Clinical Validation of the Xpert MTB/RIF test for Identification of the MTB Complex in AFB Smear-Positive MGIT broth cultures

The identification of the M. tuberculosis complex (MTBC) from smear positive broth cultures can be achieved using several methods including both lab-developed and commercially available molecular assays. In the United States, a commercially available probe-based assay has been used for over a decade by many laboratories for identification of MTBC directly from AFB smear positive broth cultures, including those recovered from the MGIT 960 system. However, recent difficulties in obtaining probe kits for identification resulted in mycobacteriology laboratories looking for alternative platforms to provide for rapid identification of MTBC and detection of rifampicin resistance. The Xpert® MTB/RIF test (Cepheid, Sunnyvale, Ca) has shown high sensitivity for the diagnosis of MTBC from pulmonary specimens but is not often used for identification directly from smear positive, MGIT 960 broth cultures (Becton Dickinson, Sparks, Md). We sought to validate the Xpert MTB/RIF test for use with AFB smear positive MGIT 960 cultures in a clinical hospital setting. Overall, the assay showed a categorical agreement of 100% for identification of MTBC and detection of rifampin resistance. No false positive results or cross-reactivity were noted. Findings indicate that the Xpert MTB/RIF test may be suitable as a rapid replacement for identification of MTBC and detection of rifampicin resistance from AFB smear positive MGIT 960 broth cultures.

Activity of Omadacycline in Rat Methicillin-Resistant Staphylococcus aureus Osteomyelitis

Omadacycline, vancomycin and rifampin as well as rifampin combination therapies were evaluated in an experimental rat model of MRSA osteomyelitis. All treatment groups had less MRSA recovered than saline-treated animals. Emergence of rifampin resistance was observed in 3 of 16 animals with rifampin monotherapy, and none with rifampin combination therapy. After treatment, the median tibial bacterial load was 6.04, 0.1, 4.81 and 5.24 log 10 cfu/g for saline-, rifampin-, vancomycin- and omadacycline-treated animals. Omadacycline or vancomycin administered with rifampin yielded no detectable MRSA. Omadacycline, administered with rifampin, deserves evaluation in humans as a potential treatment for osteomyelitis.

Transglutaminase from UV Mutated Bacillus cereus NRC215: Production, Purification, and Characterization

Transglutaminase (EC 2.3.2.13, TGase) recorded the highest activity (0.101 U/ml) in bacterial isolate NRC215. 16S rRNA sequencing revealed that NRC215 was identified as Bacillus cereus NRC215 under accession number MT229271 in the NCBI database. UV irradiation was employed to improve TGase production. Five rifampin (RIF) resistant mutants were only isolated from UV-treated Bacillus cereus NRC215 for three minutes. The best mutant, BCrif5, exhibiting induced rifampin resistance, gave TGase with higher activity (0.148 U/ml). The ISSR PCR technique was employed to detect these new rearrangements resulting from UV mutagenesis between the wild-type strain and its mutants. Moreover, TGase has been purified by three-step procedures resulting in a recovery of 28 and 34.63% for wild and BCrif5 strains, respectively. The optimal purified TGase activity was exhibited at pH 7 for wild strain while the mutant BCrif5 at pH 5.0 and 40 °C for both wild and BCrif5 strains. Bacillus cereus NRC215 TGase was activated by Ba+2 (102.50 and 107.06%), while it was inhibited by Cu+2 (30% and 22.35%) for wild and BCrif5 strains, respectively. It could be concluded that Bacillus cereus NRC215 is a promising strain for TGase production, which is beneficial as a food additive.

Genomic epidemiology of rifampicin ADP-ribosyltransferase (Arr) in the Bacteria domain

Arr is an ADP-ribosyltransferase enzyme primarily reported in association with rifamycin resistance, which has been used to treat tuberculosis in addition to Gram-positive infections and, recently, pan-resistant Gram-negative bacteria. The arr gene was initially identified on the Mycolicibacterium smegmatis chromosome and later on Proteobacteria plasmids. This scenario raised concerns on the distribution and spread of arr, considering the Bacteria domain. Based on 198,082 bacterial genomes/metagenomes, we performed in silico analysis, including phylogenetic reconstruction of Arr in different genomic contexts. Besides, new arr alleles were evaluated by in vitro analysis to assess their association with rifampin resistance phenotype. The arr gene was prevalent in thousands of chromosomes and in hundreds of plasmids from environmental and clinical bacteria, mainly from the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, this gene was identified in other and new genomic contexts. Interestingly, Arr sequences associated with rifampin resistance were distributed across all phylogeny, indicating that, despite the diversity, their association with rifampin resistance phenotype were maintained. In fact, we found that the key residues were highly conserved. In addition, other analyzes have raised evidence of another Arr function, which is related to guanidine metabolism. Finally, this scenario as a whole also suggested the Actinobacteria phylum as a potential ancestral source of arr within the Bacteria domain.

Delayed Rifampin Administration in the Antibiotic Treatment of Periprosthetic Joint Infections Significantly Reduces the Emergence of Rifampin Resistance

Rifampin is one of the most important biofilm-active antibiotics in the treatment of periprosthetic joint infection (PJI), and antibiotic regimens not involving rifampin were shown to have higher failure rates. Therefore, an emerging rifampin resistance can have a devastating effect on the outcome of PJI. The aim of this study was to compare the incidence of rifampin resistance between two groups of patients with a PJI treated with antibiotic regimens involving either immediate or delayed additional rifampin administration and to evaluate the effect of this resistance on the outcome. In this retrospective analysis of routinely collected data, all patients who presented with an acute/chronic PJI between 2018 and 2020 were recorded in the context of a single-center comparative cohort study. Two groups were formed: Group 1 included 25 patients with a PJI presenting in 2018–2019. These patients received additional rifampin only after pathogen detection in the intraoperative specimens. Group 2 included 37 patients presenting in 2019–2020. These patients were treated directly postoperatively with an empiric antibiotic therapy including rifampin. In all, 62 patients (32 females) with a mean age of 68 years and 322 operations were included. We found a rifampin-resistant organism in 16% of cases. Rifampin resistance increased significantly from 12% in Group 1 to 19% in Group 2 (p < 0.05). The treatment failure rate was 16% in Group 1 and 16.2% in Group 2 (p = 0.83). The most commonly isolated rifampin-resistant pathogen was Staphylococcus epidermidis (86%) (p < 0.05). The present study shows a significant association between the immediate start of rifampin after surgical revision in the treatment of PJI and the emergence of rifampin resistance, however with no significant effect on outcome.

Investigation of Mutations in the Rifampin-Resistance-Determining Region of the rpoB Gene of Brucella melitensis by Gene Analysis

Background: RNA polymerase beta subunit (rpoB) gene analysis in bacterial communities is known as a method for determining rifampin sensitivity and genetic diversity among Brucella spp. Detection of antibiotic resistance among Brucella isolates can be a critical approach to control brucellosis. However, rpoB gene analysis of Brucella melitensis for assessing rifampicin resistance has not yet been performed in Iran, which is considered an endemic area for brucellosis. Objectives: The aim of this study was to analyze the whole sequence of rpoB genes of different B. melitensis isolates from humans to identify the single-nucleotide polymorphisms (SNPs) and mutations related to rifampin resistance and to analyze the genetic diversity of these bacteria in Iran. Methods: Between 2017 and 2019, a total of 156 blood samples along with 12 synovial fluid specimens were collected from brucellosis patients in different Iranian provinces and subjected to bacterial culture in Brucella selective media. Brucella identification was carried out using classical biotyping and molecular examinations. Polymerase chain reaction (PCR)-based amplification of the rpoB gene was performed by specific rpoB primers for whole gene sequencing. The antimicrobial susceptibility of Brucella isolates was assessed using disk diffusion susceptibility tests and minimal inhibitory concentration (MIC) methods. The presence of rifampin-binding sites and SNPs were investigated through rpoB whole gene sequencing. Results: Clinical B. melitensis isolates were obtained from blood (13) and synovial fluid (1) samples of patients from different regions of Iran. The results of MIC and disk diffusion susceptibility tests showed that all the isolates were sensitive to rifampin except for one isolate showing intermediate rifampin resistance based on the standards defined for slow-growing bacteria by the Clinical and Laboratory Standards Institute (CLSI). Gene analysis for identifying the mutations related to rifampin resistance and investigating genetic diversity showed that none of the B. melitensis isolates had missense mutations, confirming the susceptibility of all the studied isolates to rifampin. Conclusions: The present study revealed that rpoB gene analysis could be used for the efficient and precise identifying of the mutations related to rifampin resistance, investigating rifampin binding sites, and genotyping Brucella species. Furthermore, the identification of B. melitensis isolates with intermediate resistance to rifampicin highlighted the importance of periodically carrying out antibiotic susceptibility testing. The molecular detection of rpoB mutations in different Brucella isolates may help to prevent the spread of rifampin-resistant Brucella spp. among humans and livestock.