely studied methods include assessment of β-lactamase activity by visualizing the hydrolysis of the β-lactam ring, detection of biomarkers responsible for or correlated with drug-resistance/non-susceptibility, and the comparison of proteomic profiles of bacteria incubated with or without antimicrobial drugs. Antimicrobial-resistance to a number of antibiotics belonging to different classes has been successfully tested by MALDI-TOF MS in a variety of clinically relevant bacterial species including members of family, non-fermenting Gram-negative bacteria, Gram-positive cocci, anaerobic bacteria and mycobacteria, opening this field to further clinically important developments. Early detection of drug-resistance by MALDI-TOF MS can be particularly helpful for clinicians to streamline the antibiotic therapy for a better outcome of patients with systemic infection, in all cases where a prompt and effective antibiotic treatment is essential to preserve organ function and/or patient survival.