Rifampin-resistant Isolates Research Articles

Discordant results between Xpert MTB/RIF assay and Bactec MGIT 960 culture system regarding the detection of rifampin-resistant Mycobacterium tuberculosis isolates in Wenzhou, China.

This study aimed to assess the possible causes of discordant results between Xpert MTB/RIF (Xpert) and Bactec MGIT 960 Culture System (MGIT960) regarding rifampicin (RIF) susceptibility in Mycobacterium tuberculosis. Patients with previous RIF-resistant tuberculosis who were admitted to Wenzhou Central Hospital from January 2020 to December 2022 were enrolled. The isolates obtained from these patients were subjected to RIF susceptibility tests using Xpert and MGIT960, and the minimum inhibitory concentration (MIC) of RIF was determined by the MYCOTB MIC plate test. Additionally, molecular docking and molecular dynamics (MD) simulations were performed to evaluate the binding efficacy of rpoB and RIF based on rpoB mutations detected in the isolates with discordant RIF susceptibility results. A total of 28 isolates with discordant RIF susceptibility test results were detected, 15 of them were RIF susceptible with MICs ≤ 0.5 µg/mL. Twelve out of 15 isolates contained borderline RIF resistance-associated mutations [L430P (n = 6), H445N (n = 6)], 1 isolate had D435Y and Q429H double mutation, and the remaining 2 isolates had a silent (Q432Q) mutation. Compared with the affinity of RIF toward the wild type (WT) (-45.83 kcal/mol) by MD, its affinity toward L452P (-55.52 kcal/mol), D435Y (-47.39 kcal/mol), L430P (approximately -69.72 kcal/mol), H445N (-49.53 kcal/mol), and Q429H (-55.67 kcal/mol) increased. Borderline RIF resistance-associated mutations were the main cause for the discordant RIF susceptibility results between Xpert and MGIT960, and the mechanisms of the resistance need further investigated.IMPORTANCEThis study is aimed at assessing discordant results between Xpert MTB/RIF (Xpert) assay and Bactec MGIT 960 Culture System (MGIT960) regarding the detection of rifampicin (RIF)-resistant Mycobacterium tuberculosis isolates in Wenzhou, China. The discordant results of RIF between these two assays were mainly caused by borderline RIF resistance-associated mutations, subsequently by silent mutations of rpoB. Borderline RIF resistance- associated mutations detected in our study were demonstrated to not be affected by the affinity of rpoB and RIF by molecular dynamics, and the mechanism of resistance was needed to be clarified. For the discordant results of RIF by Xpert and MGIT960 that occurred, rpoB DNA sequencing was recommended to investigate its association with resistance to RIF.

PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64M. tuberculosis and 26M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

Distribution of lineages and type II toxin-antitoxin systems among rifampin-resistant Mycobacterium Tuberculosis Isolates.

Type II toxin-antitoxin systems such as mazEF3, vapBC3, and relJK play a role in antibiotic resistance and tolerance. Among the different known TA systems, mazEF3, vapBC3, and relJK, which are type II systems, have specific roles in drug resistance. Therefore, the aim of this study was to investigate the mutations in these genes in sensitive and resistant isolates of Mycobacterium tuberculosis. Thirty-two rifampin-resistant and 121 rifampin-sensitive M. tuberculosis isolates were collected from various regions of Iran. Lineage typing was performed using the ASO-PCR method. Mutations in the rpoB gene were analyzed in all isolates by MAS-PCR. Furthermore, mutations in the mazEF3, relJK, and vapBC3 genes of the type II toxin system were assessed through PCR sequencing. These sequences were analyzed using COBALT and SnapGene 2017, and submitted to the GenBank database. Among the 153 M. tuberculosis samples, lineages 4, 3 and 2 were the most common. Lineage 2 had the highest rate of rifampin resistance. Mutations in rpoB531 were the most frequent in resistant isolates. Examination of the toxin-antitoxin system showed that rifampin-resistant isolates belonging to lineage 3 had mutations in either the toxin or antitoxin parts of all three TA systems. A mutation in nucleotide 195 (codon 65) of mazF3 leading to an amino acid change from threonine to isoleucine was detected in all rifampin-resistant isolates. M. tuberculosis isolates belonging to lineage 2 exhibited the highest rifampin resistance in our study. Identifying the mutation in mazF3 in all rifampin-resistant isolates can highlight the significance of this mutation in the development of drug resistance in M. tuberculosis. Expanding the sample size in future studies can help develop a new method for identifying resistant isolates.

RpoB Mutations are Associated with Variable Levels of Rifampin and Rifabutin Resistance in Mycobacterium tuberculosis.

To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. 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. 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.

Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

Traditionally, single critical concentrations of drugs are utilized for Mycobacterium tuberculosis (Mtb) drug susceptibility testing (DST); however, the level of drug resistance can impact treatment choices and outcomes. Mutations at the katG gene are the major genetic mutations in multidrug resistant (MDR) Mtb and usually associated with high level resistance. We assessed the minimum inhibitory concentrations (MICs) of MDR or rifampin resistant (RR) and isoniazid (INH) resistant Mtb isolates to determine the quantification of drug resistance among key anti-tuberculosis drugs. The study was conducted on stored Mtb isolates collected as part of a national drug resistance survey in Ethiopia. MIC values were determined using Sensititre™ MYCOTB plates. A line probe assay (MTBDRplus) was also performed to identify genetic determinants of resistance for all isolates. MIC testing was performed on 74 Mtb isolates including 46 MDR, 2 RR and 26 INH phenotypically resistant isolates as determined by the Löwenstein Jensen (LJ) method. Four (15%) INH resistant Mtb isolates were detected as borderline rifampin resistance (MIC = 1 μg/ml) using MYCOTB MIC plates and no rifampin resistance mutations were detected by LPA. Among the 48 MDR/RR TB cases, 9 (19%) were rifabutin susceptible (MIC was between ≤0.25 and 0.5μg/ml). Additionally, the MIC for isoniazid was between 2-4 μg/ml (moderate resistance) for 58% of MDR TB isolates and 95.6% (n = 25) of the isolates had mutations at the katG gene. Our findings suggest a role for rifabutin treatment in a subset of RR TB patients, thus potentially preserving an important drug class. The high proportion of moderate level INH resistant among MDR Mtb isolates indicates the potential benefit of high dose isoniazid treatment in a high proportion of katG gene harboring MDR Mtb isolates.

Highly Synergistic Effects of Melittin With Vancomycin and Rifampin Against Vancomycin and Rifampin Resistant Staphylococcus epidermidis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) strains are increasingly emerging as serious pathogens because they can be resistant to many antibiotics called multidrug resistance (MDR) that limit the therapeutic options. In the case of vancomycin- and rifampin-resistant MDR-MRSE, the physicians are not allowed to increase the doses of antibiotics because of severe toxicity. Accordingly, we investigated the synergistic activity of melittin antimicrobial peptide with vancomycin and rifampin against vancomycin-resistant, and rifampin-resistant MDR-MRSE isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICi), and fractional bactericidal concentration index (FBCi) of antimicrobial agents against isolates were determined. Coagulate activities and serum and salt stability as well as melittin cytotoxicity on the human embryonic kidney (HEK) 293 cells and human red blood cells (RBCs) at their synergistic concentrations. MIC and MBC values for melittin were in the range of 0.312–2.5 and 0.312–5, respectively. Results also showed that the interaction of melittin with drugs was highly synergistic in which the geometric means of FICi and FBCi were < 0.5. Induced synergism led to a decrease in melittin, rifampin, and vancomycin concentrations by 8–1,020, 2–16, and 4–16-folds, respectively. This phenomenon caused a reduction in melittin toxicity by which the synergistic concentration of melittin needed to kill bacteria did not show cytotoxicity and hemolytic activity. Besides, no coagulation activity was found for the synergistic and alone concentrations of melittin in both Prothrombin Time (PT) and Partial Thromboplastin Time (PTT). Interestingly, the antibacterial activity of melittin in Mueller Hinton Broth (MHB) containing human serum did no significant differences between MIC and MBC values of melittin in MHB and MHB containing 10% human serum. The present findings showed that the therapeutic index of melittin was improved by 32.08- and 12.82-folds when combined with vancomycin and rifampin, respectively. Taken together, the obtained data show that melittin alone was effective against MDR-MRSE isolates and this antimicrobial peptide showed highly synergistic effects with vancomycin and rifampin without causing toxicity. Therefore, the combination of melittin and traditional antibiotics could be a promising strategy for the treatment of infections caused by MDR-MRSE.

Identification of mutations in rpoB, pncA, embB, and ubiA genes among drug-resistant Mycobacterium tuberculosis clinical isolates from Iran.

Mycobacterium tuberculosis resistant to effective first-line drugs (FLDs) has challenged national and global tuberculosis control programs. This study aimed to identify mutations in 4 genes related to rifampin, pyrazinamide, and ethambutol resistance among clinical isolates of M. tuberculosis from southwestern Iran. After drug susceptibility testing of 6620 M. tuberculosis clinical isolates by proportional method, a total of 24 FLD-resistant strains were included in the study. Fragments of rpoB, pncA, embB, and ubiA genes were amplified and sequenced to mine the mutations by pairwise alignment with the corresponding M. tuberculosis H37Rv genes. Phenotypic resistance to rifampin, isoniazid, and ethambutol was detected in 67, 54, and 33% (n = 16, 13, and 8) of the isolates, respectively. Of rifampin-resistant isolates, 31% (5/16) were mono-resistant, and 56% (9/16) were multidrug-resistant (MDR). In 100% of rifampin-resistant isolates, mutations were found in the rifampin resistance-determining region (RRDR) of the rpoB, with S450L substitution being the most common, especially in MDRs (77.8%, 7/9). Resistance-conferring mutations in pncA were present in 12.5% (3/24) of FLD-resistant isolates. The embB and ubiA mutations were found in 62.5 and 12.5% (5/8 and 1/8) of ethambutol-resistant isolates, respectively, of which the embB D354A was the most common substitution (37.5%, 3/8). Sixteen distinct mutations were identified, one of which was novel. The sequence analysis of the RRDR segment was the best way to detect rifampin resistance. The rpoB S450L substitution could be a helpful molecular marker to predict MDR. In other genes, no mutation was identified as a reliable marker.

Drug resistance profiles and related gene mutations in slow-growing non-tuberculous mycobacteria isolated in regional tuberculosis reference laboratories of Iran: a three year cross-sectional study

ABSTRACT There are limited studies on the antibiotic resistance patterns of slowly growing mycobacteria (SGM) species and their related gene mutations in Iran. This study aimed to elucidate the antibiotic susceptibility profiles and the mutations in some genes that are associated with the antibiotic resistance among SGM isolates from Iran. The SGM strains were isolated from sputum samples of suspected tuberculosis (TB) patients. SGM species were identified by standard phenotypic tests and were assigned to species level by amplification and sequencing of the dnaK gene. The minimum inhibitory concentration (MIC) of eight antibiotics was determined using broth microdilution method. The mutations in rrl, rpoB, gyrA, and gyrB genes were investigated in clarithromycin, rifampin, and moxifloxacin resistant isolates using sequencing method. A total of 77 SGM isolates including 46 (59.7%) Mycobacterium kansasii, 21 (27.3%) Mycbacterium simiae, and 10 (13%) Mycobacterium avium complex (MAC) were detected. The amikacin and linezolid with the susceptibility rates of 97.4% and 1.3% were the most and the least effective antibiotics, respectively. All MAC and M. simiae isolates, and 32 (69.6%) M. kansasii strains had multiple-drug resistance (MDR) profiles. The rrl, rpoB, gyrA, and gyrB genes showed various mutations in resistant isolates. Although the current study showed an association among resistance to the clarithromycin, rifampin, and moxifloxacin with mutations in the relevant genes, further research using the whole-genome sequencing is needed to provide a clearer insight into the molecular origins of drug resistance in SGM isolates.

Gene mutations in resistant strains of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in Mazandaran and Golestan provinces of Iran - A cross-sectional study

Due to the significant prevalence of tuberculosis (TB) in Mazandaran and Golestan provinces in northern Iran, we aimed to investigate the gene mutations in resistant Mycobacterium tuberculosis isolated from pulmonary TB patients in these provinces of Iran. In this cross-sectional study, sputum of TB suspected patients were collected and cultured in a period of 3 months. The antibiotic-resistant cases identified using the resistance ratio method. Then, the DNAs of the resistant isolates were extracted manually by the CTAB-Proteinase K method and investigated for gene mutations using PCR and sequencing. Among 26 isolated M. tuberculosis, 4 isolates (15.38%) were resistant to antibiotics, while one (3.84%), 2 (7.69%), and one (3.84%) isolates were resistant to rifampin, isoniazid, and multidrug-resistant (MDR), respectively. The MDR isolate had a mutation in codon 315 of the KatG gene resulting in the conversion of serine to threonine, and a mutation in the codon 450 of the rpoB gene, causing the alteration of serine to leucine. In the rifampin-resistant isolate, a mutation occurred at the same locus converting serine to leucine. The low prevalence of antibiotic resistance in this study indicates the appropriate treatment of the patients, however, the single MDR isolate was identified in Mazandaran province.

Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis.

ABSTRACTPyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampin-resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods and explored the occurrence of pyrazinamide heteroresistance. We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole-genome sequencing (WGS), and phenotypic drug susceptibility testing. We calculated the diagnostic accuracy of the different methods and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Köser classification and expert rules. We observed 100% agreement across genotypic methods for detection of pncA fixed mutations; only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% confidence interval [CI], 93.8 to 99.3%) and specificity of 100% (95% CI, 100 to 100%), both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI, 94.6 to 99.6%) and specificity of 97.8% (95% CI, 95.7 to 99.9%), and TDS had sensitivity of 98.8% (95% CI, 97.2 to 100%) and specificity of 97.8% (95% CI, 95.7 to 99.9%). We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mycobacterium tuberculosis isolates was lower than that identified for other first-line drugs.

Genomic Profiling of Mycobacterium tuberculosis Strains, Myanmar.

Multidrug resistance is a major threat to global elimination of tuberculosis (TB). We performed phenotypic drug-susceptibility testing and whole-genome sequencing for 309 isolates from 342 consecutive patients who were given a diagnosis of TB in Yangon, Myanmar, during July 2016‒June 2018. We identified isolates by using the GeneXpert platform to evaluate drug-resistance profiles. A total of 191 (62%) of 309 isolates had rifampin resistance; 168 (88%) of these rifampin-resistant isolates were not genomically related, indicating the repeated emergence of resistance in the population, rather than extensive local transmission. We did not detect resistance mutations to new oral drugs, including bedaquiline and pretomanid. The current GeneXpert MTB/RIF system needs to be modified by using the newly launched Xpert MTB/XDR cartridge or line-probe assay. Introducing new oral drugs to replace those currently used in treatment regimens for multidrug-resistant TB will also be useful for treating TB in Myanmar.

Detection of genomic mutations in katG and rpoB genes among multidrug-resistant Mycobacterium tuberculosis isolates from Tehran, Iran

Multidrug-resistant (MDR) Mycobacterium tuberculosis strains, defined as resistant to at least isoniazid and rifampin, have emerged as a major worldwide health threat. Spontaneous point mutations in various genes of M. tuberculosis cause resistance to isoniazid, with the most frequent gene target being katG; and resistance to rifampin is usually due to mutation in the rpoB gene. The current study was aimed to detect the point mutations in the katG and rpoB regions related to isoniazid and rifampin resistance. A total of 203 respiratory specimens were collected from patients suspected of having tuberculosis respiratory infections referred to hospitals of Tehran, Iran, during 2018–2019. The isolation and identification of M. tuberculosis isolates were performed according to the WHO protocol. Drug susceptibility testing was carried out by proportional method. PCR analysis and sequencing were used to detect mutations in the selected katG and rpoB regions. Forty-four M. tuberculosis strains were isolated, of which 12 (27.3%) and 10 (22.7%) were resistant to isoniazid and rifampin, respectively. Ten isolates were resistant to both isoniazid and rifampin and were considered as MDR isolates. Of the ten MDR isolates, six (60%) carried mutations in both rpoB and katG. The most common mutations among isoniazid- and rifampin-resistant isolates were in codon 315 of the katG gene (70%) and codon 441 of the rpoB gene (50%), respectively. The results of this study indicated that MDR-TB continues to be a serious public health problem in Iran.

Drug-resistant TB prevalence study in 5 health institutions in Haiti.

Tuberculosis (TB) is the leading infectious cause of death in the world. Multi-drug resistant TB (MDR-TB) is a major public health problem as treatment is long, costly, and associated to poor outcomes. Here, we report epidemiological data on the prevalence of drug-resistant TB in Haiti. This cross-sectional prevalence study was conducted in five health centers across Haiti. Adult, microbiologically confirmed pulmonary TB patients were included. Molecular genotyping (rpoB gene sequencing and spoligotyping) and phenotypic drug susceptibility testing were used to characterize rifampin-resistant MTB isolates detected by Xpert MTB/RIF. Between April 2016 and February 2018, 2,777 patients were diagnosed with pulmonary TB by Xpert MTB/RIF screening and positive MTB cultures. A total of 74 (2.7%) patients were infected by a drug-resistant (DR-TB) M. tuberculosis strain. Overall HIV prevalence was 14.1%. Patients with HIV infection were at a significantly higher risk for infection with DR-TB strains compared to pan-susceptible strains (28.4% vs. 13.7%, adjusted odds ratio 2.6, 95% confidence interval 1.5-4.4, P = 0.001). Among the detected DR-TB strains, T1 (29.3%), LAM9 (13.3%), and H3 (10.7%) were the most frequent clades. In comparison with previous spoligotypes studies with data collected in 2000-2002 and in 2008-2009 on both sensitive and resistant strains of TB in Haiti, we observed a significant increase in the prevalence of the drug-resistant MTB Spoligo-International-Types (SIT) 137 (X2 clade: 8.1% vs. 0.3% in 2000-02 and 0.9% in 2008-09, p<0.001), 5 (T1 clade: 6.8% vs 1.9 in 2000-02 and 1.7% in 2008-09, P = 0.034) and 455 (T1 clade: 5.4% vs 1.6% and 1.1%, P = 0.029). Newly detected spoligotypes (SIT 6, 7, 373, 909 and 1624) were also recorded. This study describes the genotypic and phenotypic characteristics of DR-TB strains circulating in Haiti from April 2016 to February 2018. Newly detected MTB clades harboring multi-drug resistance patterns among the Haitian population as well as the higher risk of MDR-TB infection in HIV-positive people highlights the epidemiological relevance of these surveillance data. The importance of detecting RIF-resistant patients, as proxy for MDR-TB in peripheral sites via molecular techniques, is particularly important to provide adequate patient case management, prevent the transmission of resistant strains in the community and to contribute to the surveillance of resistant strains.

RpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China.

ObjectiveThe aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China.MethodsWe prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing.ResultsA total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR.ConclusionsThe frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

Rifampin resistance among individuals with extrapulmonary tuberculosis: 4 years of experience from a reference laboratory

Information is limited about the drug resistance patterns in extrapulmonary tuberculosis (EPTB) in Iran. This study aimed to determine the prevalence of EPTB and to investigate the drug-resistance pattern in Mycobacterium tuberculosis strains collected from extrapulmonary samples at the Tehran regional TB reference laboratory. Extrapulmonary specimens from individuals with suspected TB referred to the TB reference laboratories in five cities of Iran were collected. Both standard conventional methods (culture and direct smear microscopy) and Xpert MTB/RIF assay were used for the identification of mycobacteria. Drug susceptibility testing was done using Xpert MTB/RIF. The proportion method on Lowenstein–Jensen medium was performed for confirmation. Between 2016 and 2020, a total of 12 050 clinical specimens from individuals with suspected TB were collected, of which 10 380 (86%) were pulmonary specimens and 1670 (14%) were extrapulmonary. Of the extrapulmonary specimens, 85 (5.0%) were positive for M. tuberculosis, and the remaining 1585 (95.0%) samples were negative by standard methods. Of 85 M. tuberculosis isolates, drug susceptibility testing was performed for 32 isolates, of which 1 (3.1%, 95% CI 0.0%–9.4%) was rifampin resistant and 31 (96.9%, 95% CI 90.1%–100%) were pan-susceptible. The rifampin-resistant isolate was also resistant to isoniazid, so was assigned as a multidrug-resistant TB. Our study indicated the frequency of drug-resistance among EPTB in Iran. Establishing rapid diagnostic methods for detection of drug-resistance in EPTB, performing drug susceptibility testing for all EPTB cases to provide effective treatment, and continuous monitoring of drug resistance, are suggested for prevention and control of drug resistance in EPTB in Iran.

810. Resistance to the Minocycline-Rifampin-Chlorhexidine (MRCH) combination does not emerge in biofilms of Tetracycline and Rifampin resistant bacteria grown on MRCH catheters depleted below antimicrobially effective concentrations

BackgroundCentral Line Associated Bloodstream Infections (CLABSIs) remain a significant medical problem for critically ill cancer patients who required catheters for extended durations. Minocycline (M) -Rifampin (R) loaded catheters have shown the greatest impact on reducing CLABSIs; however, there is a risk for developing antibiotic resistant organisms when exposed to catheters whose concentration becomes depleted below antimicrobially effective levels due to extended indwells. Chlorhexidine (CH) and M-R combination catheters (MRCH) have been proposed as a next generation catheter with improved performance. Here we studied whether bacteria that were Tetracycline and Rifampin resistant became resistant to MRCH when allowed to form biofilms on MRCH catheters depleted below antimicrobially effective MRCH concentrations.MethodsMinimum inhibitory concentrations (MICs) of Tetracycline and/or Rifampin resistant stock isolates were measured by standard microbroth dilution methods. MRCH catheters were depleted to below antimicrobially effective concentrations by soaking in serum for 6 weeks. The resistant bacteria were then allowed to form biofilm for 24 hrs on the depleted catheters in broth. Following 24 hour incubation the adherent (breakthrough) bacteria were removed by sonication and MICs were remeasured. The same organisms grown on non-antimicrobial catheters were used as controls.ResultsMICs (ug/mL) of the organisms against each agent and the combination are tabulated below:MICs (ug/mL) of the organisms against each agent and the combination ConclusionThe M and R resistant bacteria did not develop in vitro resistance to the MRCH combination after forming biofilms on MRCH catheters depleted below antimicrobially effective concentrations.Disclosures Joel Rosenblatt, PhD, Cook Medical (Shareholder, Other Financial or Material Support, Inventor of the MRCH catheter technology which is owned by the University of Texas MD Anderson Cancer Center and has been licensed to Cook Medical)Novel Anti-Infective Technologies (Shareholder, Other Financial or Material Support, Inventor of the MRCH catheter technology which is owned by the University of Texas MD Anderson Cancer Center and has been licensed to Cook Medical) Issam I. Raad, MD, Citius (Other Financial or Material Support, Ownership interest)Cook Medical (Grant/Research Support)Inventive Protocol (Other Financial or Material Support, Ownership interest)Novel Anti-Infective Technologies (Shareholder, Other Financial or Material Support, Ownership interest)

Gene mutations related to rifampin resistance of tuberculosis in northwest of Iran

BackgroundMycobacterium tuberculosis causes tuberculosis disease, is a communicable disease and multidrug-resistant or rifampicin-resistant M. tuberculosis continuous to be a public health threat worldwide. Therefore, the aim of this study was the assessment of the mutations profile of rifampin-resistant and MDR M. tuberculosis in the north-west of Iran. Material and methodsThe specimens of suspicious individuals were collected and processed by direct microscopy and culture. Drug susceptibility testing was assessed using the conventional proportional method, and the rpoB genes of rifampin-resistant isolates were amplified and sequence analyses were performed. ResultsA total of 6294 individuals referred, 295 (4.68%) isolates of Mycobacterium spp. were isolated, which only 5 (1.69%) isolates were found resistant to rifampin. Three of the rifampin-resistant isolates were multidrug-resistant, originated from Azerbaijani patients, and two from Iran that were monoresistant to rifampin. Three different types of mutations were identified in the rpoB gene, involving four codons Gln510His, Asp516Val, His526Tyr and Ser531Leu. The most frequent codons affected by point mutation were codons 531 and 526. The katG315 and emb306 mutations were responsible for isoniazid- and ethambutol-resistant isolates. ConclusionThe results of this study revealed that the frequency of the mutations in the RRDR region of the rpoB gene were low, but most of the isolates were collected from patients originated from Rep. of Azerbaijan, which have a high potential for transmission of MDR tuberculosis. These results indicate the importance of policies to restrict infection distribution in the region by patients from neighbor countries. The rate of mutations in the isolates of the present study was lower than the rest of the world.

Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing.

The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.

Characteristics of compensatory mutations in the rpoC gene and their association with compensated transmission of Mycobacterium tuberculosis.

The aim of this study was to characterize rpoC gene mutations in Mycobacterium tuberculosis (MTB) and investigate the factors associated with rpoC mutations and the relation between rpoC mutations and tuberculosis (TB) transmission. A total of 245 MTB clinical isolates from patients with TB in six provinces and two municipalities in China were characterized based on gene mutations through DNA sequencing of rpoC and rpoB genes, phenotyping via standard drug susceptibility testing, and genotypic profiling by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. Approximately 36.4% of the rifampin-resistant isolates harbored nonsynonymous mutations in the rpoC gene. Twenty-nine nonsynonymous single mutations and three double mutations were identified. The rpoC mutations at locus 483 (11.3%) were predominant, and the mutations at V483G, W484G, I491V, L516P, L566R, N698K, and A788E accounted for 54.5% of the total detected mutations. Fifteen new mutations in the rpoC gene were identified. Rifampin resistance and rpoB mutations at locus 531 were significantly associated with rpoC mutations. MIRU-VNTR genotype results indicated that 18.4% of the studied isolates were clustered, and the rpoC mutations were not significantly associated with MIRU-VNTR clusters. A large proportion of rpoC mutation was observed in the rifampicin-resistant MTB isolates. However, the findings of this study do not support the association of rpoC mutation with compensated transmissibility.

In Vitro Activity of Rifampin, Rifabutin, Rifapentine, and Rifaximin against Planktonic and Biofilm States of Staphylococci Isolated from Periprosthetic Joint Infection.

The in vitro activities of rifampin, rifabutin, rifapentine, and rifaximin were tested against 200 periprosthetic joint infection (PJI)-associated staphylococci. Seven rifampin-resistant isolates had MICs of ≥4 μg/ml. Three isolates had rifampin MICs of 0.25 to 1 μg/ml and harbored an Asp471Gly RpoB variant, suggesting that the CLSI rifampin-susceptible staphylococcal breakpoint of ≤1 μg/ml may be too high. The remaining isolates had rifampin MICs of ≤0.016 μg/ml, and the rifampin, rifabutin, rifapentine, and rifaximin minimum biofilm bactericidal concentrations (MBBC) for ≥50% of isolates were 8, 1, 2, and 4 μg/ml (for S. aureus) and 2, 0.06, 0.25, and 0.5 μg/ml (for S. epidermidis), respectively, for rifampin-susceptible isolates. Nonrifampin rifamycins have promising staphylococcal activity, including antibiofilm activity.