RpoS role in antibiotic resistance, tolerance and persistence in E. coli natural isolates.

BACKGROUND: The intrinsic concentration of RpoS, the second most abundant sigma factor, varies widely across the E. coli species. Bacterial isolates that express high levels of RpoS display high resistance to environmental stresses, such as temperature, pH and osmolarity shifts, but are less nutritional competent, making them less capable of utilising alternative nutrient sources. The role of RpoS in antibiotic resistance and persistence in standard laboratory domesticated strains has been examined in several studies, most demonstrating a positive role for RpoS. RESULTS: Using disk diffusion assays we examined bacterial resistance to 15 different antibiotics, including ß -lactams (penicillins, monobactams, carbapenems and cephalosporins), aminoglycosides, quinolones and anti-folates, in a representative collection of 328 E. coli natural isolates displaying a continuum of different levels of RpoS. There was an overall trend that isolates with higher levels of RpoS were slightly more resistant to these antibiotics. In addition, the effect of RpoS on bacterial tolerance and persistence to 3 different antibiotics - ampicillin, ciprofloxacin and kanamycin was evaluated through time-kill curves. Again, there was a small beneficial effect of RpoS on tolerance and persistence to these antibiotics, but this difference was not statistically significant. Finally, a K-12 strain expressing high levels of RpoS was compared with its isogenic RpoS-null counterpart, and no significant effect of RpoS was found. CONCLUSION: Based on a representative collection of the species E. coli, RpoS was found to have a very small impact on antibiotic resistance, tolerance, or persistence.

Antimicrobial resistance patterns, virulence genes, and biofilm formation in enterococci strains collected from different sources.

BACKGROUND: Currently, antibiotic-resistant strains of Enterococcus are considered to be one of the critical health challenges globally. This study aimed to investigate the antibiotic susceptibility pattern, biofilm formation capacity, and virulence genes of enterococci isolated from different sources. METHODS: In this cross-sectional study, environmental and fecal samples were collected from the hospital environment, volunteers, and hospital staff from October 2018 to August 2019. The isolates were identified by morphological and biochemical tests (gram staining, catalase, bile resistance, esculin hydrolysis, carbohydrate fermentation, growth in 6.5% NaCl, Pyrrolidonyl arylamidase, arginine dehydrolase), and PCR for ddl gene. An antimicrobial susceptibility test was performed by the standard disk agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Quantitative microplate assays were used to assess biofilm production. The bacterial DNAs were extracted by alkaline lysis method and polymerase chain reaction technique was used detect the esp, ace, and efaA virulence genes. RESULTS: Out of 145 isolates, 84 (57.9%) were identified as E. faecalis and 61 (42.1%) as E. faecium. Resistance to kanamycin and quinupristin-dalfopristin was 82.1% (69/84) and 85.7% (72/84), respectively, in E. faecalis isolates. Out of 61 E. faecalis isolates, 38 (62.4%) were resistant to kanamycin. Among the E. faecalis isolates, esp was the most dominant virulence gene (73.80%), followed by efaA, and ace, which were detected in 60.71%, and 30.95% isolates, respectively. In total, 68.27% of the strains were biofilm producers. Further, esp and efaA genes were more frequently found among E. faecalis strains with moderate and strong biofilm biomass. CONCLUSIONS: According to the findings of our study, enterococci strains isolated from different samples possess distinctive patterns of virulence genes. The esp, ace, and efaA genes were more prevalent among E. faecalis than E. faecium. Besides, the high level antibiotic resistance of normal flora and environmental enterococci strains is alarming the researchers.

Increased expression of Mycobacterium tuberculosis Rv3737 gene associated with low-level amikacin resistance.

INTRODUCTION: As an infectious disease, tuberculosis (TB) poses a serious threat to public health. Although amikacin (AMK) is an important antibiotic for the treatment of drug-resistant TB, its resistance mechanisms are not fully understood. METHODS: To investigate the role of Rv3737 gene on AMK drug susceptibility, a Mycobacterium tuberculosis (M.tb) Rv3737 knockout strain (H37Rv△Rv3737) and a Mycobacterium smegmatis (M.sm) Rv3737 overexpressing strain (Msm/pMV261-Rv3737) were used to detect their minimal inhibitory concentrations (MICs) in this study. RESULTS: The AMK MICs of Rv3737 knockout and overexpressing strains were 4-fold lower and 2-fold higher than those of the wild-type and empty plasmid strains, respectively. The results of clinical isolates showed that no Rv3737 gene mutation was found to be associated with AMK susceptibility, while the rrs A1401G mutation remained the main mechanism of high level of AMK resistance (MIC>32 µg/ml). There was a positive correlation between Rv3737 mRNA expression level and AMK MIC. In the isolates with low-level AMK resistance (MIC = 4 µg/ml) without rrs A1401G mutation, the expression level of Rv3737 gene was significantly higher than those of susceptible isolates. CONCLUSIONS: In this study, the Rv3737 gene was reported for the first time for its effect on AMK susceptibility in M.tb. Although the rrs A1401G mutation remains the main reason of high-level AMK resistance, high expression of the Rv3737 gene was associated with low-level AMK resistance in clinical isolates.

A fluorescent sensor array based on antibiotic-stabilized metal nanoclusters for the multiplex detection of bacteria.

To address the need for facile, rapid detection of pathogens in water supplies, a fluorescent sensing array platform based on antibiotic-stabilized metal nanoclusters was developed for the multiplex detection of pathogens. Using five common antibiotics, eight different nanoclusters (NCs) were synthesized including ampicillin stabilized copper NCs, cefepime stabilized gold and copper NCs, kanamycin stabilized gold and copper NCs, lysozyme stabilized gold NCs, and vancomycin stabilized gold/silver and copper NCs. Based on the different interaction of each NC with the bacteria strains, unique patterns were generated. Various machine learning algorithms were employed for pattern discernment, among which the artificial neural networks proved to have the highest performance, with an accuracy of 100%. The developed prediction model performed well on an independent test dataset and on real samples gathered from drinking water, tap water and the Anzali Lagoon water, with prediction accuracy of 96.88% and 95.14%, respectively. This work demonstrates how generic antibiotics can be implemented for NC synthesis and used as recognition elements for pathogen detection. Furthermore, it displays how merging machine learning techniques can elevate sensitivity of analytical devices.

Evaluation of the in vitro effects of concentrations of antibiotics on three Enterobacteriaceae isolates.

Due to the misuse and overuse of antibiotics, bacteria are now exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics in various environments. In recent years, exposure of bacteria to sub-MICs of antibiotics has led to the widespread emergence of antibiotic-resistant bacteria. In this study, three bacterial species from the Enterobacteriaceae family (Raoultella ornithinolytica, Pantoea agglomerans and Klebsiella quasivariicola) were isolated from water. The antibiotic susceptibility of these bacteria to 16 antibiotics was then investigated. The effects of sub-MICs of four selected antibiotics (kanamycin, chloramphenicol, meropenem, and ciprofloxacin) on the growth, biofilm formation, surface polysaccharide production, siderophore production, morphology, and expression of the translational/transcriptional regulatory transformer gene rfaH of these bacteria were analysed. The MICs of kanamycin, chloramphenicol, meropenem, and ciprofloxacin were determined to be 1, 2, 0.03 and 0.03 µg/mL for R. ornithinolytica; 0.6, 6, 0.03 and 0.05 µg/mL for P. agglomerans; and 2, 5, 0.04 and 0.2 µg/mL for K. quasivariicola. The growth kinetics and biofilm formation ability decreased for all three isolates at sub-MICs. The surface polysaccharides of R. ornithinolytica and P. agglomerans increased at sub-MICs. There was no significant change in the siderophore activities of the bacterial isolates, with the exception of MIC/2 meropenem in R. ornithinolytica and MIC/2 kanamycin in K. quasivariicola. It was observed that the sub-MICs of meropenem and ciprofloxacin caused significant changes in bacterial morphology. In addition, the expression of rfaH in R. ornithinolytica and K. quasivariicola increased with the sub-MICs of the selected antibiotics.

Discovery and Mechanistic Analysis of Structurally Diverse Inhibitors of Acetyltransferase Eis among FDA-Approved Drugs.

Over one and a half million people die of tuberculosis (TB) each year. Multidrug-resistant TB infections are especially dangerous, and new drugs are needed to combat them. The high cost and complexity of drug development make repositioning of drugs that are already in clinical use for other indications a potentially time- and money-saving avenue. In this study, we identified among existing drugs five compounds: azelastine, venlafaxine, chloroquine, mefloquine, and proguanil as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis, a causative agent of TB. Eis upregulation is a cause of clinically relevant resistance of TB to kanamycin, which is inactivated by Eis-catalyzed acetylation. Crystal structures of these drugs as well as chlorhexidine in complexes with Eis showed that these inhibitors were bound in the aminoglycoside binding cavity, consistent with their established modes of inhibition with respect to kanamycin. Among three additionally synthesized compounds, a proguanil analogue, designed based on the crystal structure of the Eis-proguanil complex, was 3-fold more potent than proguanil. The crystal structures of these compounds in complexes with Eis explained their inhibitory potencies. These initial efforts in rational drug repositioning can serve as a starting point in further development of Eis inhibitors.

Construction of Liquid Crystal-Based Sensors Using Enzyme-Linked Dual-Functional Nucleic Acid on Magnetic Beads.

The development of liquid crystal (LC)-based sensors with superior performances such as high portability, excellent stability, great convenience, and remarkable sensitivity is highly demanded. This work proposes a new strategy for constructing the LC-based sensor using enzyme-linked dual-functional nucleic acid (d-FNA) on magnetic beads (MBs). The detection of kanamycin (KA) is demonstrated as a model. Acetylcholinesterase (AChE) is assembled onto the KA aptamer-modified MBs with a d-FNA strand that consists of an AChE aptamer and the complementary sequence of a KA aptamer. As the specific recognition of KA by its aptamer triggers the release of AChE from the MBs, the myristoylcholine (Myr) solution after incubation with the MBs causes the black image of the LCs due to the formation of the Myr monolayer at the aqueous/LC interface. Otherwise, in the absence of KA, AChE is still decorated on the MBs and causes the hydrolysis of Myr. Therefore, a bright image of LCs is obtained. The detection of KA is successfully achieved with a lower detection limit of 48.1 pg/mL. In addition, a thin polydimethylsiloxane (PDMS) layer-coated glass and a portable optical device are used to improve the stability and portability of the LC-based sensor to advance potential commercial applications. Furthermore, the detection of KA in milk with a portable device is demonstrated, showing the potential of the proposed enzyme-linked LC-based sensor.

Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Perturbation of ribosomal subunit dynamics by inhibitors of tRNA translocation.

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Diadenosine tetraphosphate modulated quorum sensing in bacteria treated with kanamycin.

BACKGROUND: The dinucleotide alarmone diadenosine tetraphosphate (Ap4A), which is found in cells, has been shown to affect the survival of bacteria under stress. RESULTS: Here, we labeled Ap4A with biotin and incubated the labeled Ap4A with the total proteins extracted from kanamycin-treated Escherichia coli to identify the Ap4A binding protein in bacteria treated with kanamycin. Liquid chromatography‒mass spectrometry (LCMS) and bioinformatics were used to identify novel proteins that Ap4A interacts with that are involved in biofilm formation, quorum sensing, and lipopolysaccharide biosynthesis pathways. Then, we used the apaH knockout strain of E. coli K12-MG1655, which had increased intracellular Ap4A, to demonstrate that Ap4A affected the expression of genes in these three pathways. We also found that the swarming motility of the apaH mutant strain was reduced compared with that of the wild-type strain, and under kanamycin treatment, the biofilm formation of the mutant strain decreased. CONCLUSIONS: These results showed that Ap4A can reduce the survival rate of bacteria treated with kanamycin by regulating quorum sensing (QS). These effects can expand the application of kanamycin combinations in the treatment of multidrug-resistant bacteria.

Synergistic effect of kanamycin and amikacin with setomimycin on biofilm formation inhibition of Listeria monocytogenes.

Listeria monocytogenes, a foodborne pathogen that causes listeriosis with high fatality rate, exhibits multidrug resistance (MDR) known to be progressively increasing. Alternative antibacterial strategies are in high demand for treating this well-known pathogen. Anti-biofilm and anti-virulence strategies are being explored as novel approaches to treat bacterial infections. In this study, one rare antibacterial named setomimycin was isolated from Streptomyces cyaneochromogenes, which showed potent antibacterial activity against L. monocytogenes. Next, the inhibition of biofilm formation and listeriolysin O (LLO) production against L. monocytogenes were investigated at sub-minimal inhibitory concentrations (sub-MICs) of setomimycin alone or combined with kanamycin and amikacin. Crystal violet staining confirmed that setomimycin combining with kanamycin or amikacin could dramatically reduce biofilm formation against L. monocytogenes at sub-MICs, which was further evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). In the meantime, sub-MICs of setomimycin could significantly suppress the secretion of LLO. Furthermore, the transcription of genes associated with biofilms and main virulence factors, such as LLO, flagellum, and metalloprotease, were suppressed by setomimycin at sub-MICs. Hence, the study provided a deep insight into setomimycin as an alternative antibacterial agent against L. monocytogenes.

High efficient electrochemical biosensor based on exonuclease-â ¢-assisted dual-recycling amplification for ultrasensitive detection of kanamycin.

A target-triggered and exonuclease-Ⅲ-assisted strand displacement, dual-recycling amplification reaction-based biosensor was developed for the rapid, ultrasensitive and accurate detection of kanamycin. The robust profiling platform was constructed using high conductive MXene/VS2 for the electrode surface modification and high active CeCu2O4 bimetallic nanoparticles as nanozyme to improve the sensitivity as well as the catalytic signal amplification of the biosensor. Using the dual supplementary recycling of primer DNA and hairpin DNA, the electrochemical platform could accurately detect kanamycin to as low as 0.6 pM from the range of 5 pM to 5 µM. By profiling five other antibiotics, this platform exhibited high specificity, enhanced repeatability and reproducibility. Based on these intrinsic characteristics and by utilizing milk and water samples, the as-designed biosensor offers a remarkable strategy for antibiotic detection due to its favorable analytical accuracy and reliability, thereby demonstrating potential application prospect for various antibiotic biosensing in food quality control, water contamination detection and biological safety analysis.

A simple tag-free fluorometric aptasensing assay for sensitive detection of kanamycin.

A novel label-free and enzyme-free fluorescence aptasensing assay that uses Sybr Green I (SGI) as the signal indicator for the kanamycin determination was designed. An aptamer-complementary strand (Apt/CP) conjugate was formed, which provided the intercalation sites for SGI and, therefore, a considerable fluorescent signal. The introduction of the target led to the separation of Apt from CP due to the high affinity of Apt toward kanamycin. Hence, the suitable intercalation gaps reduced, which resulted in a decrease in the generated fluorescent signal. Under optimized conditions, a broad linear concentration range from 0.05 µM to 20 µM and a limit of detection of 11.76 nM were obtained, confirming the ability of the fabricated aptasensor for sensitive and specific kanamycin detection in real samples such as milk and human serum. The aptasensing method has the potential to be extensively employed in the food industry and veterinary science due to its simplicity, sensitivity, user-friendly, and capability of on-site detection of kanamycin.

Adenanthin Is an Efficient Inhibitor of Peroxiredoxins from Pathogens, Inhibits Bacterial Growth, and Potentiates Antibiotic Activities.

The emergence and re-emergence of bacterial strains resistant to multiple drugs represent a global health threat, and the search for novel biological targets is a worldwide concern. AhpC are enzymes involved in bacterial redox homeostasis by metabolizing diverse kinds of hydroperoxides. In pathogenic bacteria, AhpC are related to several functions, as some isoforms are characterized as virulence factors. However, no inhibitor has been systematically evaluated to date. Here we show that the natural ent-kaurane Adenanthin (Adn) efficiently inhibits AhpC and molecular interactions were explored by computer assisted simulations. Additionally, Adn interferes with growth and potentializes the effect of antibiotics (kanamycin and PMBN), positioning Adn as a promising compound to treat infections caused by multiresistant bacterial strains.

A reusable fiber-embedded microfluidic chip for rapid and sensitive on-site detection of kanamycin residues in water environments.

The overuse and abuse of antibiotics have led to increased pollution in water environments. Thus, it is crucial to develop a rapid, high-frequency, and cost-effective method for on-site detection of antibiotics. In this regard, a reusable fiber-embedded microfluidic chip was constructed by combining a microfluidic chip with a functionalized fiber bioprobe that served as both a biorecognition element and an optical transducer. The fiber-embedded microfluidic chip enabled the quantitative detection of kanamycin (KANA) by integrating a portable all-fiber evanescent wave fluorescence detection device. Under optimized conditions, quantitative KANA detection was achieved with a detection limit of 0.03 µg L-1 and a linear detection range of 0.21-10.3 µg L-1. The accurate detection of KANA in various water samples can be completed within 25 min without pretreatment. The functionalized fiber-embedded microfluidic chip could be reused more than 200 times without significant performance loss. To demonstrate its suitability for practical applications, the fiber-embedded microfluidic chip was used to investigate KANA residues in surface waters obtained from the Qinghe River in Beijing, China. The results were compared with those of a traditional enzyme-linked immunosorbent assay, which showed a high correlation. Compared to conventional optical microfluidic chips, the proposed fiber-embedded microfluidic chip has several advantages, including its ease of use, miniaturization, cost-effectiveness, reusability, and high flexibility. It is an ideal alternative for rapid, sensitive on-site detection of antibiotics and other trace substances in environmental, food, and medical fields.

A label-free G-quadruplex aptamer fluorescent aptasensor for visual and real-time kanamycin detection in lake and human samples.

Antibiotic abuse is considered a serious problem affecting human health, necessitating that great attention be paid to explore robust, simple and sensitive methods for rapid evaluation. In this paper, we developed a fluorescent aptasensor for visual and real-time kanamycin detection by taking advantage of the label-free strategy based on H-aggregate disassembly of a chiral cyanine dye induced by a G-quadruplex aptamer. The good sensitivity and selectivity enabled this aptasensor to have a detection limit as low as 43 nM and have high specificity for kanamycin recognition. Furthermore, this assay was successfully applied for the detection of kanamycin in lake water and urine with excellent recoveries.

Deglycosylation Inactivation Initiated by a Novel Periplasmic Dehydrogenase Complex Provides a Novel Strategy for Eliminating the Recalcitrant Antibiotic Kanamycin.

Biodegradation using enzyme-based systems is a promising approach to minimize antibiotic loads in the environment. Aminoglycosides are refractory antibiotics that are generally considered non-biodegradable. Here, we provide evidence that kanamycin, a common aminoglycoside antibiotic, can be degraded by an environmental bacterium through deglycosylation of its 4'-amino sugar. The unprecedented deglycosylation inactivation of kanamycin is initiated by a novel periplasmic dehydrogenase complex, which we designated AquKGD, composed of a flavin adenine dinucleotide-dependent dehydrogenase (AquKGDα) and a small subunit (AquKGDγ) containing a twin-arginine signal sequence. We demonstrate that the formation of the AquKGDα-AquKGDγ complex is required for both the degradation activity of AquKGD and its translocation into the periplasm. Native AquKGD was successfully expressed in the periplasmic space of Escherichia coli, and physicochemical analysis indicated that AquKGD is a stable enzyme. AquKGD showed excellent degradation performance, and complete elimination of kanamycin from actual kanamycin manufacturing waste was achieved with immobilized AquKGD. Ecotoxicity and cytotoxicity tests suggest that AquKGD-mediated degradation produces less harmful degradation products. Thus, we propose a novel enzymatic antibiotic inactivation strategy for effective and safe treatment of recalcitrant kanamycin residues.

Genotypic and phenotypic comparison of drug resistance profiles of clinical multidrug-resistant Mycobacterium tuberculosis isolates using whole genome sequencing in Latvia.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) remains a major public health problem in many high tuberculosis (TB) burden countries. Phenotypic drug susceptibility testing (DST) take several weeks or months to result, but line probe assays and Xpert/Rif Ultra assay detect a limited number of resistance conferring gene mutations. Whole genome sequencing (WGS) is an advanced molecular testing method which theoretically can predict the resistance of M. tuberculosis (Mtb) isolates to all anti-TB agents through a single analysis. METHODS: Here, we aimed to identify the level of concordance between the phenotypic and WGS-based genotypic drug susceptibility (DS) patterns of MDR-TB isolates. Overall, data for 12 anti-TB medications were analyzed. RESULTS: In total, 63 MDR-TB Mtb isolates were included in the analysis, representing 27.4% of the total number of MDR-TB cases in Latvia in 2012-2014. Among them, five different sublineages were detected, and 2.2.1 (Beijing group) and 4.3.3 (Latin American-Mediterranean group) were the most abundant. There were 100% agreement between phenotypic and genotypic DS pattern for isoniazid, rifampicin, and linezolid. High concordance rate (> 90%) between phenotypic and genotypic DST results was detected for ofloxacin (93.7%), pyrazinamide (93.7%) and streptomycin (95.4%). Phenotypic and genotypic DS patterns were poorly correlated for ethionamide (agreement 56.4%), ethambutol (85.7%), amikacin (82.5%), capreomycin (81.0%), kanamycin (85.4%), and moxifloxacin (77.8%). For capreomycin, resistance conferring mutations were not identified in several phenotypically resistant isolates, and, in contrary, for ethionamide, ethambutol, amikacin, kanamycin, and moxifloxacin the resistance-related mutations were identified in several phenotypically sensitive isolates. CONCLUSIONS: WGS is a valuable tool for rapid genotypic DST for all anti-TB agents. For isoniazid and rifampicin phenotypic DST potentially can be replaced by genotypic DST based on 100% agreement between the tests. However, discrepant results for other anti-TB agents limit their prescription based solely on WGS data. For clinical decision, at the current level of knowledge, there is a need for combination of genotypic DST with modern, validated phenotypic DST methodologies for those medications which did not showed 100% agreement between the methods.

The differences in drug resistance between drug-resistant tuberculosis patients with and without diabetes mellitus in northeast China: a retrospective study.

BACKGROUND: Diabetes mellitus (DM) and drug-resistant tuberculosis (DR-TB) are serious global public health problems. This study aimed to explore the differences in drug resistance between DR-TB patients with and without DM. Risk factors for developing multidrug-resistant tuberculosis (MDR-TB) were also investigated among DR-TB patients. METHODS: The patient's basic demographic,　clinical characteristics, and drug susceptibility testing (DST) data were collected from the Chinese Disease Control Information System. Descriptive statistics were used to estimate the frequency and proportion of included variables. Categorical variables were compared using the Chi-square test or Fisher's exact test. Chi-square tests for trends were used to determine changes and trends in MDR-TB and pre-extensively drug-resistantTB (pre-XDR-TB) patterns over time. Univariate and multivariate logistic regression analysis was used to explore the risk factors of MDR-TB. RESULTS: Compared with DR-TB patients with DM, DR-TB patients without DM had significantly higher rates of mono-resistant streptomycin (SM) and any resistance to kanamycin (KM), but significantly lower rates of any resistance to protionamide (PTO) and mono-resistance to levofloxacin (LFX), and pre-XDR-TB (P<0.05). The proportion of resistance to other anti-TB drugs was not statistically different between the DR-TB with and without DM. Among DR-TB patients without and with DM, the proportion of patients with MDR-TB and pre-XDR-TB patterns showed a significant downward trend from 2016 to 2021 (P<0.05). Among DR-TB patients without DM, male, previously treated DR-TB cases, and immigration were risk factors for MDR-TB (P<0.05). In DR-TB patients with DM, a negative sputum smear is a risk factor for MDR-TB (P<0.05). CONCLUSION: There was no statistical difference in resistance patterns between DR-TB with and without DM, except in arbitrary resistance to PTO and KM, mono-resistant SM and LFX, and pre-XDR-TB. Great progress has been made in the prevention and control of MDR-TB and pre-XDR-TB. However, DR-TB patients with and without DM differ in their risk factors for developing MDR-TB.

Molecular Characterization of Resistance to Second-Line Anti-Mycobacterial Drugs among Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis.

BACKGROUND: The emergence of multidrug resistance and extensively drug-resistant tuberculosis is a serious public health crisis. Using rapid and inexpensive molecular methods such as HRM assay in the detection of second-line drugs resistance in M. tuberculosis would be helpful in the treatment and control of XDR tuberculosis cases. METHODS: MDR-TB isolates were collected from Iranian tuberculosis laboratories. Drug susceptibility test performed via the indirect proportion method utilizing LJ Medium. Susceptibility to ciprofloxacin, ofloxacin, amikacin, kanamycin, and capreomycin, as second-line anti-tuberculosis agents were assessed. Single point mutations in gyrA, rrs and eis genes were detected via HRM assay and DNA sequencing. RESULTS: A DST test was performed for 56 MDR isolates and at least 27 (48.2%) isolates were resistant to CIP or OFL. Also, 14 (25%), 12 (21.4%), and 15 (26.7%) isolates were resistant to capreomycin, amikacin, and kanamycin, respectively. D94G, A90V, and G88C mutations were the most frequent mutations in gyrA gene. Also, A1401G mutation was detected more than the other mutations in rrs gene. CONCLUSIONS: The frequency of CIP/OFL and AMK/CAP/KAN-resistant TB is considerable among Iranian tuberculosis cases. HRM assay is a rapid and inexpensive test and can detect important mutation-based drug resistance in MDR-TB and XDR-TB isolates.