Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

The NeutraClear® Needleless Connector is Equally Effective against Catheter Colonization Compared to MicroClave®

Introduction Neutral-valve closed-system connectors can reduce the frequency of catheter colonization. Commercially available closed system connectors need to be tested and compared with each other to assess how they protect against contamination. We aimed to compare, in vitro, the efficacy of connectors NeutraClear® and MicroClave® against contamination under conditions of daily clinical practice. Methods The model consisted of a set of 200 blood culture bottles (BCBs) with a cannula inserted (100 closed with NeutraClear® and 100 closed with MicroClave®) that were assessed in two experiments while instilling 1 mL of saline: manipulation based on the standard of care and manipulation using gloves impregnated with a 0.05 McFarland Staphylococcus aureus solution. The BCBs were incubated in a BACTEC System at 37°C under continuous shaking for up to 7 days. When a bottle turned positive, 100 µL of the fluid was cultured. The positivity rate and time to positivity of the BCB in each experiment was compared. Results In the aseptic model in the NeutraClear® and MicroClave® groups, only 1 BCB and 2 BCBs were positive, respectively, (p = 0.55). In the contaminated model, all BCBs were positive in both groups at the end of the incubation time. We did not find differences for the MTP between NeutraClear® and MicroClave® (36.04 vs. 20.13 hours, p = 0.09). Conclusions The NeutraClear® needleless connector proved to be as efficient as the MicroClave® connector in the prevention of catheter colonization and migration of S. aureus from the surface to the inside of the hub in an in vitro model.

Sonication of Explanted Prosthesis Combined with Incubation in BD Bactec Bottles for Pathogen-Based Diagnosis of Prosthetic Joint Infection

Prosthetic joint infection (PJI) is a rare but refractory complication of arthroplasty. Accurate identification of pathogens is a key step for successful treatment of PJI, which remains a challenge for clinicians and laboratory workers. We designed a combined culture method with sonication of implants and incubation in a BD Bactec system to improve the effectiveness of pathogen diagnosis in PJI. The aims of this study were to investigate the diagnostic accuracy of sonicate fluid cultures in the BD Bactec system and to compare the results with those of synovial fluid cultures in the BD Bactec system. The prosthetic components removed were sonicated in Ringer's solution, and then sonicate fluid was incubated in Bactec bottles for 5 days. Synovial fluid was incubated in Bactec bottles for 5 days as a control. Synovial fluid cultures with Bactec bottles and sonicate fluid cultures with Bactec bottles showed sensitivities of 64% and 88%, respectively (P= 0.009), with specificities of 98% and 87% (P= 0.032), respectively. Sonicate fluid cultures with Bactec bottles were more sensitive than synovial fluid cultures with Bactec bottles regardless of whether antimicrobial agents were used within 14 days before surgery (81% versus 52%;P= 0.031) or not (93% versus 72%;P= 0.031). Sonication of explanted prostheses followed by incubation of the resulting sonicate fluid in Bactec bottles detected many more pathogens than did synovial fluid cultures with Bactec bottles. This method is also effective in cases with antibiotic treatment before surgery.

Comparison of recoveries of Mycobacterium tuberculosis using the automated BACTEC MGIT 960 System and Lowenstein – Jensen medium

We examined whether the BACTEC/ Mycobacteria Growth Indicator Tube (MGIT) System alone could supplant the use of a supplemental Lowenstein–Jensen (LJ) slant for routine recovery of M. tuberculosis from clinical specimens. A total of 392 specimens of sputum were included in the study, collected from 196 patients. Specimens were processed with standard N-acetyl- L- Cysteine (NALC-NaOH) method, then inoculated onto BACTEC MGIT 960 and onto LJ media. The recovery rates of M.tuberculosis were 100 % (256/256) with BACTEC MGIT 960 and 72.6%(186/256) with LJ. The rates of contamination for each of the system were 4.8%with BACTEC MGIT 960 and 5.3%with LJ. The TTD for M. tuberculosis was 11.3 days with BACTEC System and 30.8 days with LJ. The difference in TTD between smear positive and smear negative specimens for M.tuberculosis with BACTEC MGIT 960 was not statistically significant. This study shows that the BACTEC System demonstrates better sensitivity for the recovery of M. tuberculosis from clinical specimens.

Isolation of pathogenic aerobic bacteria from the blood of septicaemic neonatal calves and the susceptibility of isolates to various antibiotics

An automated blood culture system (BACTEC 9240) was used for the isolation of aerobic bacteria from the blood of septicaemic neonatal calves. Blood samples were collected from 90 clinically septicaemic and 20 healthy neonatal calves and inoculated into blood culture bottles. There were 89 significant isolates from 90 positive blood cultures using the BACTEC system. Escherichia coli was the most common pathogen detected accounting for 56 (63 %) out of 89 isolates. The other pathogens were β-haemolytic streptococci (15.7 %), Staphylococcusaureus (10.1 %), Klebsiella sp. (5.6 %) and Corynebacterium sp. (5.6 %). All isolates showed a susceptibility rate of 100 % to enrofloxacin, cefepim, cefoperazone / sulbactam, imipenem and meropenem while some of them were ranged from 75 to 91.7 % susceptible to amoxicillin / clavulanic acid, ampicillin / sulbactam, gentamicin and cephalosporins.

Mefloquine Is Active In Vitro and In Vivo againstMycobacterium avium Complex

ABSTRACT Despite the development of several agents, new classes of antimicrobials with activity against the Mycobacterium avium complex (MAC) are needed. Based on a broad screening of compounds, we found that mefloquine has MICs of 8 to 16 μg/ml by the BACTEC system and 16 μg/ml by broth microdilution for five MAC strains tested. An expansion of the screening with broth microdilution to 24 macrolide-susceptible strains and 6 macrolide-resistant strains determined that the MIC for all strains was 16 μg/ml. To determine the intracellular activity of mefloquine, U937 macrophage monolayers infected with MAC strain 101, 100, or 109 (serovars 1, 8, and 4) were treated with mefloquine daily, and the number of intracellular bacteria was quantitated after 4 days. Significant growth inhibition against the three MAC strains at concentrations greater than or equal to 10 μg/ml (P < 0.05) was obtained. Due to the encouraging anti-MAC activity, in vivo efficacy in beige mice infected with MAC 101 was evaluated. Animals were treated with 5, 10, 20, or 40 mg/kg of body weight daily, three times a week, twice a week, or once a week for 4 weeks, and bacteria were quantitated in blood, liver, and spleen. No toxicity was observed with any of the treatment regimens. Mefloquine had borderline bactericidal activity at a dosage of 40 mg/kg daily (100% inhibition compared with a 1-week control), and significant inhibition was obtained at dosages of 40 mg/kg three times a week, as well as 20 mg/kg daily. Mefloquine had no significant effect on bacteremia. A combination of mefloquine and ethambutol showed significantly more activity than did either drug alone in liver, spleen, and blood; the combination was also bactericidal againstM. avium. Although safety is a potential concern, mefloquine and related compounds deserve further investigation as anti-MAC therapies.