Conventional Phenotypic Tests Research Articles

Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.

Simultaneous detection of phylogroups and ESBL genes in E. coli using Multiplex PCR

Background: Escherichia coli (E. coli) strains harbor various allelic versions of beta lactamase genes and their identification using conventional phenotypic tests is a tedious and time consuming task. In the present study, multiplex PCR is performed for the simulatanoues detection of E. coli phylogroups and extended-spectrum β-lactamase- (ESBL) genes.Methods: A total of 128 E. coli isolates from urine samples were screened for antibiotic resistance and expression of ESBL activity using phenotypic and genotypic methods. Uniplex and multiplex PCRs were used to detect E. coli phylogroup detrminants and blaCTX-M-15, blaOXA-1 and TEM1 genes. Chi Square test of independence was employed for evaluating significant levels at P value < 0.05. Results: Phylogroup B2 was detected as the predominant group (36%) followed by group D (30%), A (25%) and B1 (9%). The highest resistance was seen against nalidixic acid (100%) and the lowest against amoxicillin-clavulanic acid (55%). Significant P values were observed for resistance against cefotaxime and cefepime in the phylogroup B2 while resistance against cefoxitin, sulfamethoxazole and fosfomycin was significantly associated with group D. Combination disc diffusion test (CDDT) showed ESBL activity in 42% E. coli isolates. A significant association of blaCTX-M-15 gene was observed for phylogroup B2 (P = 0.007). Moreover, a combination genotype of blaCTX-M-15 and TEM1 was also found statistically prevalent in phylogroup B2 (P = 0.006).Conclusion: The study highlights the alarming rise in antibiotic resistance and delineates B2 a predominant phylogoup with a high prevalence of blaCTX-M-15 and TEM1 genes in urinary E. coli isolates.Keywords: UTI; E. Coli; Phylogenetic groups; ESBL genes; Multiplex PCR

Rapid detection of multidrug resistance in tuberculosis using nanopore-based targeted next-generation sequencing: a multicenter, double-blind study.

Resistance to anti-tuberculous drugs is a major challenge in the treatment of tuberculosis (TB). We aimed to evaluate the clinical availability of nanopore-based targeted next-generation sequencing (NanoTNGS) for the diagnosis of drug-resistant tuberculosis (DR-TB). This study enrolled 253 patients with suspected DR-TB from six hospitals. The diagnostic efficacy of NanoTNGS for detecting Mycobacterium tuberculosis and its susceptibility or resistance to first- and second-line anti-tuberculosis drugs was assessed by comparing conventional phenotypic drug susceptibility testing (pDST) and Xpert MTB/RIF assays. NanoTNGS can be performed within 12 hours from DNA extraction to the result delivery. NanoTNGS showed a remarkable concordance rate of 99.44% (179/180) with the culture assay for identifying the Mycobacterium tuberculosis complex. The sensitivity of NanoTNGS for detecting drug resistance was 93.53% for rifampicin, 89.72% for isoniazid, 85.45% for ethambutol, 74.00% for streptomycin, and 88.89% for fluoroquinolones. Specificities ranged from 83.33% to 100% for all drugs tested. Sensitivity for rifampicin-resistant tuberculosis using NanoTNGS increased by 9.73% compared to Xpert MTB/RIF. The most common mutations were S531L (codon in E. coli) in the rpoB gene, S315T in the katG gene, and M306V in the embB gene, conferring resistance to rifampicin, isoniazid, and ethambutol, respectively. In addition, mutations in the pncA gene, potentially contributing to pyrazinamide resistance, were detected in 32 patients. Other prevalent variants, including D94G in the gyrA gene and K43R in the rpsL gene, conferred resistance to fluoroquinolones and streptomycin, respectively. Furthermore, the rv0678 R94Q mutation was detected in one sample, indicating potential resistance to bedaquiline. NanoTNGS rapidly and accurately identifies resistance or susceptibility to anti-TB drugs, outperforming traditional methods. Clinical implementation of the technique can recognize DR-TB in time and provide guidance for choosing appropriate antituberculosis agents.

Comparison of the MeltPro TB assay and whole-genome sequencing assay for rapid molecular diagnosis of drug resistant tuberculosis in guangdong province

BackgroundRifampicin (RIF) and multidrug-resistant tuberculosis (TB) are major public health threats. As conventional phenotypic drug susceptibility testing requires two–eight weeks, molecular diagnostic assays are widely used to determine drug resistance. MethodsClinical Mycobacterium tuberculosis isolates with consistent drug susceptibility results, tested using microbroth dilution and proportion methods in Löwenstein-Jensen medium from patients with TB in Guangdong province were utilized to evaluate MeltPro TB and whole-genome sequencing (WGS) assays in detecting resistance to RIF, isoniazid (INH), ethambutol (EMB), fluoroquinolones (FQ), and streptomycin (SM). Solid phenotypic drug susceptibility testing was used as the gold standard to evaluate the detection capacity of MeltPro TB on clinical sputum samples of patients with TB. ResultsSimilar to WGS, MeltPro TB successfully detected RIF, INH, and SM resistance with sensitivities of 86.3, 84.8, and 86.6 %, respectively. However, the resistant isolate detection rates were only 58.1 and 69.6 % for EMB and FQ-resistant strains. For clinical specimens, MeltPro TB still showed good detectable rates of RIF and INH resistance, with sensitivities of 82.4 % and 95.2 %, respectively. Detectable rates of FQ and EMB resistance were low: 77.8 % and 35.3 %, respectively. ConclusionsMeltPro TB can detect known DNA mutations associated with drug resistance in Mycobacterium tuberculosis strains with comparable efficacy to WGS. For FQ and EMB resistance testing, MeltPro TB requires optimization and is unsuitable for general use. MeltPro TB can be used for diagnosis of RIF and multidrug-resistant tuberculosis to rapidly initiate appropriate anti-TB drug therapy.

Value of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) in Discriminating Candida Species

Abstract Background Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI TOF-MS) allows rapid and accurate identification of Candida species. Phenotypic identification of Candida species remains labor- and time consuming, and the results may sometimes be inconclusive. Rapid and reliable species identification of Candida is essential for antifungal treatment due to speciesspecific susceptibility patterns. Objectives We evaluated the performance of MALDI TOF-MS (VITEK MS) compared with conventional VITEK 2 YST (V2C) for identification of clinically important Candida strains. Subject and Methods A total of 50 Candida isolates collected from different clinical samples submitted to the Main Microbiology Laboratory Ain Shams University Hospitals, were included in this study. Candida species were identified by conventional phenotypic tests as Gram stain, culture morphology and with automated V2C panels as well as with VITEK MS. Results There was a total agreement of (100%) between VITEK MS and V2C at the level of the genus and (96%) at the level of the species. The most common isolates identified were C.tropicalis followed by C. albicans in urinary samles. Conclusion VITEK MS is a rapid and reliable method in comparison to V2C for identification of Candida strains in Microbiology Clinical Laboratory.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes

Background: Foodborne infections like Listeriosis cover several disorders and are a worldwide public health emergency. Listeria monocytogenes has been isolated from various foodstuffs, including milk and fishes. MALDI-TOF mass spectrometry technique is known for its rapid and accurate identification of bacterial organisms. Methods: In the current research, a total of 360 samples comprising raw milk (130), milk products (125) and freshwater fishes (105) were screened for the detection of pathogenic Listeria species by using the USDA method. The recovered Listeria isolates were characterized using conventional set of biochemical analysis along with sugar fermentation tests and further confirmed by MALDI-TOF MS. The virulent nature of pathogenic Listeria isolates was also assessed by in vitro tests like hemolysis on blood agar, CAMP and PI-PLC assay. Result: In current study, on screening 360 animal origin food samples, three isolates were recovered from raw milk samples and identified as Listeria monocytogenes indicating an overall prevalence of Listeria monocytogenes to the tune of 0.83%. Excellent correlation was observed with identification of Listeria species using conventional phenotypic tests and advanced molecular tool Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. The results depict dependability of advanced technique for rapid and reliable identification of Listeria species.

Budget impact of next-generation sequencing for diagnosis of TB drug resistance in Moldova.

BACKGROUND Diagnosing drug resistance is critical for choosing effective TB treatment regimens. Next-generation sequencing (NGS) represents an alternative approach to conventional phenotypic drug susceptibility testing (pDST) for diagnosing TB drug resistance.METHODS We undertook a budget impact analysis estimating the costs of introduction and routine use of NGS in the Moldovan National TB Programme. We conducted an empirical costing study and collated price and operating characteristics for NGS platforms. We examined multiple NGS scenarios in comparison to the current approach (pDST) for pre-treatment drug resistance testing over 2021-2025.RESULTS Annual testing volume ranged from 912 to 1,926 patients. For the pDST scenario, we estimated total costs of US$362,000 (2021 USD) over the 5-year study period. Total costs for NGS scenarios ranged from US$475,000 to US$1,486,000. Lowest cost NGS options involved targeted sequencing as a replacement for pDST, and excluded individuals diagnosed as RIF-susceptible on Xpert® MTB/RIF. For all NGS scenarios, the majority (55-80%) of costs were devoted to reagent kits. Start-up costs of NGS were small relative to routine costs borne each year.CONCLUSION NGS adoption will require expanded resources compared to conventional pDST. Further work is required to better understand the feasibility of NGS in settings such as Moldova.

P099 Molecular identification of dermatophyte species from Eastern Assam, Northeast India

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesDermatophyte infections occur worldwide both in developing as well as developed countries. However, species of dermatophytes may vary based on geographical region. Studies on dermatophytes from northeast India are rare. This study was done to know the various species of dermatophytes that are commonly associated with infection in this part of the country.MethodsThis study was done from 2020-2021. A total of 49 consecutive isolates of dermatophytes isolated from clinically suspected cases attending Assam Medical College and Hospital, a tertiary care hospital were subjected to molecular identification by using PCR and sequencing of the ITS region of the ribosomal RNA gene as well as using MALDI-TOF (VITEK MS). Samples from active margin of lesions from skin, nail, and hair were collected and primary identification was done by culture and microscopy as well as conventional phenotypic tests. Culture was done in Sabouraud Dextrose agar, Sabouraud Dextrose agar with chloramphenicol and cycloheximide, and dermatophyte test medium which was followed by genotypic confirmation by PCR of the ITS region and sequencing of PCR amplicons using already published protocols.ResultsThe species isolated were T. rubrum (36.7%), T. interdigitale (32.6%), T. mentagrophytes complex (14.2%), T. tonsurans (8%), M. gypseum (6%), T. violaceum (2%). The cases were clinically found to be T. corporis (44.89%), T. manuum (12.24%), T. pedis (12.24%), T. cruris (10.20%), T. faciei (8.16%), T. capitis (8.16%), and T. unguium (4.08%).Conclusion T. rubrum, T. interdigitale, T. mentagrophytes, and T. tonsurans complex were the predominant species isolated.

Accelerated antibiotic susceptibility testing of pseudomonas aeruginosa by monitoring extracellular electron transfer on a 3-D paper-based cell culture platform

Pseudomonas aeruginosa is the most important opportunistic pathogen leading to serious and life-threatening infections, especially in immunocompromised patients. Because of its remarkable capacity to resist antibiotics, the selection of the right antibiotics with the exact dose for the appropriate duration is critical to effectively treat the infections and prevent antibiotic resistance. Although conventional genotypic and phenotypic antibiotic susceptibility testing (AST) methods have been dramatically advanced, they have suffered from many technical and operational issues as a generalized antibiotic stewardship program. Furthermore, given that most microbial infections are caused by their biofilms, the existing AST methods do not provide evidence-based antibiotic prescribing guidance for biofilm-based infections because the results are based on individual bacteria traditionally grown in their planktonic form. In this work, we create an innovative susceptibility testing technique for P. aeruginosa that offers clinically relevant guidelines and widely adaptable stewardship to effectively treat the infections and minimize antibiotic resistance. Our approach evaluates the antibiotic efficacy by continuously monitoring the accumulated electrical outputs from the bacterial extracellular electron transfer (EET) process in the presence of antibiotics. Our innovative paper-based culturing 3-D scaffold promotes surface-associated growth of bacterial colonies and biofilms. The platform replicates a natural habitat for P. aeruginosa where it can grow similarly to sites it infects. Our technique enables an all-electrical, real-time, easy-to-use, portable AST that can be easily translatable to clinical settings. The entire procedure takes 96 min to provide evidence-based antimicrobial prescribing guidance for biofilm-based infections.

Automated extraction of genes associated with antibiotic resistance from the biomedical literature.

The detection of bacterial antibiotic resistance phenotypes is important when carrying out clinical decisions for patient treatment. Conventional phenotypic testing involves culturing bacteria which requires a significant amount of time and work. Whole-genome sequencing is emerging as a fast alternative to resistance prediction, by considering the presence/absence of certain genes. A lot of research has focused on determining which bacterial genes cause antibiotic resistance and efforts are being made to consolidate these facts in knowledge bases (KBs). KBs are usually manually curated by domain experts to be of the highest quality. However, this limits the pace at which new facts are added. Automated relation extraction of gene-antibiotic resistance relations from the biomedical literature is one solution that can simplify the curation process. This paper reports on the development of a text mining pipeline that takes in English biomedical abstracts and outputs genes that are predicted to cause resistance to antibiotics. To test the generalisability of this pipeline it was then applied to predict genes associated with Helicobacter pylori antibiotic resistance, that are not present in common antibiotic resistance KBs or publications studying H. pylori. These genes would be candidates for further lab-based antibiotic research and inclusion in these KBs. For relation extraction, state-of-the-art deep learning models were used. These models were trained on a newly developed silver corpus which was generated by distant supervision of abstracts using the facts obtained from KBs. The top performing model was superior to a co-occurrence model, achieving a recall of 95%, a precision of 60% and F1-score of 74% on a manually annotated holdout dataset. To our knowledge, this project was the first attempt at developing a complete text mining pipeline that incorporates deep learning models to extract gene-antibiotic resistance relations from the literature. Additional related data can be found at https://github.com/AndreBrincat/Gene-Antibiotic-Resistance-Relation-Extraction

Diagnosis of Multidrug-Resistant Pathogens of Pneumonia.

Hospital-acquired pneumonia and ventilator-associated pneumonia that are caused by multidrug resistant (MDR) pathogens represent a common and severe problem with increased mortality. Accurate diagnosis is essential to initiate appropriate antimicrobial therapy promptly while simultaneously avoiding antibiotic overuse and subsequent antibiotic resistance. Here, we discuss the main conventional phenotypic diagnostic tests and the advanced molecular tests that are currently available to diagnose the primary MDR pathogens and the resistance genes causing pneumonia.

1251. Comparative Evaluation of Phenotypic Synergy Tests vs. Resist-4 O.K.N.V® and NG Test Carba 5® Lateral Flow Immunoassays for the Detection and Differentiation of Carbapenemases in Enterobacterales and Pseudomonas aeruginosa

BackgroundThe spread of carbapenem resistant Pseudomonas aeruginosa and carbapenemase-producing Enterobacterales (CPE) has had a great impact on morbidity and mortality. COVID-19 pandemic has favoured the selection of these microorganisms because of the excessive and prolonged use of broad-spectrum antibiotics and the outbreaks related to patient transfer between hospitals and inadequate use of personal protective equipment. Therefore, detection is considered essential for their control. Our aim was to compare conventional phenotypic synergy tests and two lateral flow immunoassays for detecting carbapenemases in Enterobacterales and P. aeruginosa.MethodsWe analysed 100 carbapenem-resistant Gram-negative bacilli isolates, 80 Enterobacterales and 20 Pseudomonas aeruginosa, (86 isolates producing KPC, NDM, OXA-48, IMP and VIM carbapenemases and 14 non-carbapenemase-producing isolates). We performed a modified Hodge test, boronic acid and ethylenediaminetetraacetic acid (EDTA) synergy tests, and two lateral flow immunoassays: RESIST-4 O.K.N.V (Coris Bioconcept®) and NG Test Carba 5® (NG Biotech®).ResultsIn total, 76 KPC, 7 VIM, 1 NDM, 1 OXA-48 and 1 isolate coproducing KPC + NDM enzymes were included. The concordance of different methods estimated by Kappa index was 0.432 (Standard error: 0.117), thus showing a high variability with the synergy tests with boronic acid and EDTA and reporting 16 false negatives that were detected by the two immunochromatographic methods. Co-production was only detected using immunoassays. Figure 1. Errors in synergy tests. 1) KPC-producing K. pneumoniae not detected with boronic acid and EDTA (1a). Positive result adjusting distance to 5 mm (1b) and positive result by NG Test Carba 5® and RESIST-4 O.K.N.V® immunoassays (1c). 2) VIM -producing P. aeruginosa with inconclusive EDTA result (2a), Positive result adjusting distance to 5 mm (2b), confirmation of VIM by immunoassay (2c). 3) K. pneumoniae with KPC and NDM co-production without synergistic effect with boronic acid or EDTA (3a), positivity for both enzymes by immunoassay (3b). 4) Impact of distance between disks: False negative for serine-carbapenemases with 15 mm (4a) positive result at 10 mm (4b). Differences in boronic acid tests for serin-carbapenemases at 10 mm, 5 mm and 0 mm distance (4c, 4d). Differences in synergy between imipenem, meropenem and ertapenem discs at 0 mm distance (4e).Table 2. Results of synergy methods and immunoassay tests for Enterobacterales and Pseudomonas aeruginosa MHTModified Hodge Test, APB: boronic acid synergy; EDTA: EDTA synergy; Pos: positive; Neg: negative. KPC (Klebsiella pneumoniae Carbapenemase), VIM (Verona integron-mediated metallo- β-lactamase), NDM (New Delhi metallo-β-lactamase), OXA (oxacillinase-48-like carbapenemase (OXA-48))ConclusionConventional phenotypic synergy tests with boronic acid and EDTA used for detecting carbapenemases are suboptimal and their routine use should be reconsidered. They depend on the degree of enzyme expression and the distance between disks. Lateral flow immunoassay tests are a rapid and cost-effective tool to detect and differentiate carbapenemases, improving clinical outcomes through targeted therapy and promoting infection prevention measures. Disclosures Diego Josa, Msc, ALIFAX (Speaker’s Bureau) German Esparza, n/a, Biomerieux (Consultant)Pfizer (Speaker’s Bureau) Luis Reyes, n/a, MSD (Speaker’s Bureau)

Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019

ABSTRACT Objectives: Since January 2019, the Belgian National Reference Center for Mycobacteria (NRC) has switched from conventional typing to prospective whole-genome sequencing (WGS) of all submitted Mycobacterium tuberculosis complex (MTB) isolates. The ISO17025 validated procedure starts with semi-automated extraction and purification of gDNA directly from the submitted MGIT tubes, without preceding subculturing. All samples are then sequenced on an Illumina MiSeq sequencer and analyzed using an in-house developed and validated bioinformatics workflow to determine the species and antimicrobial resistance. In this study, we retrospectively compare results obtained via WGS to conventional phenotypic and genotypic testing, for all Belgian MTB strains analyzed in 2019 (n = 306). Results: In all cases, the WGS-based procedure was able to identify correctly the MTB species. Compared to MGIT drug susceptibility testing (DST), the sensitivity and specificity of genetic prediction of resistance to first-line antibiotics were respectively 100 and 99% (rifampicin, RIF), 90.5 and 100% (isoniazid, INH), 100 and 98% (ethambutol, EMB) and 61.1 and 100% (pyrazinamide, PZA). The negative predictive value was above 95% for these four first-line drugs. A positive predictive value of 100% was calculated for INH and PZA, 80% for RIF and 45% for EMB. Conclusions: Our study confirms the effectiveness of WGS for the rapid detection of M. tuberculosis complex and its drug resistance profiles for first-line drugs even when working directly on MGIT tubes, and supports the introduction of this test into the routine workflow of laboratories performing tuberculosis diagnosis.

Isolation and characterization of Campylobacter massiliensis sp. nov., a novel Campylobacter species detected in a gingivitis subject.

A Gram-negative bacterium, designated strain Marseille-Q3452T, was isolated from subgingival dental plaque of a subject suffering from dental plaque biofilm-induced gingivitis on an intact periodontium in Marseille, France. The strain was characterized by 16S rRNA and atpA gene sequence analysis and by conventional phenotypic and chemotaxonomic testing. The average nucleotide identity (ANI) and core genome phylogeny were determined using whole-genome sequences. Although strain Marseille-Q3452T showed 99.72 % 16S rRNA gene sequence similarity with Campylobacter showae strain ATCC 51146T, atpA and ANI analyses revealed divergence between the two strains. The two species could also be distinguished phenotypically on the basis of the absence of flagella and nitrate reduction. On the basis of the results from phenotypic, chemotaxonomic, genomic and phylogenetic analyses and data, we concluded that strain Marseille-Q3452T represents a novel species of the genus Campylobacter, for which the name Campylobacter massiliensis sp. nov. is proposed (=CSUR Q3452=CECT 30263).

Antibiotic Resistance and Plasmid Profile Analysis of Salmonella Enteritidis Isolated in Siberia and the Far East of Russia between 1990 and 2017.

Salmonella is one of the major causes of foodborne disease outbreaks globally. Specifically, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the major causes of zoonotic Salmonella infection in humans worldwide. In this study, we present data on antimicrobial resistance (AMR) and plasmid profiles of S. Enteritidis strains isolated from patients, food, and the environment in Siberia and the Far East of Russia obtained during Salmonella monitoring between 1990 and 2017. A total of 345 S. Enteritidis isolates were tested by the disk diffusion method with a set of 15 antibiotics using EUCAST breakpoints v. 10 and by plasmid profile analysis using the alkaline lysis method. The results have shown a substantial decrease in susceptibility to aminoglycosides and quinolones during the study period. No significant differences were found in the susceptibility of strains between regions as well as in the its correlation with different plasmid types of the pathogen. Several S. Enteritidis strains were found to be resistant to ampicillin, kanamycin, tetracycline, chloramphenicol, and cephalosporins. All tested S. Enteritidis strains were susceptible only to imipenem. In this study, we observed a relatively low level of AMR in S. Enteritidis strains isolated in Siberia and the Far East of Russia. Nevertheless, it is important to continue the molecular genetic monitoring and AMR surveillance of S. Enteritidis to track further increases in AMR using conventional phenotypic susceptibility testing and by introducing whole-genome sequencing to identify AMR mechanisms.

A 3D-printed magnetic digital microfluidic diagnostic platform for rapid colorimetric sensing of carbapenemase-producing Enterobacteriaceae

Carbapenemase-producing Enterobacteriaceae (CPE) are a group of drug-resistant Gram-negative pathogens that are classified as a critical threat by the World Health Organization (WHO). Conventional methods of detecting antibiotic-resistant pathogens do not assess the resistance mechanism and are often time-consuming and laborious. We have developed a magnetic digital microfluidic (MDM) platform, known as MDM Carba, for the identification of CPE by measuring their ability to hydrolyze carbapenem antibiotics. MDM Carba offers the ability to rapidly test CPE and reduce the amount of reagents used compared with conventional phenotypic testing. On the MDM Carba platform, tests are performed in droplets that function as reaction chambers, and fluidic operations are accomplished by manipulating these droplets with magnetic force. The simple droplet-based magnetic fluidic operation allows easy system automation and simplified hands-on operation. Because of the unique “power-free” operation of MDM technology, the MDM Carba platform can also be operated manually, showing great potential for point-of-care testing in resource-limited settings. We tested 27 bacterial isolates on the MDM Carba platform, and the results showed sensitivity and specificity that were comparable to those of the widely used Carba NP test. MDM Carba may shorten the overall turnaround time for CPE identification, thereby enabling more timely clinical decisions for better clinical outcomes. MDM Carba is a technological platform that can be further developed to improve diagnostics for other types of antibiotic resistance with minor modifications.

Identification of Veillonella spp. on Tongue Plaque and Saliva Using Real-Time PCR

Veillonella spp., Gram-negative obligate anaerobic cocci bacteria, amounts to 3% in the oral cavity, relies on the fermentation of lactate as a carbon and energy source for growth. The bacteria are considered anti-cariogenic as they metabolize lactic acid into propionic acid which increases oral environment’s pH and reduces demineralization rate of tooth structure. Identification of Veillonella spp. using traditional methods is difficult due to the lack of conventional phenotypic and biochemical tests. Thus, the biomolecular methods are suitable for the specific detection and identification of Veillonella spp. One of the biomolecular methods that can be used is real-time Polymerase Chain Reaction (PCR), which the results can be qualitative and quantitative. This study aimed to identify Veillonella spp. in tongue plaque’s and saliva’s samples using Real -time PCR. The DNA of Veillonella spp. derived from 36 samples, 18 samples of tongue plaque and 18 samples of saliva, were extracted using a freeze-thaw method and then quantified by real-time PCR using forward primer 5’-CCG TGA TGG GAT GGA AAC TGC-3’ and reverse primer 5’-CCT TCG CCA CTG GTG TTC TTC-3’. Veillonella spp. in 18 samples of tongue plaque was 3,06 x 107 CFU/ml and in 18 saliva samples was 1,51 x 105 CFU/ml. It was concluded real-time PCR can detect Veillonella spp. from all tongue plaque’s and saliva’s samples.

Early detection of drug-resistant Streptococcus pneumoniae and Haemophilus influenzae by quantitative flow cytometry

Early detection of drug resistance contributes to combating drug-resistant bacteria and improving patient outcomes. Microbial testing in the laboratory is essential for treating infectious diseases because it can provide critical information related to identifying pathogenic bacteria and their resistance profiles. Despite these clinical requirements, conventional phenotypic testing is time-consuming. Additionally, recent rapid drug resistance tests are not compatible with fastidious bacteria such as Streptococcus and Haemophilus species. In this study, we validated the feasibility of direct bacteria counting using highly sensitive quantitative flow cytometry. Furthermore, by combining flow cytometry and a nucleic acid intercalator, we constructed a highly sensitive method for counting viable fastidious bacteria. These are inherently difficult to measure due to interfering substances from nutrients contained in the medium. Based on the conventional broth microdilution method, our method acquired a few microliter samples in a time series from the same microplate well to exclude the growth curve inconsistency between the samples. Fluorescent staining and flow cytometry measurements were completed within 10 min. Therefore, this approach enabled us to determine antimicrobial resistance for these bacteria within a few hours. Highly sensitive quantitative flow cytometry presents a novel avenue for conducting rapid antimicrobial susceptibility tests.

Genexpert rapid detection of rifampicin resistance among patients with pulmonary tuberculosis

The routine conventional phenotypic bacteriological tests available for pulmonary tuberculosis (PTB) diagnosis, such as microscopy by Ziehl Neelsen staining, and Lowenstein Jensen method of solid culture, requires a long time for revealing positivity, resulting in a delayed diagnosis. After Mycobacterium tuberculosis (MTB) genome was discovered, nucleic acid amplification techniques were developed with more rapid detection and identification of rifampicin resistance in respiratory and extra-pulmonary specimens. GeneXpert is a DNA-polymerase chain reaction technique based on NAA, which allows an accurate genotypic method for a quick diagnosis of PTB diagnosis. The aim of the study was to assess retrospectively the yield of genotypic assay GeneXpert in revealing multidrug resistant TB compare to phenotypic confirmation by solid culture. 512 patients with positive and negative smears of suspected PTB were investigated by conventional phenotypic assays. PTB diagnosis assessed by positive genotypic assay and confirmed by phenotypic conventional solid culture method was 78.12%. The yield of GeneXpert in revealing rifampicin resistance was high (n=79/512; 15.43%) with 12.5% additional resistance against isoniazid revealed by positive solid cultures and drug susceptibility test. GeneXpert assay is a very useful method for rapid detection of MTB and drug resistance, which facilitates timely diagnosis and appropriate management of pulmonary tuberculosis.