Molecular Characteristics and Prevalence of Rifampin Resistance in Staphylococcus aureus Isolates from Patients with Bacteremia in South Korea.

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.

Tuberculosis Variant with Rifampin Resistance Undetectable by Xpert MTB/RIF, Botswana.

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.

Xpert MTB/RIF in the Diagnosis of Mediastinal Tuberculous Lymphadenitis by Endoscopic Ultrasound-Guided Needle Aspiration Techniques: A Systematic Review and Meta-Analysis.

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.

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

Objective: 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.

International Spread of Multidrug-Resistant Rhodococcus equi.

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.

Environmental Dependence of Competitive Fitness in Rifampin-Resistant rpoB Mutants of Bacillus subtilis.

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 ß-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. RifrrpoB 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.

Dihydroartemisinin-Loaded Chitosan Nanoparticles Inhibit the Rifampicin-Resistant Mycobacterium tuberculosis by Disrupting the Cell Wall.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a deadly infection, and increasing resistance worsens an already bad scenario. In this work, a new nanomedicine antibacterial agent, based on dihydroartemisinin (DHA) and chitosan (CS), has been successfully developed to overcome MTB's drug-resistant. To enhance DHA's solubility, we have prepared nanoparticles of DHA loaded CS by an ionic crosslinking method with sodium tripolyphosphate (STPP) as the crosslinking agent. The DHA-CS nanoparticles (DHA-CS NPs) have been fully characterized by scanning electron microscopy, Fourier transforms infrared spectroscopy, dynamic light scattering, and ultraviolet spectrophotometry. DHA-CS NPs show an excellent antibacterial effect on the rifampicin (RFP)-resistant strain (ATCC 35838) and, at a concentration of 8.0 µg/ml, the antibacterial impact reaches up to 61.0 ± 2.13% (n = 3). The results of Gram staining, acid-fast staining, auramine "O" staining and electron microscopy show that the cell wall of RFP-resistant strains is destroyed by DHA-CS NPs (n = 3), and it is further verified by gas chromatography-mass spectrometry. Since all the metabolites identified in DHA-CS NPs treated RFP-resistant strains indicate an increase in fatty acid synthesis and cell wall repair, it can be concluded that DHA-CS NPs act by disrupting the cell wall. In addition, the resistance of 12 strains is effectively reduced by 8.0 µg/ml DHA-CS NPs combined with RFP, with an effective rate of 66.0%. The obtained results indicate that DHA-CS NPs combined with RFP may have potential use for TB treatment.

rpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

OBJECTIVE: 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.

Detecting Mycobacterium tuberculosis complex and rifampicin resistance via a new rapid multienzyme isothermal point mutation assay.

Simple, rapid, and accurate detection of the Mycobacterium tuberculosis complex (MTBC) and drug resistance is critical for improving patient care and decreasing the spread of tuberculosis. To this end, we have developed a new simple and rapid molecular method, which combines multienzyme isothermal rapid amplification and a lateral flow strip, to detect MTBC and simultaneously detect rifampin (RIF) resistance. Our findings showed that it has sufficient sensitivity and specificity for discriminating 118 MTBC strains from 51 non-tuberculosis mycobacteria strains and 11 of the most common respiratory tract bacteria. Further, compared to drug susceptibility testing, the assay has a sensitivity, specificity, and accuracy of 54.1%, 100.0%, and 75.2%, respectively, for detection of RIF resistance. Some of the advantages of this assay are that no special instrumentation is required, a constant low temperature of 39 °C is sufficient for the reaction, the turnaround time is less than 20 min from the start of the reaction to read out and the result can be seen with the naked eye and does not require specialized training. These characteristics of the new assay make it particularly useful for detecting MTBC and RIF resistance in resource-limited settings.

Generation of Tetracycline and Rifamycin Resistant Chlamydia Suis Recombinants.

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 ß-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.

Status of rpoB gene mutation associated with rifampicin-resistant Mycobacterium tuberculosis isolated in a rural setting in Nepal.

Mycobacterium tuberculosis ranks among the top 10 causes of deaths in Nepal despite the country having a long history of national tuberculosis prevention programmes that have proved very successful in the control of tuberculosis. Several cases of active or latent tuberculosis are still missing despite that the number of infected individuals is increasing each year. Microscopy has its own limitations and factors like low bacterial load, quality of sample, quality of smear, experience of microscopist etc. influence the overall sensitivity of the test. The implementation of a molecular technique-based rapid, point-of-care testing system offers higher sensitivity in the early diagnosis of tuberculosis. Cepheid GeneXpert is the most commonly used molecular technology in Nepal. It is a cartridge-based semi-quantitative, nested real-time PCR-based diagnostic system. It detects mutations in the beta-subunit of RNA polymerase (rpoB) gene that lead to rifampicin resistance (RR) in M. tuberculosis complex. The present study aims to increase our understanding of the epidemiology of mutations in the rpoB gene in tuberculosis-positive patients by using the Xpert MTB/RIF assay in a rural setting in Pyuthan Hospital, Nepal. Sputum from 2733 patients was tested for the diagnosis of tuberculosis using the Cepheid GeneXpert system between July 2018 and January 2020 at Pyuthan Hospital. Two hundred and ninety-seven of these samples (10.86 %) were positive for M. tuberculosis , of which 3.3 % (10/297) were rifampicin-resistant. Among rifampicin-resistant tuberculosis (RR-TB) patients, 50.0 % (5/10) showed mutations located in codons 529-533 (probe E) of the rpoB gene, followed by others. The GeneXpert system can be a convenient, highly sensitive, rapid and accurate tool for the diagnosis of tuberculosis, also identifying RR-TB and at the same time determining the molecular epidemiology of rifampin resistance-associated mutations in rural and/or resource-limited laboratory settings.

Performance Assessment of Xpert MTB/RIF Assay for Detecting Pulmonary Tuberculosis and Rifampin Resistance in a Tertiary Care Hospital in Korea.

In this study, we aimed to assess the performance of the Xpert MTB/RIF assay for the detection of pulmonary tuberculosis compared to the acid-fast bacilli (AFB) smear and culture analysis, and the incidence of rifampin resistance using the drug susceptibility test. The specimens referred for AFB smear and culture analysis and Xpert MTB/RIF assay from April 2015 to March 2018 were retrospectively reviewed. The sensitivity, specificity, and mean cycle threshold (Ct) values obtained in Xpert MTB/RIF assay and for rifampin resistance were analyzed. The results of Xpert MTB/RIF assay for pulmonary tuberculosis were evaluated based on the AFB smear grade. Among 3,840 specimens, 491 were positive in Xpert MTB/RIF assay and 626 in culture analysis. The sensitivity and specificity of Xpert MTB/RIF assay were 75.6% and 99.4%, respectively. The sensitivity of Xpert MTB/RIF assay for smear-positive/culture-positive specimens was 98.6% and that of smear-negative and -trace/culture-positive specimens was 63.1%. The positivity of Xpert MTB/RIF assay for culture-positive specimens was 89.9%, 98.6%, 95.7%, 100.0%, and 100.0% for the smear grades trace, 1+, 2+, 3+, 4+, respectively. The Ct values of 491 specimens significantly decreased as the AFB smear grade increased (P < 0.0001). The Ct values of smear-positive, -trace, and -negative specimens were 21.7 ± 4.2, 26.5 ± 3.9, and 27.4 ± 3.6, respectively. Rifampin resistance evaluated using Xpert MTB/RIF assay and culture analysis exhibited a correlation of 98.3%. The region covered by probe E was the most frequently mutated region (50.0%). Xpert MTB/RIF assay demonstrated reliable performance in detecting pulmonary tuberculosis from smear-positive and culture-positive specimens; however, further improvements are still required to detect smear-negative and culture-positive specimens.

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

OBJECTIVE: The aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China. METHODS: We 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. RESULTS: A 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. CONCLUSIONS: The 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.

Spread of Multidrug-Resistant Rhodococcus equi, United States.

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.

Probing the Molecular Mechanism of Rifampin Resistance Caused by the Point Mutations S456L and D441V on Mycobacterium tuberculosis RNA Polymerase through Gaussian Accelerated Molecular Dynamics Simulation.

Rifampin is the first-line antituberculosis drug, with Mycobacterium tuberculosis RNA polymerase as the molecular target. Unfortunately, M. tuberculosis strains that are resistant to rifampin have been identified in clinical settings, which limits its therapeutic effects. In clinical isolates, S531L and D516V (in Escherichia coli) are two common mutated codons in the gene rpoB, corresponding to S456L and D441V in M. tuberculosis However, the resistance mechanism at the molecular level is still elusive. In this work, Gaussian accelerated molecular dynamics simulations were performed to uncover the resistance mechanism of rifampin due to S456L and D441V mutations at the atomic level. The binding free energy analysis revealed that the reduction in the ability of two mutants to bind rifampin is mainly due to a decrease in electrostatic interaction, specifically, a decrease in the energy contribution of the R454 residue. R454 acts as an anchor and forms stable hydrogen bond interaction with rifampin, allowing rifampin to be stably incorporated in the center of the binding pocket. However, the disappearance of the hydrogen bond between R454 and the mutated residues increases the flexibility of the side chain of R454. The conformation of R454 changes, and the hydrogen bond interaction between it and rifampin is disrupted. As result, the rifampin molecule moves to the outside of the pocket, and the binding affinity decreases. Overall, these findings can provide useful information for understanding the drug resistance mechanism of rifampin and also can give theoretical guidance for further design of novel inhibitors to overcome the drug resistance.

[Optimizing Mycobacterium Recombineering System (pJV53) to Promote the Screening of the Mycobacterium Mutants].

OBJECTIVE: To establish a way for screening Mycobacterium mutants through adding the screening markers into pJV53. METHODS: The sucrose counter selection gene SacB and mutant hygromycin-resistant gene hygS were inserted into pJV53; The recovery of the hygromycin-resistance indicated the successful homologous recombination in Mycobacterium smegmatis (Ms), which could serve as mutant screening marker; The sucrose counter selection could be used to screen the plasmid-free mutants. RESULTS: The recombinant plasmid pJV53-SacB-hygS were successfully constructed. The rifampin-resistant rpoB D516Y and rpoB H526Q mutants and MSMEG_4487 G188A mutant were efficiently screened out. All mutants had shed the plasmid successfully. CONCLUSION: pJV53-SacB-hygS can efficiently contribute to construct and screen the mutants and to get the mutants shedding the plasmid self, which has high value of extensive application; the D516Y and H526Q mutations in gene rpoB of Mycobacterium tuberculosis contribute to its rifampin-resistance.

Clonal Confinement of a Highly Mobile Resistance Element Driven by Combination Therapy in Rhodococcus equi.

Antibiotic use has been linked to changes in the population structure of human pathogens and the clonal expansion of multidrug-resistant (MDR) strains among healthcare- and community-acquired infections. Here we present a compelling example in a veterinary pathogen, Rhodococcus equi, the causative agent of a severe pulmonary infection affecting foals worldwide. We show that the erm(46) gene responsible for emerging macrolide resistance among equine R. equi isolates in the United States is part of a 6.9-kb transposable element, TnRErm46, actively mobilized by an IS481 family transposase. TnRErm46 is carried on an 87-kb conjugative plasmid, pRErm46, transferable between R. equi strains at frequencies up to 10-3 The erm(46) gene becomes stabilized in R. equi by pRErm46's apparent fitness neutrality and wholesale TnRErm46 transposition onto the host genome. This includes the conjugally exchangeable pVAPA virulence plasmid, enabling the possibility of cotransfer of two essential traits for survival in macrolide-treated foals in a single mating event. Despite its high horizontal transfer potential, phylogenomic analyses show that erm(46) is paradoxically confined to a specific R. equi clone, 2287. R. equi 2287 also carries a unique rpoBS531F mutation conferring high-level resistance to rifampin, systematically administered together with macrolides against rhodococcal pneumonia on equine farms. Our data illustrate that under sustained combination therapy, several independent "founder" genetic events are concurrently required for resistance, limiting not only its emergence but also, crucially, horizontal spread, ultimately determining multiresistance clonality.IMPORTANCE MDR clades arise upon acquisition of resistance traits, but the determinants of their clonal expansion remain largely undefined. Taking advantage of the unique features of Rhodococcus equi infection control in equine farms, involving the same dual antibiotic treatment since the 1980s (a macrolide and rifampin), this study sheds light into the determinants of multiresistance clonality and the importance of combination therapy in limiting the dissemination of mobile resistance elements. Clinically effective therapeutic alternatives against R. equi foal pneumonia are currently lacking, and the identified macrolide-rifampin MDR clone 2287 has serious implications. Still at early stages of evolution and local spread, R. equi 2287 may disseminate globally, posing a significant threat to the equine industry and, also, public health due to the risk of zoonotic transmission. The characterization of the 2287 clone and its resistance determinants will enable targeted surveillance and control interventions to tackle the emergence of MDR R. equi.

Rifampin resistance and its fitness cost in Riemerella anatipestifer.

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.