Antimicrobial Susceptibility Testing Methods Research Articles

Assessment of the Antibacterial Effects of Bismuth Nanoparticles against Enterococcus faecalis.

Introduction Enterococcus faecalis (E. faecalis) is the most important species in dentistry and plays a significant role in the etiology of persistent apical lesions after root canal treatment. Up to date, the intracanal application of 2% chlorhexidine for 7 days is the best way to eliminate E. faecalis. However, due to the ability of this bacterium to persist and survive in harsh environments, many studies have been directed towards finding an alternative strategy for prevention or eradication of it. This study was conducted to investigate the effect of bismuth nanoparticles on E. faecalis, as an etiologic factor in recurrent root canal infections. Methods Forty patients, referred to Endodontic Ward of Shiraz University of Medical Science for endodontic pretreatment, provided root canal samples. First, all samples were transferred in Enterococcosel broth and incubated. Then, samples which showed growth were plated on blood agar plates and incubated for further PCR procedure. Nanoparticle powder was dissolved in high-purity water, and the final concentration of bismuth nanoparticles (BiNPs) was measured by the spectrophotometer. Minimum inhibitory concentration (MIC) of BiNPs against E. faecalis was determined by microbroth dilution method according to methods for antimicrobial susceptibility tests. Also, bactericidal assays were conducted in Mueller-Hinton broth medium and reported as the concentration of BiNPs that reduced the viable bacterial count by 99.9%. Results Of all samples, 77.5% revealed the presence of E. faecalis by PCR. Also, E. faecalis growth inhibition was observed at concentrations ranging from 0.625 μg/ml to 20 μg/ml (geometric mean: 2.337 μg/ml), and the MBC values were between 1.25 μg/ml and 40 μg/ml (geometric mean: 4.781 μg/ml), which in comparison with chlorhexidine, these values were about one-eighth of chlorhexidine. Conclusion The experimental data suggest that bismuth nanoparticles could be an interesting alternative to combat E. faecalis, which, in view of the advantages mentioned for bismuth nanoparticle like inhibiting Streptococcus mutans biofilm formation and higher antibacterial activity compared to chlorhexidine, can be suggested to be used in different fields of dentistry.

Is it time to move away from polymyxins?: evidence and alternatives.

Increasing burden of carbapenem resistance and resultant difficult-to-treat infections are of particular concern due to the lack of effective and safe treatment options. More recently, several new agents with activity against certain multidrug-resistant (MDR) and extensive drug-resistant (XDR) Gram-negative pathogens have been approved for clinical use. These include ceftazidime-avibactam, meropenem-vaborbactam, imipenem-cilastatin-relebactam, plazomicin, and cefiderocol. For the management of MBL infections, clinically used triple combination comprising ceftazidime-avibactam and aztreonam is hindered due to non-availability of antimicrobial susceptibility testing methods and lack of information on potential drug-drug interaction leading to PK changes impacting its safety and efficacy. Moreover, in several countries including Indian subcontinent and developing countries, these new agents are yet to be made available. Under these circumstances, polymyxins are the only last resort for the treatment of carbapenem-resistant infections. With the recent evidence of suboptimal PK/PD particularly in lung environment, limited efficacy and increased nephrotoxicity associated with polymyxin use, the Clinical and Laboratory Standards Institute (CLSI) has revised both colistin and polymyxin B breakpoints. Thus, polymyxins 'intermediate' breakpoint for Enterobacterales, P. aeruginosa, and Acinetobacter spp. are now set at ≤ 2mg/L, implying limited clinical efficacy even for isolates with the MIC value 2mg/L. This change has questioned the dependency on polymyxins in treating XDR infections. In this context, recently approved cefiderocol and phase 3 stage combination drug cefepime-zidebactam assume greater significance due to their potential to act as polymyxin-supplanting therapies.

Agreement of Quantitative and Qualitative Antimicrobial Susceptibility Testing Methodologies: The Case of Enrofloxacin and Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the causal agent of colibacillosis, one of the most common bacterial infections in the poultry sector. Antimicrobial susceptibility testing (AST) is essential for rational and prudent antimicrobial therapy. Subsequently, uniformity in test results from the various testing methodologies used in diagnostic laboratories is pivotal. The aim of this study was therefore to evaluate the agreement between different AST methods in determining fluoroquinolone resistance in APEC. Twenty APEC isolates were selected and subjected to four different susceptibility tests: the quantitative microbroth dilution, agar dilution and gradient strip tests, and the qualitative disk diffusion method. The experiments were performed in triplicate. Categorical agreement, essential agreement and different errors were assessed. Moreover, agreement was also evaluated by calculating intraclass correlation coefficients (ICCs) for the quantitative tests and determining the Pearson correlation coefficients for the agreement between the disk diffusion method and the quantitative tests. Categorical agreement and essential agreement when compared with the microbroth technique ranged from 85–95% and 85–100%, respectively. No very major errors (false susceptible) and only one major error (false resistant) and minor errors (results involving an intermediary category) were detected. The calculated ICC values of the three quantitative tests fluctuated around 0.970 (range 0.940–0.988). There was a high negative correlation between the disk diffusion method and the other tests (correlation coefficients ranging from −0.979 to −0.940), indicating a clear inverse relationship between the minimum inhibitory concentration value and the zone diameter of growth inhibition. In conclusion, the overall agreement between the four different testing methodologies was very high. These results confirm the reliability of the disk diffusion and gradient strip test methods as substantiated alternatives, next to the gold standard agar and microbroth dilution, for fluoroquinolone susceptibility testing of APEC isolates.

Comparison of two methods for cell count determination in the course of biocide susceptibility testing

The inoculum density is an important parameter for numerous experimental approaches in bacteriology, including antimicrobial susceptibility testing (AST), biocide susceptibility testing (BST) and biocide efficacy testing (BET). Methods to determine the inoculum density commonly refer to cell counts and have been described for BET according to the German Medical Veterinary Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) and for AST according to the Clinical and Laboratory Standards Institute (CLSI). In this study, the DVG method using 1000 μL volumes of two different dilution steps and the AST method according to CLSI using a 100 μL volume of a single dilution step from the inoculum suspension were compared. For this, each of the four reference strains, Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442, was comparatively tested 28 times using the inoculum preparation according to DVG. The results were statistically analysed using Bland-Altman plots and 95 % limits of agreement (AL). Moreover, cell counts were correlated with the optical density of the bacterial suspensions used. In comparison, the CLSI method measured lower values for colony-forming units (CFU) of -0.12 log10 compared to the DVG method. Overall, both methods returned an AL of -0.52 to 0.27 log10. Since the variations observed between the two methods were within one log10 step and the measured CFUs did not differ systematically, both methods proved to be suitable for cell count determination. Therefore, the CLSI method, which is less complex and less time-consuming, is recommended.

Impact of Rapid Antimicrobial Susceptibility Testing in Gram-Negative Rod Bacteremia: a Quasi-experimental Study.

Clinical justification for rapid antimicrobial susceptibility testing (AST) in Gram-negative rod (GNR) bacteremia is compelling; however, evidence supporting its value is sparse. We investigated the impact of rapid AST on clinical and antimicrobial stewardship outcomes in real-world practice. We performed a before-and-after quasi-experimental study from February 2018 to July 2019 at a tertiary hospital of the 24-h/day, 7-day/week implementation of the direct Vitek 2 AST method from positive blood culture broth for GNR bacteremia with electronic isolate-specific de-escalation comments and daytime antibiotic stewardship program (ASP) intervention. The primary outcome was time to appropriate antibiotic escalation or de-escalation, and secondary outcomes included time to oral antibiotic stepdown, hospital length of stay (LOS), all-cause 30-day mortality, 7-day incidence of acute kidney injury (AKI), and 30-day incidence of Clostridioides difficile infection (CDI). A total of 671 GNR isolates were included from 643 adult patients. Among patients for whom antibiotic change occurred after rapid AST result, rapid AST was associated with a trend in decreased time to escalation or de-escalation (hazard ratio, 1.22; 95% confidence interval [CI], 0.99 to 1.51; P = 0.06), with median times of 52.3 versus 42.2 h. Secondary outcomes were similar in both groups and include median time to oral antibiotic stepdown, LOS, all-cause mortality, and incidence of AKI and CDI. Rapid AST led to improved stewardship measures but did not impact clinical patient outcomes. These results highlight that multiple variables in addition to the timing of the AST result contribute to clinical outcome and that further intervention may be required to clinically justify rapid AST implementation.

Comprehensive phenotypic and genotypic characterization and comparison of virulence, biofilm, and antimicrobial resistance in urinary Escherichia coli isolated from canines

Urinary tract infections (UTIs) affect nearly half of women and an estimated 14 % of the canine companion animal population at least once in their lifetime. As with humans, Escherichia coli is the most commonly isolated bacteria from canine UTIs and infections are dominated by specific phylogenetic groups with notable virulence attributes. In this study, we evaluated uropathogenic E. coli (UPEC) (n = 69) isolated from canine UTIs phenotypically and genotypically for virulence factors, biofilm formation and antimicrobial resistance profiles. Biofilm formation in UPEC strains was positively associated with common virulence factors including papG (p = 0.006), fimH (p < 0.0001), sfaS (p = 0.004), focA (p = 0.004), cnf-1 (p = 0.009) and hlyA (p = 0.006). There was a negative association between biofilm formation and phenotypic antimicrobial resistance for ampicillin (p < 0.0004), ciprofloxacin (p < 0.0001), and trimethoprim-sulfamethoxazole (p < 0.02), as well as multidrug resistance (isolates resistant to ≥ 3 classes of antimicrobials) (p < 0.0002), and the presence of extended spectrum beta-lactamase (ESBL)-producing genes (p < 0.05). In conclusion, UPECs isolated from clinical cases of canine UTIs show a broad negative association between antimicrobial resistance and biofilm formation, and this observation is supported both by phenotypic and genotypic endpoints. As the biofilm formation may result in antimicrobial tolerance, this could be a secondary evasive tactic of UPEC lacking traditional antimicrobial resistance traits. This observation is important for veterinary practitioners to consider when treating puzzling chronic intractable and/or recurrent cases of UTI that appear to be susceptible to antimicrobial therapy via traditional antimicrobial susceptibility testing (AST) methods.

Electrical antimicrobial susceptibility testing based on aptamer-functionalized capacitance sensor array for clinical isolates

To prescribe effective antibiotics to patients with bacterial infections in a timely manner and to avoid the misuse of antibiotics, a rapid antimicrobial susceptibility test (AST) is essential. However, conventional AST methods require more than 16 h to provide results; thus, we developed an electrical AST (e-AST) system, which provides results within 6 h. The proposed e-AST is based on an array of 60 aptamer-functionalized capacitance sensors that are comparable to currently available AST panels and a pattern-matching algorithm. The performance of the e-AST was evaluated in comparison with that of broth microdilution as the reference test for clinical strains isolated from septic patients. A total of 4,554 tests using e-AST showed a categorical agreement of 97% with a minor error of 2.2%, major error of 0.38%, and very major error of 0.38%. We expect that the proposed e-AST could potentially aid antimicrobial stewardship efforts and lead to improved patient outcomes.

Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry.

Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: (i) to assess the antibiotic susceptibility of uropathogenic Escherichia coli isolates, exhibiting different antibiotic resistance phenotypes, directly in artificially contaminated urine samples using a flow cytometry (FC) based protocol; (ii) to optimize the protocol on urine samples deliberately contaminated with bacterial suspensions prepared from uropathogenic E. coli strains. Results: The results of the FC based antimicrobial susceptibility testing (AST) protocol were compared with the reference AST methods results (disk diffusion and broth microdilution) for establishing the sensitivity and specificity. The proposed FC protocol allowed the detection and quantification of uropathogenic E. coli strains susceptibility to nitrofurantoin, trimethoprim–sulfamethoxazole, ciprofloxacin, and ceftriaxone within 4 h after the inoculation of urine specimens. The early availability of preliminary antibiotic susceptibility results provided by direct analysis of clinical specimens could essentially contribute to a more targeted emergency therapy of UTIs in the anticipation of AST results obtained by reference methodology. Conclusions: This method will increase the therapeutic success rate and help to prevent the emergence and dissemination of drug resistant pathogens.

Synthesis and antibacterial properties of novel Al2O3-Ag anodised composite coating

ABSTRACT In this research, Al2O3-Ag anodic coating was synthesised by a single-step anodising and the antibacterial properties were investigated. The microstructure of the coatings was studied by FESEM and XRD analyses. The hardness test was also carried out to assess the mechanical properties of the coatings. Antimicrobial susceptibility and colony count test (CFU) methods were used to evaluate the antibacterial behaviour. The results showed that the microstructure of the coatings were amorphous with a columnar structure containing the longitudinal porosities. The average pore diameter was obtained about 55 nm and 13 nm for Al2O3-Ag and Al2O3 coatings, respectively. Moreover, the antibacterial results also demonstrated that the composite anodic films had the antibacterial activity whilst the Gram-negative bacteria were more sensitive against silver nanoparticles in comparison with the Gram-positive bacteria.

Rapid Detection of mecA and femA Genes by Loop-Mediated Isothermal Amplification in a Microfluidic System for Discrimination of Different Staphylococcal Species and Prediction of Methicillin Resistance.

Staphylococcal infection is one of the most pressing problems in modern medicine due to the increasing antibiotic resistance with the overuse of antibiotics. Conventional methods for identification and antimicrobial susceptibility testing (AST) generally take 3–7 days and require skilled technicians. In this study, a microfluidic device based on loop-mediated isothermal amplification (LAMP) was developed, which could discriminate Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis and predict their methicillin resistance by targeting the mecA and femA genes within 70 min including the hands-on time. Multiplex and real-time detection was achieved in a closed system without aerosol contamination. The limits of detection (LODs) for S. aureus, S. epidermidis, S. hominis, and methicillin-resistant S. aureus (MRSA) were 20 CFU/reaction, while that for S. haemolyticus was 200 CFU/reaction. A total of 102 positive cultures of cerebrospinal fluid (CSF) were also tested, and the results were in good agreement with those from conventional methods. Furthermore, mixed cultures were readily identified by our method. The portable and integrated device is rapid, accurate, and easy to use, which can provide information for prompt institution of proper antimicrobial therapy and has great potential for clinical applications, especially in resource-limited settings.

Antimicrobial resistance pattern of Acinetobacter; a multicenter study, comparing European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI); evaluation of susceptibility testing methods for polymyxin

Introduction: Acinetobacter species in clinical isolates cause severe infections including meningitis, bloodstream infection, ventilator-associated pneumonia, and surgical site infections. Objectives: In the present study, we evaluated Acinetobacter drug resistance using both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) antimicrobial susceptibility test methods. Materials and Methods: Clinical specimens of 128 patients who were admitted in three referral tertiary care teaching hospitals were enrolled in 2014. Blood and other sterile fluid samples, endotracheal secretion, ulcer, urine and other clinical specimen cultures were included, and microbial resistance of Acinetobacter isolates was determined and compared with disk diffusion and E-test antimicrobial susceptibility methods, using both the EUCAST and CLSI standards. Cohen’s kappa coefficient was also reported. Results: The highest percentage of resistance (96.9%) was found for meropenem and imipenem antimicrobials, and the lowest resistance (82.8%) was found for amikacin. The highest kappa agreement coefficient was for ciprofloxacin (kappa coefficient = 0.783), and the lowest kappa was for amikacin (kappa coefficient = 0.21). Conclusion: According to the results, it is better to consider amikacin as a choice in combination with another effective antimicrobial for treatment of drug resistant Acinetobacter.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy.

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.

Comparison of Microdilution Method with Agar Dilution Method for Antibiotic Susceptibility Test of Neisseria gonorrhoeae.

IntroductionAntimicrobial resistance (AMR) of Neisseria gonorrhoeae (N. gonorrhoeae) becomes a grave public health problem in the world. A strengthened Antimicrobial Resistance Surveillance Program is needed to track the trend of AMR development. However, the lack of a proper antimicrobial susceptibility test (AST) method is a barrier to expand the AMR surveillance in China. Traditional agar dilution (AD) method is laborious and E-test strips have no approval license for clinical use. Herein, a Chinese group modified the microdilution (MD) method for clinical ASTs. The objective of this study is to compare the MD method with the AD method for N. gonorrhoeae AST.Materials and MethodsA total of 166 clinical isolates were tested for antimicrobial susceptibility of ceftriaxone, spectinomycin, azithromycin, ciprofloxacin, tetracycline, and penicillin using MD and AD method simultaneously. Results of MD method were read manually or automatically. Rates of essential agreement (EA), category agreement (CA), minor error, and very major error were compared.ResultsThe total EAs (compared with results read manually) of penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, and azithromycin were 90.4%, 97.0%, 85.5%, 100.0%, 94%, and 72.3%; and CAs were 82.5%, 94.0%, 100%, 100%, 95.2%, and 94%, respectively.ConclusionWe conclude that the MD method might be an alternative for clinical AST of N. gonorrhoeae in China. In particular, MD method has the potency of accurate differentiation of isolates resistant to ceftriaxone or azithromycin, which were empirically recommended for gonococcal treatment, but its quality remained suboptimal, and further improvement is needed for clinical use.

Polymyxin B resistance rates in carbapenem-resistant Pseudomonas aeruginosa isolates and a comparison between Etest® and broth microdilution methods of antimicrobial susceptibility testing.

Polymyxin B has been considered to be the last line of defense for life-threatening infections caused by multiple drug resistant gram-negative pathogens, including carbapenem-resistant Pseudomonas aeruginosa (CRPA). The present study analyzed CRPA resistance to polymyxin B in the Suzhou district of China. Additionally, polymyxin B resistance rates were compared in different parts of the world to determine global trends. The present study also assessed the reliability and effectiveness of the Etest® in a clinical setting, as laboratories lack a reliable and efficient susceptibility test for polymyxin B. The susceptibility rate of polymyxin B reached 96.0%, which is in accordance with results obtained from the United States of America, Europe, Africa and the majority of Asian countries. However, the rate of polymyxin B non-susceptibility (resistant or intermediate) in Singapore is 0.53 (95% confidence interval, 0.12-0.93). In addition, the susceptibility rate of polymyxin B determined via Etest® was not significantly increased compared with that determined via broth microdilution (98.0 vs. 96.0%; P=0.558). Essential and categorical agreement rates reached 98.0%. In conclusion, the polymyxin B resistance rate of CRPA isolates is relatively low in the majority of countries, with the exception of Singapore. Furthermore, Etest® may be a reliable clinical method for the measurement of polymyxin B resistance in CRPA isolates.

Eight rules for improving the quality of papers on the antimicrobial susceptibility of bacteria isolated from aquatic animals.

The methods for antimicrobial susceptibility testing of bacteria, although relatively simple, are not robust. As a consequence, if the data generated in such tests are to be commensurate, all susceptibility tests must be performed using standard protocols. A review of the published literature of antimicrobial susceptibility testing of bacteria from aquatic animals revealed a frequent occurrence of significant errors, particularly with regard to testing methodology, quality control and the use of appropriate interpretive criteria in the performance and reporting of susceptibility tests. This opinion piece provides a set of rules that, if followed, would help authors to avoid these frequently detected shortcomings.

Comparison of Automated and Manual Methods for Antimicrobial Susceptibility Testing

Background: Carbapenems are considered the broadest-spectrum β-lactam agents and are often required for treatment of severe hospital-acquired infections caused by multidrug-resistant Gram-negative organisms. Minimum inhibitory concentrations (MICs) are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents.&#x0D; Aims and Objectives: To compare the MIC obtained by Broth Microdilution method (BMD) with that of Vitek-2(automated method) for recovered isolates of Klebsiella pneumoniae.&#x0D; Materials and Methods: Prospective study conducted over a period of one year. It included all isolates of Klebsiella pneumoniae recovered from blood culture of the patients. The identification and antimicrobial susceptibility was done on Vitek-2.These Isolates were subjected to Microbroth dilution method for MIC determination.&#x0D; Results: Out of the 55 meropenem resistant isolates by vitek-2, 20(36.3%) had MIC of ≥256 µg/ml followed by 18(32.7%) isolates with a MIC of 128 µg/ml, followed by 11(20%) isolates with MIC of 64 µg/ml and 6(10.9%) isolates with MIC of 32 µg/ml. Also among 15 meropenem sensitive isolates by Vitek-2, 13(86.7%) had MIC of ≤0.5 µg/ml, followed by two (13.3%) isolates with MIC of 2 µg/ml. Results obtained by vitek 2 were compared with those from BMD(the reference method), which showed a 13.3% minor error rate and no major or very major error rate.&#x0D; Conclusion: Overall, the Vitek 2 performance was comparable to that of BMD for testing a limited number of Klebsiella pneumoniae isolates.

Altitude Effect on the Properties of Honeys from the Region of Jijel (Algeria)

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Metagenomic Next-Generation Sequencing in Clinical Microbiology

Identification of causative pathogens in infectious disease is a critical component of health care, as infectious diseases continue to be a leading cause of mortality and morbidity worldwide. In addition, the detection of drug resistance by traditional and novel antimicrobial susceptibility testing methods is becoming ever more important as antimicrobial resistance continues to emerge and spread. While culture-based methods are the current reference standard for identifying microbes, the time required to achieve results and the difficulty in culturing certain fastidious organisms have led to the development of multiple alternatives. Alternatives to culture, such as polymerase chain reaction (PCR) assays, serologic assays, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene sequencing have been used in clinical laboratories, and positive impacts on patient care have been documented. Nonetheless, such methods require either presumption about the type of microbes present in the sample or growth of the organism(s) before analysis. Subsequently, there are some documented limitations in the routine detection methods for clinically relevant organisms.In contrast to the routine alternatives listed above, modern nucleic acid sequencing platforms support sequencing of random DNA strands, an approach known as “shotgun” sequencing of the DNA, also known as metagenomic next-generation sequencing (mNGS) present in the sample. The mNGS approach offers an unbiased and hypothesis-free approach to pathogen identification with the future potential to (i) achieve results in 12 to 24 hours; (ii) avoid the challenges associated with growth of fastidious organisms; (iii) avoid the biased growth that occurs when only routine culture medium is used; (iv) detect viral, fungal, and parasitic organisms in the same assay; and (v) detect the presence of drug resistance genes. Advances in mNGS technology and data analysis have reduced testing costs to the point where the potential advantages of mNGS over culture and other methods warrant its development for use in clinical settings. In this review, the mNGS approach is discussed, along with a comparison to other methods, limitations, and suggestions for further development and overcoming hurdles to adoption in the clinical setting.

Validation of an antimicrobial susceptibility testing protocol for Brachyspira hyodysenteriae and Brachyspira pilosicoli in an international ring trial

Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.

Detection of Antibiotic Susceptibility by Colorimetric Minimum Inhibitory Concentration in Staphylococcal Isolates

Abstract Background Staphylococcus species are implicated in a wide range of community and hospital-acquired infections, which necessitates rapid provision of antibiotic susceptibility results for patients at risk. The current study evaluated the usefulness of applying broth microdilution (BMD) and agar dilution methods for antimicrobial susceptibility testing of Staphylococci using tetrazolium salts as redox indicators. Methods Minimum inhibitory concentration (MICs) of eight antimicrobials representing different antibiotic classes as recommended by CLSI; vancomycin (VA), linezolid (LZD), oxacillin (OX), gentamicin (CN), tetracycline (TE), ciprofloxacin (CIP), erythromycin (E) and clindamycin (DA) was investigated for 80 isolates of Staphylococci by a modified BMD with the addition of dimethyl thiazole diphenyl tetrazolium bromide (MTT). As well as agar dilution with the addition of MTT &amp; triphenyl tetrazolium chloride (TTC) at the standard bacterial concentration together with addition of MTT at an experimental bacterial concentration. Results BMD (MTT) showed the highest agreement regarding most antibiotics among different isolates in comparison with the standard BMD. All performed methods showed significant agreement except agar dilution (TTC) using TE, DA, E, and agar dilution (MTT 108) using DA as well as agar dilution (MTT 104) using E among S. aureus. Conclusions Colorimetric BMD was rapid, easy to interpret and showed the highest agreement with the standard BMD. Colorimetric agar dilution (MTT) was less tedious than BMD. The colorimetric MIC method using MTT may be a useful surrogate of antimicrobial susceptibility testing among Staphylococcus isolates.