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Cases of diphtheria, even in immunized individuals, are still reported in several parts of the world, including in Brazil. New outbreaks occur in Europe and other continents. In this context, studies on Corynebacterium diphtheriae infections are highly relevant, both for a better understanding of the pathogenesis of the disease and for controlling the circulation of clones and antimicrobial resistance genes. Here we present a case of cutaneous infection by multidrug-resistant Corynebacterium diphtheriae and provide its whole-genome sequencing. Genomic analysis revealed resistance genes, including tet(W), sul1, cmx, rpoB2, rbpA and mutation in rpoB. We performed phylogenetic analyzes and used the BRIG to compare the predicted resistance genes with those found in genomes from other significant isolates, including those associated with some outbreaks. Virulence factors such as spaD, srtBC, spaH, srtDE, surface-anchored pilus proteins (sapD), nonfimbrial adhesins (DIP0733, DIP1281, and DIP1621), embC and mptC (putatively involved in CdiLAM), sigA, dtxR and MdbA (putatively involved) in post-translational modification, were detected. We identified the CRISPR-Cas system in our isolate, which was classified as Type II-U based on the database and contains 15 spacers. This system functions as an adaptive immune mechanism. The strain was attributed to a new sequence type ST-928, and phylogenetic analysis confirmed that it was related to ST-634 of C. diphtheriae strains isolated in French Guiana and Brazil. In addition, since infections are not always reported, studies with the sequence data might be a way to complement and inform C. diphtheriae surveillance. [ABSTRACT FROM AUTHOR]

GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre-extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.

Introduction: Pakistan has the highest burden of Tuberculosis (TB) worldwide and the 5th highest prevalence of multi- drug resistant (MDR) TB worldwide. There is scarce data on the socioeconomic characteristics and factors associated with the patients presenting with drug resistant-TB in Pakistani context. Aims & Objectives: To determine the association of different variables with rifampin resistance (RR) in new and previously treated cases of pulmonary (PTB) and extra-pulmonary TB (EPTB) Place and Duration of Study: It was a cross-sectional study conducted at Gulab Devi Hospital Lahore, a tertiary care trust hospital. Data was collected from Oct 2021 to December 31, 2021. Material & Method: By applying convenient sampling technique, 202 individuals were interviewed, and data was recorded on a self-constructed questionnaire. Data was analyzed using SPSS version 24.0. Frequency tables were generated for sociodemographic variables as well as the site of TB and Chi square test was applied to find association between categorical variables. P value of

Rifampin resistance (RIF-R) in Staphylococcus aureus (S. aureus) with rpoB mutations as one of its resistance mechanisms has raised concern about clinical treatment and infection prevention strategies. Data on the prevalence and molecular epidemiology of RIF-R S. aureus blood isolates in South Korea are scarce. We used broth microdilution to investigate RIF-R prevalence and analyzed the rpoB gene mutation in 1615 S. aureus blood isolates (772 methicillin-susceptible and 843 methicillin-resistant S. aureus (MRSA)) from patients with bacteremia, between 2008 and 2017. RIF-R prevalence and antimicrobial susceptibility were determined. Multilocus sequence typing was used to characterize the isolate's molecular epidemiology; Staphylococcus protein A (spa), staphylococcal cassette chromosome mec (SCCmec), and rpoB gene mutations were detected by PCR. Among 52 RIF-R MRSA isolates out of 57 RIF-R S. aureus blood isolates (57/1615, 0.4%; 5 methicillin-susceptible and 52 MRSA), ST5 (44/52, 84.6%), SCCmec IIb (40/52, 76.9%), and spa t2460 (27/52, 51.9%) were predominant. rpoB gene mutations with amino acid substitutions showed that A477D (17/48, 35.4%) frequently conferred high-level RIF resistance (MIC > 128 mg/L), followed by H481Y (4/48, 8.3%). RIF-R S. aureus blood isolates in South Korea have unique molecular characteristics and are closely associated with rpoB gene mutations. RIF-R surveillance through S. aureus–blood isolate epidemiology could enable effective therapeutic management. [ABSTRACT FROM AUTHOR]

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Ma-Chao Li,1,* Xiao-Yue Wang,1,* Tong-Yang Xiao,2,* Shi-Qiang Lin,3 Hai-Can Liu,1 Cheng Qian,4 Da Xu,1 Gui-Lian Li,1 Xiu-Qin Zhao,1 Zhi-Guang Liu,1 Li-Li Zhao,1 Kang-Lin Wan1 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China; 2Department of Clinical Laboratory, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong, People’s Republic of China; 3Department of Bioinformatics, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province, People’s Republic of China; 4Beijing Center for Disease Control and Prevention, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li-Li Zhao; Kang-Lin Wan, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing, 102206, People’s Republic of China, Tel/Fax +86 10 58900779, Email zhaolili@icdc.cn; wankanglin@icdc.cnObjective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis).Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays.Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified.Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.Keywords: Mycobacterium tuberculosis, rifampin resistance, rifabutin resistance, mutation

Background: Lymphadenopathy is one of the most prevalent clinical manifestations of extrapulmonary tuberculosis. Endosonography is the recommended technique in the diagnostic work-up of mediastinal lymphadenopathies. Xpert MTB/RIF assay is a self-contained cartridge-based fully automated DNA testing platform which can accurately detect both tuberculosis and mycobacterial resistance to rifampicin. A few studies assessed its accuracy for mediastinal lymph node aspirates collected using endosonography. A systematic review of observational studies was performed to provide a pooled estimate of sensitivity and specificity of Xpert MTB/RIF in the diagnosis of mediastinal tuberculous lymphadenitis using endoscopic ultrasound-guided needle aspiration techniques. Methods: A search of the scientific evidence was carried out using PubMed, Embase, and Scopus. Articles describing observational studies on Xpert MTB/RIF in the diagnosis of mediastinal tuberculous lymphadenitis using endoscopic ultrasound-guided needle aspiration techniques were selected. Results: Eight studies met the inclusion criteria. The overall pooled sensitivity was 61% (95% CI = 55–68%; I2 = 66.3%; p = 0.004), overall pooled specificity was 89% (95% CI = 85–91%; I2 = 90.1%; p < 0.0001). Area under the sROC curve was 0.68. Only one study reported data on rifampin resistance detection and showed a sensitivity of 83.3% and a specificity of 16%. Conclusions: Xpert MTB/RIF shows a good accuracy in the diagnosis of mediastinal mycobacterial lymphadenitis by endosonographic needle aspiration techniques. It should be always recommended for suspected mediastinal tuberculosis. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis is considered one of the major infectious diseases especially in the developing countries and a significant diagnostic and therapeutic challenge worldwide. Gene Xpert MTB/RIF assay is a diagnostic test which depends on a real-time polymerase chain reaction for rapid diagnosis of Mycobacterium Tuberculosis and rifampicin resistance in a single test. Aim: To evaluate the diagnostic utility of Gene Xpert MTB/RIF assay for rapid diagnosis of pulmonary and extrapulmonary tuberculosis as well as the detection of rifampin resistance in specimens obtained from suspected tuberculous patients in Abbassia Chest Hospital. Patients and Methods: Pulmonary and extrapulmonary samples from 77 cases with suspected TB were collected. All retrieved samples were examined with: direct smear microscopy for AFB, culture for Mycobacterium tuberculosis (L.J. media) along with Drug susceptibility testing (D.S.T.) for positive cultures. As well as the gene Xpert MTB/RIF assay and their results were com-pared to those of both culture and D.S.T. (as gold standard reference methods). Results: Gene Xpert MTB/RIF assay showed the highest sensitivity, negative predictive value (NPV) and diagnostic accuracy in detection of pulmonary TB (96.4, 96.4 and 93.1% respectively), with 90% specificity. Xpert MTB/RIF assay showed the lowest sensitivity in diagnosing TB pleural effusion (66.6%) but with the highest specificity and positive predictive value (PPV) (100% for both). As regards diagnosing TB lymphadenitis, gene Xpert MTB/RIF assay showed 75% sensitivity and the lowest specificity (33.3%). Considering rifampin resistance, gene Xpert MTB/RIF assay showed 100% specificity in detection of RIF- Resistance with variable sensitivity according to the type of TB, being the highest in pulmonary TB (90.9% sensitivity). Conclusion: Gene Xpert MTB/RIF assay could be used as the first line diagnostic tool for diagnosis of pulmonary TB because of its high diagnostic yield. Yet, it's not recommended to replace the culture for diagnosing extrapulmonary TB especially in diagnosing tuberculous pleural effusion (pauci-bacillary specimens) which has limited diagnostic utility. Also, the assay showed 100% specificity in diagnosing rifampicin resistance guiding physicians to justifiably starting second line anti-TB treatment. [ABSTRACT FROM AUTHOR]

Rifampin resistance (RIF-R) in Staphylococcus aureus (S. aureus) with rpoB mutations as one of its resistance mechanisms has raised concern about clinical treatment and infection prevention strategies. Data on the prevalence and molecular epidemiology of RIF-R S. aureus blood isolates in South Korea are scarce. We used broth microdilution to investigate RIF-R prevalence and analyzed the rpoB gene mutation in 1615 S. aureus blood isolates (772 methicillin-susceptible and 843 methicillin-resistant S. aureus (MRSA)) from patients with bacteremia, between 2008 and 2017. RIF-R prevalence and antimicrobial susceptibility were determined. Multilocus sequence typing was used to characterize the isolate’s molecular epidemiology; Staphylococcus protein A (spa), staphylococcal cassette chromosome mec (SCCmec), and rpoB gene mutations were detected by PCR. Among 52 RIF-R MRSA isolates out of 57 RIF-R S. aureus blood isolates (57/1615, 0.4%; 5 methicillin-susceptible and 52 MRSA), ST5 (44/52, 84.6%), SCCmec IIb (40/52, 76.9%), and spa t2460 (27/52, 51.9%) were predominant. rpoB gene mutations with amino acid substitutions showed that A477D (17/48, 35.4%) frequently conferred high-level RIF resistance (MIC > 128 mg/L), followed by H481Y (4/48, 8.3%). RIF-R S. aureus blood isolates in South Korea have unique molecular characteristics and are closely associated with rpoB gene mutations. RIF-R surveillance through S. aureus–blood isolate epidemiology could enable effective therapeutic management.

A multidrug-resistant clone of the animal and human pathogen Rhodococcus equi, MDR-RE 2287, has been circulating among equine farms in the United States since the 2000s. We report the detection of MDR-RE 2287 outside the United States. Our finding highlights the risk for MDR-RE spreading internationally with horse movements. [ABSTRACT FROM AUTHOR]

Ma-chao Li,1,&ast; Jie Lu,2,&ast; Yao Lu,1,3,&ast; Tong-yang Xiao,4,&ast; Hai-can Liu,1 Shi-qiang Lin,5 Da Xu,1 Gui-lian Li,1 Xiu-qin Zhao,1 Zhi-guang Liu,1 Li-li Zhao,1 Kang-lin Wan1 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China; 2Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, MOE Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, People’s Republic of China; 3School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, People’s Republic of China; 4Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, Shenzhen Third People’s Hospital, Southern University of Science and Technology, Shenzhen, People’s Republic of China; 5Department of Bioinformatics, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Li-li Zhao; Kang-lin WanNational Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing, 102206, People’s Republic of ChinaTel/Fax +86 10 58900779Email zhaolili@icdc.cn; wankanglin@icdc.cnObjective: To investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.Methods: We sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.Results: Results revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.Conclusion: Some mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.Keywords: Mycobacterium tuberculosis, rifampin resistance, structure, mutation

RNA polymerase (RNAP) is a highly conserved macromolecular machine that contributes to the flow of genetic information from genotype to phenotype. In Bacillus subtilis, mutations in the rpoB gene encoding the b-subunit of RNAP have been shown to alter a number of global phenotypes, including growth, utilization of unusual nutrient sources, sporulation, germination, and production of secondary metabolites. In addition, the spectrum of mutations in rpoB leading to rifampin resistance (Rifr) can change dramatically depending upon the environment to which B. subtilis cells or spores are exposed. Rifr rpoB mutations have historically been associated with slower growth and reduced fitness; however, these assessments of fitness were conducted on limited collections of mutants in rich laboratory media that poorly reflect natural environments typically inhabited by B. subtilis. Using a novel deep-sequencing approach in addition to traditional measurements of growth rate, lag time, and pairwise competitions, we demonstrated that the competitive advantages of specific rpoB alleles differ depending on the growth environment in which they are determined. IMPORTANCE Microbial resistance to antibiotics is a growing threat to public health across the world. Historically, resistance to antibiotics has been associated with reduced fitness. A growing body of evidence indicates that resistance to rifampin, a frontline antibiotic used to treat mycobacterial and biofilm-associated infections, may increase fitness given an appropriate environment even in the absence of the selective antibiotic. Here, we experimentally confirm this phenomenon by directly comparing the fitness of multiple rifampin-resistant mutants of Bacillus subtilis in rich LB medium and an asparagine minimal medium. Our research demonstrates that the fitness cost of rifampin resistance can vary greatly depending upon the environment. This has important implications for understanding how microbes develop antimicrobial resistance in the absence of antibiotic selection. [ABSTRACT FROM AUTHOR]

The Chlamydiaceae are a family of obligate intracellular, gram-negative bacteria known to readily exchange DNA by homologous recombination upon co-culture in vitro, allowing the transfer of antibiotic resistance residing on the chlamydial chromosome. Among all the obligate intracellular bacteria, only Chlamydia (C.) suis naturally integrated a tetracycline resistance gene into its chromosome. Therefore, in order to further investigate the readiness of Chlamydia to exchange DNA and especially antibiotic resistance, C. suis is an excellent model to advance existing co-culture protocols allowing the identification of factors crucial to promote homologous recombination in vitro. With this strategy, we co-cultured tetracycline-resistant with rifamycin group-resistant C. suis, which resulted in an allover recombination efficiency of 28%. We found that simultaneous selection is crucial to increase the number of recombinants, that sub-inhibitory concentrations of tetracycline inhibit rather than promote the selection of double-resistant recombinants, and identified a recombination-deficient C. suis field isolate, strain SWA-110 (1-28b). While tetracycline resistance was detected in field isolates, rifampicin/rifamycin resistance (RifR) had to be induced in vitro. Here, we describe the protocol with which RifR C. suis strains were generated and confirmed. Subsequent whole-genome sequencing then revealed that G530E and D461A mutations in rpoB, a gene encoding for the β-subunit of the bacterial RNA polymerase (RNAP), was likely responsible for rifampicin and rifamycin resistance, respectively. Finally, whole-genome sequencing of recombinants obtained by co-culture revealed that recombinants picked from the same plate may be sibling clones and confirmed C. suis genome plasticity by revealing variable, apparently non-specific areas of recombination.

Abstract Background Riemerella anatipestifer (R. anatipestifer) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer. Results Comparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10− 8. A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 μg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h. Conclusions Mutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo.

Multidrug resistance has been detected in the animal and zoonotic human pathogen Rhodococcus equi after mass macrolide/rifampin antibioprophylaxis in endemically affected equine farms in the United States. Multidrug-resistant (MDR) R. equi emerged upon acquisition of pRERm46, a conjugative plasmid conferring resistance to macrolides, lincosamides, streptogramins, and, as we describe, tetracycline. Phylogenomic analyses indicate that the increasing prevalence of MDR R. equi since it was first documented in 2002 is caused by a clone, R. equi 2287, attributable to coselection of pRErm46 with a chromosomal rpoBS531F mutation driven by macrolide/rifampin therapy. pRErm46 spillover to other R. equi genotypes has given rise to a novel MDR clone, G2016, associated with a distinct rpoBS531Y mutation. Our findings illustrate that overuse of antimicrobial prophylaxis in animals can generate MDR pathogens with zoonotic potential. MDR R. equi and pRErm46-mediated resistance are currently disseminating in the United States and are likely to spread internationally through horse movements. [ABSTRACT FROM AUTHOR]

Background and Objectives: The incidence of rifampin-resistant strains of Mycobacterium tuberculosis has attracted more attention than the tuberculosis infection due to laborious treatment and control. Recognizing the Mycobacterium tuberculosis genotypes involving in drug resistance via multiplex PCR, a simple and rapid genotyping method, is an emergency for better treatment and control of tuberculosis. This study was designed to specify the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from patients by multiplex allele-specific Polymerase Chain Reaction assay (MAS-PCR).Methods: In this study, 88 Mycobacterium tuberculosis positive samples were included from Qaem Hospital, Mashhad. MAS-PCR was used to detect the rifampin resistance associated mutations in rpoB gene. Results: Mutations in three codons of rpoB gene causing rifampin resistance were detected in 51 isolates (58.96%). The detected mutations in codons 531, 526, and 516 were 55.68%, 38.63%, and 13.63%, respectively. The simultaneous mutations were detected in 11 isolates (12.50%) in codons 531, 526 and 516, in 21 isolates (23.86%) in codons 531 and 526, and in one isolate (1.13%) in codons 526 and 516. Conclusion: According to the results of this study, the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from Khorasan province patients (North-East of Iran) was high. The developed MAS-PCR assay can be used for rapid detection in clinical diagnostic laboratories in areas with high prevalence of multidrug-resistant Mycobacterium tuberculosis strains. In this respect, MAS-PCR is simple, rapid, and highly sensitive method for drug susceptibility tests for detecting multidrug-resistant Mycobacterium tuberculosis.

Background: A simple and cost-effective method is needed for the detection of rifampicin resistance in Mycobacterium tuberculosis in resource-limited settings. We suggest a broth medium-based method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC) for detection of rifampin resistance of tubercle bacilli within a reasonable time frame.Methods: The type strain (M. tuberculosis H37Rv) and 45 cultured clinical strains of M. tuberculosis (35 rifampin-susceptible and 10 rifampin-resistant) were used. Phenotypes of rifampicin resistance were tested by the Korea Institute of tuberculosis, and confirmed by GenoType MTBDRplus (Hain Lifescience, Germany). Susceptibility tests were performed using STC-containing OADC-enriched Middlebrook 7H9 broth (BD, USA).Results: All tests were finished in 3 to 6 days. The same results were obtained with the standard and current methods for all 45 clinical isolates (100% sensitivity and specificity for resistance detection).Conclusion: The current method using STC is a good alternative for detecting M. tuberculosis rifampin resistance in a cost-effective and timely fashion, which is particularly important in resource-limited settings. (Ann Clin Microbiol 2018;21:47-50)

Yu-Tsung Huang,1–3 Chun-Hsing Liao,1,4 Shey-Ying Chen,5 Chia-Jui Yang,1,4 Hsin-Sui Hsu,1 Lee-Jene Teng,2,3 Po-Ren Hsueh2,6 1Department of Internal Medicine, Far Eastern Memorial Hospital, New Taipei City, Taiwan; 2Department of Laboratory Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan; 3Department of Clinical Laboratory Science and Medical Biotechnology College of Medicine, National Taiwan University, Taipei, Taiwan; 4Department of Internal Medicine, College of Medicine, Yang-Ming University, Taipei, Taiwan; 5Department of Emergency Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan; 6Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan Objectives: This study investigated molecular characteristics of rifampin (RIF)-resistant (RIF-R) Staphylococcus aureus isolates recovered from patients receiving RIF-containing regimens for tuberculosis (TB). Patients and methods: Patients with TB who received RIF-containing regimens from November 2009 to May 2011 at a medical center were enrolled. Nasal swabs for S. aureus culture were obtained at the time of enrollment, and then every two months until two months after RIF treatment had been completed. Genetic relatedness of the isolates was determined using pulsed-field gel electrophoresis, multilocus sequence typing, and gene typing of spa and SCCmec. The presence of RIF resistance-associated mutations in rpoB, and fusidic acid resistance genes fusB and fusC in the S. aureus isolates were analyzed. Results: Among the 200 patients enrolled in this study, 152 completed follow-ups during treatment, and 114 completed two months of follow-up after discontinuing use of RIF. At enrollment, ten patients (5%) had nasal colonization with S. aureus, namely eight with methicillin-susceptible S. aureus (MSSA), and two with methicillin-resistant S. aureus (MRSA, ST59-SCCmecIV-RIF-susceptible). All these patients were decolonized after RIF usage. Two patients with MSSA colonization at enrollment showed recolonization with genetically unrelated MSSA strains two months after completion of RIF treatment. There were five ST45-SCCmecVT-RIF-R strains from two patients isolated during RIF exposure. Sequencing of rpoB in the RIF-R S. aureus isolates revealed different mutation sites between the MSSA and MRSA isolates. Conclusion: RIF-R S. aureus strains are more likely to result in persistent nasal carriage in TB patients receiving RIF treatment. Monitoring of emergence and possible dissemination of the MRSA ST45 strains among TB patients treated with RIF in Taiwan is warranted. Keywords: Staphylococcus aureus, methicillin-resistant S. aureus, rifampin resistance, tuberculosis, nasal carriage, Taiwan

ABSTRACT: Background and objective: Delayed diagnosis and treatment of tuberculosis (TB) contribute to poor outcomes, especially for endobronchial TB (EBTB), which typically leads to tracheobronchial stenosis. Finding rapid and accurate diagnostic tools for EBTB is crucial. GeneXpert Mycobacterium tuberculosis (MTB)/rifampin (RIF) was recommended by the World Health Organization (WHO) as a standard molecular biological diagnostic technique for MTB. The aim of this study was to evaluate the efficacy of GeneXpert MTB/RIF for diagnosing EBTB and for evaluating RIF resistance. Methods: Biopsy tissue and bronchial brushings from EBTB patients were prospectively assessed with GeneXpert MTB/RIF. The diagnostic yields of auramine O‐stained sputum smears and bronchial brush smears were obtained, and the results were compared with the cultures of sputum and biopsy tissues for MTB. Results: In 61 confirmed cases of EBTB, the sensitivities of sputum smear, bronchial brush smear, sputum culture and tissue culture to diagnose EBTB were 13.1%, 32.8%, 36.1% and 68.9%, respectively. For bronchial brushings and biopsies, our data showed sensitivities of 57.4% and 63.9%, respectively, and a specificity of 100% for GeneXpert MTB/RIF, and these results were superior to those of sputum smears, bronchial brush smears and sputum culture. GeneXpert MTB/RIF for bronchial brushings and biopsies showed complementarity in its diagnostic performance. Resistance to RIF was identified in 17.4% (8/46) of GeneXpert MTB‐positive cases. Conclusion: GeneXpert MTB/RIF may enable more rapid EBTB diagnosis and determination of RIF resistance, which are crucial for timely treatment. [ABSTRACT FROM AUTHOR]

Background and Objectives: The incidence of rifampin-resistant strains of Mycobacterium tuberculosis has attracted more attention than the tuberculosis infection due to laborious treatment and control. Recognizing the Mycobacterium tuberculosis genotypes involving in drug resistance via multiplex PCR, a simple and rapid genotyping method, is an emergency for better treatment and control of tuberculosis. This study was designed to specify the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from patients by multiplex allele-specific Polymerase Chain Reaction assay (MAS-PCR). Methods: In this study, 88 Mycobacterium tuberculosis positive samples were included from Qaem Hospital, Mashhad. MAS-PCR was used to detect the rifampin resistance associated mutations in rpoB gene. Results: Mutations in three codons of rpoB gene causing rifampin resistance were detected in 51 isolates (58.96%). The detected mutations in codons 531, 526, and 516 were 55.68%, 38.63%, and 13.63%, respectively. The simultaneous mutations were detected in 11 isolates (12.50%) in codons 531, 526 and 516, in 21 isolates (23.86%) in codons 531 and 526, and in one isolate (1.13%) in codons 526 and 516. Conclusion: According to the results of this study, the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from Khorasan province patients (North-East of Iran) was high. The developed MAS-PCR assay can be used for rapid detection in clinical diagnostic laboratories in areas with high prevalence of multidrug-resistant Mycobacterium tuberculosis strains. In this respect, MAS-PCR is simple, rapid, and highly sensitive method for drug susceptibility tests for detecting multidrug-resistant Mycobacterium tuberculosis. [ABSTRACT FROM AUTHOR]

In a fluctuation experiment, the number of cells existing in a culture immediately before plating (commonly known as N t ) varies across the parallel cultures. However, most existing mathematical models for fluctuation assay data do not recognize the variation in N t . Despite repeated attempts in the past to integrate this source of variability in the estimation of microbial mutation rates, several questions of practical importance remain unanswered. The present investigation finds that the variation needs accounting for only when the coefficient of variation for N t is large, and experimental data suggest that the coefficient of variation is often moderate or small. Moreover, an increase in the inoculum size can reduce the coefficient of variation. Through extensive simulation, several existing methods that accommodate the variation in N t are compared. It was found that a newly devised likelihood method based on the existing gamma mixture model outperforms other existing methods. The investigation focuses on the estimation of mutation rates using the Lea–Coulson model, under which mutation is selectively neutral; however, the paper also explores the major findings' implications for the comparison of mutation rates using the likelihood ratio test, and for the estimation of mutation rates using the Mandelbrot–Koch model that allows for non-neutral mutations. [ABSTRACT FROM AUTHOR]

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae ( M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy. [ABSTRACT FROM AUTHOR]

Introduction: In most cases of extrapulmonary tuberculosis, its etiological diagnosis is very seldom acomplished. The meningeal form of tuberculosis requires an early diagnosis and treatment because of its high rate of mortality and neurological sequelae. In this study, we report the results obtained with the use of a polymerase chain reaction and rifampin resistance technique (GeneXpert MTB/RIF)TM in 693 patients with the presuntive diagnosis of extrapulmonary tuberculosis, in comparison with the conventional techniques of stains, culture, tipification and antimicrobial susceptibility. Material and methods: We make a retrospective analysis of the results obtained in 693 samples of the same number of patients with presuntive clinical diagnosis of tuberculosis using the Ziehl-Neelsen stain technique, two culture media: Lowestein Jensen (BioMerieux) and Middle-brook broth base 7H9 (MGIT Becton Dickinson), two tipification techniques: Gen Probe (Accuprobe), Genotype MTBRD (Hain Lifescience), two antimicrobial susceptibility methods: Genotype MTBRD (Hain Lifescience), MGIT (Becton Dickinson) and a rapid technique of polymerase chain reaction and rifampin resistance (GeneXpert MTB/RIF (Cepheid), in the lapse of five years. Results: Most of the samples studied correspond to CSF and pleural effusions. 101 samples were positive by culture. 74 of the isolations correspond to the Mycobacterium tuberculosis complex, 22 to Mycobacterium avium, and five to Mycobacterium kansasii. The most sensitive method was the culture in the MGIT medium (86.12%), followed by the technique of GeneXpert (73.2%). None of the strains of Mycobacterium tuberculosis complex showed resistance to rifampin or to the other antimicrobials studied. The mean time for the obtention of a positive result was 34 days for the culture in the Lowestein Jensen media, 14 for the MGIT media, and 2.5 hours for the GeneXpert test. Conclusions: The GeneXpert MTB/RIF TestTM used in the study of 693 patients with the presuntive diagnosis of extrapulmonary tuberculosis showed numerous advantages over the conventional techniques: shorter time of obtention of results (hours versus days). High sensitivity (73.2%) and specificity (100%) --its sensitivity, considering only the isolations of Mycobacterium tuberculosis complex, was 100%--, the additional determination of rifampin resistance, and its technical simplicity. Its disadvantages were: no detection of strains other than the Mycobacterium tuberculosis complex (26.8% of the cases) and its high cost. [ABSTRACT FROM AUTHOR]