Rapid Gene Silencing Followed by Antimicrobial Susceptibility Testing for Target Validation in Antibiotic Discovery.

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB)-an ancient yet widespread global infectious disease to which 1.6 million people lost their lives in 2021. Antimicrobial resistance (AMR) has been an ongoing crisis for decades; 4.95 million deaths were associated with antibiotic resistance in 2019. While AMR is a multi-faceted problem, drug discovery is an urgent part of the solution and is at the forefront of modern research.The landscape of drug discovery for TB has undoubtedly been transformed by the development of high-throughput gene-silencing techniques that enable interrogation of every gene in the genome, and their relative contribution to fitness, virulence, and AMR. A recent advance in this area is CRISPR interference (CRISPRi). The application of this technique to antimicrobial susceptibility testing (AST) is the subject of ongoing research in basic science.CRISPRi technology can be used in conjunction with the high-throughput SPOT-culture growth inhibition assay (HT-SPOTi) to rapidly evaluate and assess gene essentiality including non-essential, conditionally essential (by using appropriate culture conditions), and essential genes. In addition, the HT-SPOTi method can develop drug susceptibility and drug resistance profiles.This technology is further useful for drug discovery groups who have designed target-based inhibitors rationally and wish to validate the primary mechanisms of their novel compounds' antibiotic action against the proposed target.

Modified HT-SPOTi: An Antimicrobial Susceptibility Testing to Evaluate Formulated Therapeutic Combinations Against Bacterial Growth and Viability.

Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system.

Evaluation of the in vitro activity of ampicillin-sulbactam and cefoperazone-sulbactam against A. Baumannii by the broth disk elution test.

To evaluate the in vitro activity of ampicillin-sulbactam and cefoperazone-sulbactam against A. baumannii using the broth disk elution testing, a total of 150 A. baumannii isolates were collected from across China between January 2019 and January 2021, including 51 carbapenem-susceptible and 99 carbapenem-resistant isolates. Broth disk elution (BDE) and the broth microdilution (BMD) method were performed for all strains. The concentration range of the BDE was 10/10 µg/mL, 20/20 µg/mL, and 30/30 µg/mL for ampicillin-sulbactam, and 37.5/15 µg/mL, 75/30 µg/mL, 112.5/45 µg/mL, and 150/60 µg/mL for cefoperazone-sulbactam, respectively. Compared with BMD, the BDE results of ampicillin-sulbactam and cefoperazone-sulbactam showed a categorical agreement of 83.3% (125/150) and 95.3% (143/150), with minor errors of 16.7% (25/150) and 4.7% (7/150), respectively. No major error or very major errors were detected. The sensitivity differences by BDE of carbapenem-resistant A. baumannii (CRAb) to different concentrations of ampicillin-sulbactam showed statistically significant (p < 0.017), while those to cefoperazone-sulbactam at 37.5/15 µg/mL, 75/30 µg/mL, and 112.5/45 µg/mL were significant (p < 0.008). However, no significant difference in sensitivity was observed between 112.5/45 µg/mL and 150/60 µg/mL (p > 0.008). In conclusion, the BDE is a reliable and convenient method to detect the in vitro activity of cefoperazone-sulbactam against A. baumannii, and the results could serve as a clinical reference value when deciding whether or not to use high-dose sulbactam for the treatment of A. baumannii infections.

Clinical Characteristics and Risk Factors for Infection and Death in Critically Ill Patients with Pulmonary Infection with Elizabethkingia Spp.

Purpose: Elizabethkingia spp. infections have recently increased, and they are difficult to treat because of intrinsic antimicrobial resistance. This study aimed to investigate the clinical characteristics of patients with pulmonary infection with Elizabethkingia spp. and reveal the risk factors for infection and death. Patients and Methods: In this retrospective case-control study, patients were divided into infection and control groups based on the bacterial identification results. Patients in the infection group were further divided into survival and death groups according to their hospital outcomes. Clinical characteristics between different groups were compared. We further analyzed antimicrobial susceptibility testing results of the isolated strains. Results: A total of the 316 patients were divided into infection (n = 79), 23 of whom died, and control (n = 237) groups. Multivariate logistic regression analysis showed that glucocorticoid consumption (OR: 2.35; 95% CI: 1.14-4.81; P = 0.02), endotracheal intubation (OR: 3.74; 95% CI: 1.62-8.64; P = 0.002), and colistin exposure (OR: 2.50; 95% CI: 1.01-6.29; P = 0.046) were significantly associated with pulmonary infection with Elizabethkingia spp. Advanced age (OR: 1.07, 95% CI: 1.00-1.15; P = 0.046), high acute physiology and chronic health evaluation (APACHE) II score (OR: 1.21; 95% CI: 1.01-1.45; P = 0.037), and low albumin level (OR: 0.73, 95% CI: 0.56-0.96; P = 0.025) were significantly associated with in-hospital mortality of infected patients. Elizabethkingia spp. was highly resistant to cephalosporins, carbapenems, macrolides, and aminoglycoside, and was sensitive to fluoroquinolones, minocycline, and co-trimoxazole in vitro. Conclusion: Glucocorticoid consumption, tracheal intubation, and colistin exposure were associated with pulmonary infection with Elizabethkingia spp. for critically ill patients. Patients with advanced age, high APACHE II score, and low albumin level had higher risk of death from infection.

Genomic and phenotypic inconsistencies in Pseudomonas aeruginosa resistome among intensive care patients.

Objective: Pseudomonas aeruginosa, a difficult-to-manage nosocomial pathogen, poses a serious threat to clinical outcomes in intensive care (ICU) patients due to its high antimicrobial resistance (AMR). To promote effective management, it is essential to investigate the genomic and phenotypic differences in AMR expression of the isolates. Methods: A prospective observational study was conducted from July 2022 to April 2023 at Liepaja Regional Hospital in Latvia. The study included all adult patients who were admitted to the ICU and had a documented infection with P. aeruginosa, as confirmed by standard laboratory microbiological testing and short-read sequencing. Since ResFinder is the only sequencing-based database offering antibacterial susceptibility testing (AST) data for each antibiotic, we conducted a comparison of the resistance profile with the results of phenotypic testing, evaluating if ResFinder met the US Food and Drug Administration (FDA) requirements for approval as a new AMR diagnostic test. Next, to improve precision, AST data from ResFinder was compared with two other databases - AMRFinderPlus and RGI. Additionally, data was gathered from environmental samples to inform the implementation of appropriate infection control measures in real time. Results: Our cohort consisted of 33 samples from 29 ICU patients and 34 environmental samples. The presence of P. aeruginosa infection was found to be associated with unfavourable clinical outcomes. A third of the patient samples were identified as multi-drug resistant isolates. Apart from resistance against colistin, significant discrepancies were observed when phenotypic data were compared to genotypic data. For example, the aminoglycoside resistance prediction of ResFinder yielded a major errors value of 3.03% for amikacin, which was marginally above the FDA threshold. Among the three positive environmental samples, one sample exhibited multiple AMR genes similar to the patient samples in its cluster. Conclusion: Our findings underscore the importance of utilizing a combination of diagnostic methods for the identification of resistance mechanisms, clusters, and environmental reservoirs in ICUs.

How to verify and validate a Clinical Microbiology test before it can be used in routine diagnostics, a practical guide.

BACKGROUND: Before a new test can be routinely used in your laboratory, its reliability must be established in the laboratory where it will be used. International standards demand validation and verification procedures for new tests. The International Organization for Standardization (ISO) 15189 was recently updated, and the European Commission's In Vitro Diagnostic Regulation (IVDR) came into effect. These events will likely increase the need for validation and verification procedures. OBJECTIVES: This paper aims to provide practical guidance in validating or verifying microbiology tests, including antimicrobial susceptibility tests in a clinical microbiology laboratory. SOURCES: It summarizes and interprets standards, such as ISO 15189: 20222 and regulations, such as IVDR 2017/745. CONTENT: The reasons for choosing a new test and the outline of the validation and verification plan are discussed. Further, the following topics are touched upon: the choice of reference standard, number of samples, testing procedures, how to solve the discrepancies between results from new test and reference standard, and acceptance criteria. Arguments for selecting certain parameters (such as reference standard and samples size) and examples are given. IMPLICATIONS: With the expected increase in validation and verification procedures due to implementation of IVDR, this paper may aid in planning and executing these procedures.

Care practices of contact lens solutions and microbial contamination among wearers in Ghana.

PURPOSE: This study sought to assess contact lens solutions care practices, and their microbial contamination among contact lens wearers in Ghana and to profile their antibiotic susceptibility pattern. METHODS: The study employed a biphasic approach which involved a cross-sectional design that investigated participants' habits related to care for the solutions with a two-part questionnaire and a microbiological analysis of samples of contact lens care solutions of the participants for microbial contamination. A snowball sampling method provided access to 32 different contact lens wearers in four care facilities in Ghana. In most cases, the participants had no pre-existing familial relationship with each other or with the care facilities. RESULTS: Out of 32 samples of contact lens solutions, 30 were tested for microbial contamination. A total of 23 (76.67 %) samples of contact lens solution were found to be contaminated with Enterobacter sp. (34.80 %), Pseudomonas sp. (21.70 %), Bacilli sp. (21.70 %), Klebsiella sp. (17.20 %), and Escherichia coli (4.60 %). The duration of solution storage in the open bottle and nonadherence to manufacturer instructions for solution storage showed a statistically significant association with microbial contamination (p ≤ 0.05). CONCLUSION: Contact lens care solutions have been found to harbour multiple antibiotic-resistant bacteria that are potentially pathogenic to the corneal surface. The contamination is associated with some unhealthy solution-care practices among wearers.

External quality assessment schemes in bacteriology support public health in Germany-results from 2006 to 2023.

External Quality Assessment schemes (EQAS) are mandatory to ensure quality standards in diagnostic methods and achieve laboratory accreditation. As host institution for two German culture-based bacteriology EQAS (RV-A and RV-B), we investigated the obtained data of 590 up to 720 surveys per year in RV-A and 2,151 up to 2,929 in RV-B from 2006 to 2023. As educational instruments, they function to review applied methodology and are valuable to check for systemic- or method-dependent failures in microbiology diagnostics or guidelines. Especially, containment of multi-resistant bacteria in times of rising antibiotic resistance is one major point to assure public health. The correct identification and reporting of these strains is therefore of high importance to achieve this goal. Moreover, correct antimicrobial susceptibility testing (AST) per se is important for selecting appropriate therapy, to restrict broad-spectrum antibiotics and minimize resistance development. The reports of participating laboratories displayed a high level of correct identification results in both schemes with mostly consistent failure rates around 2.2% (RV-A) and 3.9% (RV-B) on average. In contrast, results in AST revealed increasing failure rates upon modification of AST requirements concerning adherence to standards and subsequent bacterial species-specific evaluation. Stratification on these periods revealed in RV-A a moderate increase from 1.3% to 4.5%, while in RV-B failure rates reached 14% coming from 4.3% on average. Although not mandatory, subsequent AST evaluation and consistent reporting are areas of improvement to benefit public health.

European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

Prevalence of Helicobacter pylori resistance to certain antibiotics at An-Najah University Hospital: a cross-sectional study.

Antibiotic resistance among bacteria is recognized as the primary factor contributing to the failure of treatment. In this research, our objective was to examine the prevalence of antibiotic resistance in H. pylori bacteria in Palestine. We enlisted 91 individuals suffering from dyspepsia, comprising 49 females and 42 males. These participants underwent esophagogastroduodenoscopy procedures with gastric biopsies. These biopsies were subsequently subjected to microbiological assessments and tested for their susceptibility to various antimicrobial drugs. Among the 91 patients, 38 (41.7%) exhibited the presence of H. pylori. Notably, Ciprofloxacin displayed the highest efficacy against H. pylori, followed by Levofloxacin, Moxifloxacin, and Amoxicillin, with resistance rates of 0%, 0%, 2.6%, and 18.4%, respectively. On the contrary, Metronidazole and Clarithromycin demonstrated the lowest effectiveness, with resistance percentages of 100% and 47.4%, respectively. The outcomes of this investigation emphasize that H. pylori strains within the Palestinian patient group exhibit substantial resistance to conventional first-line antibiotics like clarithromycin and metronidazole. However, alternative agents such as fluoroquinolones and amoxicillin remain efficacious choices. Consequently, we recommend favoring quinolone-based treatment regimens for H. pylori infections and adopting a more judicious approach to antibiotic usage among the Palestinian population.

Comparison of available methods to evaluate cefiderocol susceptibility in Acinetobacter spp.

Recently, considerable uncertainty has arisen concerning the appropriate susceptibility testing for cefiderocol in gram-negative bacilli, particularly in the context of its application to Acinetobacter spp. The optimal method for assessing the susceptibility levels of Acinetobacter spp. to cefiderocol remains a subject of debate due to substantial disparities observed in the values obtained through various testing procedures. This study employed four minimum inhibitory concentration (MIC) methodologies and the disk diffusion to assess the susceptibility of twenty-seven carbapenem resistant (CR)-Acinetobacter strains to cefiderocol. The results from our study reveal significant variations in the minimum inhibitory concentration (MIC) values obtained with the different methods and in the level of agreement in interpretation categories between the different MIC methods and the disk diffusion test. Among the MIC methods, there was relatively more consistency in reporting the interpretation categories. For European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the categorical agreement (CA) for MIC methods ranged between 66.7 and 81.5%. On the other hand, the essential agreement (EA) values were as low as 18.5-29.6%. The CA between MIC methods and disk diffusion was 81.5%. These results emphasize the need for a reliable, accurate, and clinically validated methodology to effectively assess the susceptibility of Acinetobacter spp. to cefiderocol. The wide variability observed in our study highlights the importance of standardizing the susceptibility testing process for cefiderocol to ensure consistent and reliable results for clinical decision-making.

Short-term culture for rapid identification by mass spectrometry and automated antimicrobial susceptibility testing from positive bottles.

BACKGROUND: Early and appropriate antibiotic treatment improves the clinical outcome of patients with sepsis. There is an urgent need for rapid identification (ID) and antimicrobial susceptibility testing (AST) of bacteria that cause bloodstream infection (BSI). Rapid ID and AST can be achieved by short-term incubation on solid medium of positive blood cultures using MALDI-TOF mass spectrometry (MS) and the BD M50 system. The purpose of this study is to evaluate the performance of rapid method compared to traditional method. METHODS: A total of 124 mono-microbial samples were collected. Positive blood culture samples were short-term incubated on blood agar plates and chocolate agar plates for 5 ∼ 7 h, and the rapid ID and AST were achieved through Zybio EXS2000 MS and BD M50 System, respectively. RESULTS: Compared with the traditional 24 h culture for ID, this rapid method can shorten the cultivation time to 5 ∼ 7 h. Accurate organism ID was achieved in 90.6% of Gram-positive bacteria (GP), 98.5% of Gram-negative bacteria (GN), and 100% of fungi. The AST resulted in the 98.5% essential agreement (EA) and 97.1% category agreements (CA) in NMIC-413, 99.4% EA and 98.9% CA in PMIC-92, 100% both EA and CA in SMIC-2. Besides, this method can be used for 67.2% (264/393) of culture bottles during routine work. The mean turn-around time (TAT) for obtaining final results by conventional method is approximately 72.6 ± 10.5 h, which is nearly 24 h longer than the rapid method. CONCLUSIONS: The newly described method is expected to provide faster and reliable ID and AST results, making it an important tool for rapid management of blood cultures (BCs). In addition, this rapid method can be used to process most positive blood cultures, enabling patients to receive rapid and effective treatment.

Evaluation of an automated rapid phenotypic antimicrobial susceptibility testing (ASTar, Q-linea AB) applied directly on blood cultures bottles positive for Gram-negative pathogens.

We evaluated the performance of a new rapid phenotypic antimicrobial susceptibility test (ASTar; Q-linea AB) on Gram-negative bacilli, directly from positive blood cultures bottles. MIC values obtained by the routine reference method (Microscan, Beckman Coulter) were compared to the ones provided by the tested method (ASTar). ASTar demonstrated an overall essential agreement of 98% and a category agreement of 96.1%. The overall rate of major errors and very major errors was 2.5% and 3.3%, respectively. ASTar can represent a rapid, simple, and reliable method to speed up information about antimicrobial susceptibility of Gram-negative pathogens from positive blood culture bottles.

Rapid Direct Identification of Microbial Pathogens and Antimicrobial Resistance Genes in Positive Blood Cultures Using a Fully Automated Multiplex PCR Assay.

This study assessed the performance of the BioFire Blood Culture Identification 2 (BCID2) panel in identifying microorganisms and antimicrobial resistance (AMR) profiles in positive blood cultures (BCs) and its influence on turnaround time (TAT) compared with conventional culture methods. We obtained 117 positive BCs, of these, 102 (87.2%) were correctly identified using BCID2. The discordance was due to off-panel pathogens detected by culture (n = 13), and additional pathogens identified by BCID2 (n = 2). On-panel pathogen concordance between the conventional culture and BCID2 methods was 98.1% (102/104). The conventional method detected 19 carbapenemase-producing organisms, 14 extended-spectrum beta-lactamase-producing Enterobacterales, 18 methicillin-resistant Staphylococcus spp., and four vancomycin-resistant Enterococcus faecium. BCID2 correctly predicted 53 (96.4%) of 55 phenotypic resistance patterns by detecting AMR genes. The TAT for BCID2 was significantly lower than that for the conventional method. BCID2 rapidly identifies pathogens and AMR genes in positive BCs.

Multicenter comparison of Etest, Vitek2 and BD Phoenix to broth microdilution for beta-lactam susceptibility testing of Streptococcus pneumonia.

PURPOSE: To assess performance of Etest®, Vitek®2 and BD Phoenix™ to determine the susceptibility of Streptococcus pneumoniae strains to penicillin, ampicillin and cefotaxime. METHODS: Sixty unique S. pneumoniae challenge strains were selected to cover a wide range of penicillin, ampicillin and cefotaxime minimal inhibitory concentrations (MICs). Strains were analyzed in four different Belgian laboratories. Etest® benzylpenicillin (BEN), ampicillin/amoxicillin (AMP) and cefotaxime (CTA) (bioMérieux), Vitek®2 AST-ST03 (bioMérieux) and BD Phoenix™ SMIC/ID-11 testing were each performed in two different labs. Results were compared to Sensititre® broth microdilution (BMD) (Thermo Fisher Scientific) results. MIC results were interpreted using EUCAST non-meningitis breakpoints (v 13.0). RESULTS: Essential agreement (EA) was ≥ 90% for all methods compared to BMD, except for Etest® BEN on Oxoid plate (58.3%), Etest® AMP (both on Oxoid (65.8%) and BD BBL plate (84.2%)). Categorical agreement (CA) for penicillin was only ≥ 90% for Vitek®2, for other methods CA ranged between 74 and 84%. CA for AMP was for all methods < 90% (range 75.8-88.3%) and CA for CTA was between 87 and 90% for all methods except for Etest on Oxoid plate (79.2%). CONCLUSIONS: Our study indicates that Vitek®2 and BD Phoenix™ are reliable for providing accurate pneumococcal susceptibility results for BEN, AMP and CTA. Using Etest BEN or AMP on Oxoid plate carries a risk of underestimating the MIC and should be interpreted with caution, especially when the obtained MIC is 1 or 2 doubling dilutions below the S or R clinical breakpoint.

Evaluation of the QMAC-dRAST System Version 2.5 for Rapid Antimicrobial Susceptibility Testing of Gram-Negative Bacteria From Positive Blood Culture Broth and Subcultured Colony Isolates.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) for bloodstream infections (BSIs) facilitates the optimization of antimicrobial therapy, preventing antimicrobial resistance and improving patient outcomes. QMAC-dRAST (QuantaMatrix Inc., Korea) is a rapid AST platform based on microfluidic chip technology that performs AST directly using positive blood culture broth (PBCB). This study evaluated the performance of QMAC-dRAST for Gram-negative bacteria using PBCB and subcultured colony isolates, comparing it with that of VITEK 2 (bioMérieux, France) using broth microdilution (BMD) as the reference method. METHODS: We included 141 Gram-negative blood culture isolates from patients with BSI and 12 carbapenemase-producing clinical isolates of Enterobacterales spiked into blood culture bottles. QMAC-dRAST performance was evaluated using PBCB and colony isolates, whereas VITEK 2 and BMD were tested only on colony isolates. RESULTS: For PBCB, QMAC-dRAST achieved 92.1% categorical agreement (CA), 95.3% essential agreement (EA), with 1.8% very major errors (VMEs), 3.5% major errors (MEs), and 5.2% minor errors (mEs). With colony isolates, it exhibited 92.5% CA and 95.1% EA, with 2.0% VMEs, 3.2% MEs, and 4.8% mEs. VITEK 2 showed 94.1% CA and 96.0% EA, with 4.3% VMEs, 0.4% MEs, and 4.3% mEs. QMAC-dRAST yielded elevated error rates for specific antimicrobial agents, with high VMEs for carbapenems and aminoglycosides. The median time to result for QMAC-dRAST was 5.9 h for PBCB samples and 6.1 h for subcultured colony isolates. CONCLUSIONS: The QMAC-dRAST system demonstrated considerable strengths and comparable performance to the VITEK 2 system; however, challenges were discerned with specific antimicrobial agents, underlining a necessity for improvement.

Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese.

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Evaluation of Direct Antimicrobial Susceptibility Testing from Positive Flagged Blood Cultures in Sepsis Patients.

Background: Presently, many laboratories are equipped with automated system for antimicrobial susceptibility testing (AST) for minimum inhibitory concentration-based reporting which enables the clinician to choose the right antimicrobial for timely treatment of sepsis. The study aimed to assess performance of direct AST from blood culture positive broth using automated AST system for accuracy and time taken to release the report. Materials and methods: The present study conducted in a 25-bedded ICU in North India for 12 months. Single morphotype of bacteria on gram stain from positively flagged blood culture bottles were included, which was directly identified (using an in-house protocol) with MALDI-TOF-MS from positive blood culture broths. DAST was carried out from 200 such blood culture broths and results were compared with reference AST (RAST) which was also done using VITEK-2 using overnight grown bacterial colonies as per standard protocol. Results: Among 60 isolates of Enterobacterales, 99% categorical agreement for both E. coli and K. pneumoniae observed by two methods were tested for AST. Among non-fermenters, Pseudomonas aeruginosa showed a categorical agreement of 99.6%, as compared with Acinetobacter spp. and exotic GNBs, which showed 95-96% agreement. A significant difference of 18-24 hours was noted in time to release the report between DAST and RAST, for GNB and GPC both. Conclusion: Direct AST from positive flagged blood culture bottles can significantly reduce the time to release the bacterial susceptibility report by up to 24 hours, at the same time maintaining the accuracy. How to cite this article: Singh V, Agarwal J, Nath SS, Sharma A. Evaluation of Direct Antimicrobial Susceptibility Testing from Positive Flagged Blood Cultures in Sepsis Patients. Indian J Crit Care Med 2024;28(4):387-392.

How New Regulation of Laboratory-Developed Antimicrobial Susceptibility Tests Will Affect Infectious Diseases Clinical Practice.

Antimicrobial resistance (AMR) affects 2.8 million Americans annually. AMR is identified through antimicrobial susceptibility testing (AST), but current and proposed regulatory policies from the United States Food and Drug Administration (FDA) jeopardize the future availability of AST for many microorganisms. Devices that perform AST must be cleared by the FDA using their susceptibility test interpretive criteria, also known as breakpoints. The FDA list of breakpoints is relatively short. Today, laboratories supplement FDA breakpoints using breakpoints published by the Clinical and Laboratory Standards Institute, using legacy devices and laboratory-developed tests (LDTs). FDA proposes to regulate LDTs, and with no FDA breakpoints for many drug-bug combinations, the risk is loss of AST for key clinical indications and stifling innovation in technology development. Effective solutions require collaboration between manufacturers, infectious diseases clinicians, pharmacists, laboratories, and the FDA.