CRISPR Interference-Mediated Silencing of the mmpL3 Gene in Mycobacterium smegmatis and Its Impact on Antimicrobial Susceptibility.

BACKGROUND: The discovery of novel therapeutic agents, especially those targeting mycobacterial membrane protein large 3 (mmpL3), has shown promise. In this study, the CRISPR interference-Streptococcus thermophilus nuclease-deactivated Cas9 (CRISPRi-dCas9Sth1) system was utilized to suppress mmpL3 expression in Mycobacterium smegmatis, and its impacts on susceptibility to antimicrobial agents were evaluated. METHODS: The repression of the mmpL3 gene was confirmed by RT-qPCR. The essentiality, growth curve, viability, and antimicrobial susceptibility of the mmpL3 knockdown strain were investigated. RESULTS: mmpL3 silencing was achieved by utilizing 0.5 and 1 ng/mL anhydrotetracycline (ATc), resulting in reductions in the expression of 60.4% and 74.4%, respectively. mmpL3 silencing led to a significant decrease in bacterial viability when combined with one-half of the minimal inhibitory concentrations (MICs) of rifampicin, rifabutin, ceftriaxone, or isoniazid, along with 0.1 or 0.5 ng/mL ATc (p < 0.05). However, no significant difference was observed for clarithromycin or amikacin. CONCLUSIONS: The downregulation of the mmpL3 gene in mycobacteria was achieved through the use of CRISPRi-dCas9Sth1, resulting in growth deficiencies and resensitization to certain antimicrobial agents. The impact was dependent upon the level of gene expression.

Diagnostic performance of the Anyplex MTB/NTM real-time PCR in detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria from pulmonary and extrapulmonary specimens.

Accurate and rapid diagnosis of mycobacterial infections is significant for appropriate treatment. In this study, we retrospectively evaluated the performance of the Anyplex MTB/NTM real-time detection assay (Anyplex MTB/NTM) compared to mycobacterial culture in detecting Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) in 9,575 clinical specimens. For MTBC detection, the sensitivity, specificity, PPV, NPV, and percent agreement of the Anyplex MTB/NTM were 79.7%, 94.5%, 64.4%, 97.4%, and 92.9%, respectively. In pediatric patient (age ≤15) specimens, the Anyplex MTB/NTM demonstrated 84.8% sensitivity and 95.8% specificity. For NTM detection, the sensitivity, specificity, PPV, NPV, and percent agreement were 44.9%, 97.7%, 36.7%, 98.4%, and 96.2%, respectively. The sensitivity of the Anyplex MTB/NTM was enhanced in acid-fast bacilli (AFB) smear-positive specimens which was 97.7% and 80% for MTBC and NTM detection, respectively. The Anyplex MTB/NTM is a rapid tool for detection and differentiation of MTBC and NTM in clinical specimens.

Lactobacillus rhamnosus attenuates Thai chili extracts induced gut inflammation and dysbiosis despite capsaicin bactericidal effect against the probiotics, a possible toxicity of high dose capsaicin.

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-ß-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02-2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

Evaluation of Anyplex™ II MTB/MDR kit's performance to rapidly detect isoniazid and rifampicin resistant Mycobacterium tuberculosis from various clinical specimens.

To determine the accuracy of multiplex real-time PCR (Anyplex™ II MTB/MDR kit) in detecting Isoniazid (INH)- and Rifampin (RIF)-resistant Mycobacterium tuberculosis strains from various clinical specimens. The performance of Anyplex™ II MTB/MDR kit in detecting INH- and RIF-resistant M. tuberculosis compared to the conventional drug susceptibility tests by Mycobacterial Growth Indicator Tube (MGIT). A total of 430 clinical samples had positive results for M. tuberculosis from both Anyplex™ II MTB/MDR kit assay and mycobacterial cultures by MGIT method. When compared to MGITs, the sensitivity and specificity of Anyplex™ II MTB/MDR kit in detecting INH-resistant TB were 85.71% and 99.75%, respectively. For the detection of MDR-TB, the sensitivity and specificity of the test were 82.35% and 99.76%, respectively. The positive predictive values and negative predictive values to detect INH-resistant TB were 96.77% and 98.75%, respectively. Anyplex™ II MTB/MDR kit can be used to rapidly detect isoniazid and rifampicin resistances. It has a high sensitivity, specificity and PPV in detecting INH-resistant TB and MDR-TB. This test can be used as an alternative test to Xpert MTB/RIF because it can rapidly detect both INH-resistant TB and RIF-resistant TB.

PROSPECTIVE EVALUATION OF A NOVEL TWO-STEP PROTOCOL FOR SCREENING OF CLOSTRIDIUM DIFFICILE INFECTION IN HOSPITALIZED ADULT PATIENTS.

Abstract. Clostridium difficile infection (CDI) is one of the most common nosocomial infections in Thailand and worldwide. The clinical spectrum ranges from annoy- ing diarrhea to severe life-threatening disease. Enzyme-linked immunofluorescent assay for cytotoxins A/B (cytotoxins A/B ELFA), which has been widely used in our institute, generally is considered as having low sensitivity for diagnosis of CDI. The study was a prospective evaluation of a novel two-step diagnostic algorithm, in which the first step involved concurrent cytotoxins A/B ELFA and enzyme immunoassay for glutamate dehydrogenase (GDH EIA) for CDI, followed by PCR assay of tcdA and tcdB in samples with discordant results. Of the 91 adult patients (37 males and 54 females, mean age of 60.0 ± 19.5 years) with suspected CDI hospitalized at King Chulalongkorn Memorial Hospital, Bangkok, Thailand from December 2012 to February 2013, 22 were diagnosed with CDI by the gold standard PCR test for tcdA and tcdB, among whom 21 were positive by GDH EIA, accounting for a sensitivity of 95%. Of the 69 patients without CDI, GDH EIA was negative in 46 patients, accounting for a specificity of 67%. The positive predic- tive value (PPV), negative predictive value (NPV) and accuracy of GDH EIA was 48%, 98% and 74%, respectively, whereas sensitivity, specificity, PPV, NPV, and accuracy of cytotoxins A/B ELFA was 73%, 96%, 84%, 92% and 92%, respectively. Some 30% of specimens required the more expensive PCR assay. However, this two-step protocol detected 20% more patients with CDI than the currently used cytotoxins A/B ELFA method.