Drug-susceptible Isolates Research Articles

Genotypic and phenotypic diversity of Mycobacterium tuberculosis complex genotypes prevalent in West Africa.

Findings from previous comparative genomics studies of the Mycobacterium tuberculosis complex (MTBC) suggest genomic variation among the genotypes may have phenotypic implications. We investigated the diversity in the phenotypic profiles of the main prevalent MTBC genotypes in West Africa. Thirty-six whole genome sequenced drug susceptible MTBC isolates belonging to lineages 4, 5 and 6 were included in this study. The isolates were phenotypically characterized for urease activity, tween hydrolysis, Thiophen-2-Carboxylic Acid Hydrazide (TCH) susceptibility, nitric oxide production, and growth rate in both liquid (7H9) and solid media (7H11 and Löwenstein–Jensen (L-J)). Lineage 4 isolates showed the highest growth rate in both liquid (p = 0.0003) and on solid (L-J) media supplemented with glycerol (p<0.001) or pyruvate (p = 0.005). L6 isolates optimally utilized pyruvate compared to glycerol (p<0.001), whereas L5 isolates grew similarly on both media (p = 0.05). Lineage 4 isolates showed the lowest average time to positivity (TTP) (p = 0.01; Average TTP: L4 = 15days, L5 = 16.7days, L6 = 29.7days) and the highest logCFU/mL (p = 0.04; average logCFU/mL L4 = 5.9, L5 = 5.0, L6 = 4.4) on 7H11 supplemented with glycerol, but there was no significant difference in growth on 7H11 supplemented with pyruvate (p = 0.23). The highest release of nitrite was recorded for L5 isolates, followed by L4 and L6 isolates. However, the reverse was observed in the urease activity for the lineages. All isolates tested were resistant to TCH except for one L6 isolate. Comparative genomic analyses revealed several mutations that might explain the diverse phenotypic profiles of these isolates. Our findings showed significant phenotypic diversity among the MTBC lineages used for this study.

Synergism of Zinc Oxide Quantum Dots with Antifungal Drugs: Potential Approach for Combination Therapy against Drug Resistant Candida albicans

Candidiasis caused by Candida albicans is one of the most common microbial infections. Azoles, polyenes, allylamines, and echinocandins are classes of antifungals used for treating Candida infections. Standard drug doses often become ineffective due to the emergence of multidrug resistance (MDR). This leads to the use of higher drug doses for prolonged duration, resulting in severe toxicity (nephrotoxicity and liver damage) in humans. However, combination therapy using very low concentrations of two or more antifungal agents together, can lower such toxicity and limit evolution of drug resistance. Herein, 4–6 nm zinc oxide quantum dots (ZnO QDs) were synthesized and their in vitro antifungal activities were assessed against drug-susceptible (G1, F1, and GU4) and resistant (G5, F5, and GU5) isolates of C. albicans. In broth microdilution assay, ZnO QDs exhibited dose dependent growth inhibition between 0 – 200 µg/ml and almost 90% growth was inhibited in all Candida strains at 200 µg/ml of ZnO QDs. Synergy between ZnO QDs and antifungal drugs at sub-inhibitory concentrations of each was assessed by checkerboard analysis and expressed in terms of the fractional inhibitory concentration (FIC) index. ZnO QDs were used with two different classes of antifungals (azoles and polyenes) against Candida isolates: combination 1 (with fluconazole); combination 2 (with ketoconazole); combination 3 (with amphotericin B), and combination 4 (with nystatin). Results demonstrated that the potency of combinations of ZnO QDs with antifungal drugs even at very low concentrations of each was higher than their individual activities against the fungal isolates. The FIC index was found to be less than 0.5 for all combinations in the checkerboard assay, which confirmed synergism between sub-inhibitory concentrations of ZnO QDs (25 µg/ml) and individual antifungal drugs. Synergism was further confirmed by spot assay where cell viabilities of Candida strains were significantly reduced in all combinations, which was clearly evident from the disappearance of fungal cells on agar plates containing antifungal combinations. For safer clinical use, the in vitro cytotoxic activity of ZnO QDs was assessed against HeLa cell line and it was found that ZnO QDs were non-toxic at 25 µg/ml. Results suggested that the combination of ZnO QDs with drugs potentiate antimicrobial activity through multitargeted action. ZnO QDs could therefore offer a versatile alternative in combination therapy against MDR fungal pathogens, wherein lowering drug concentrations could reduce toxicity and their multitargeted action could limit evolution of fungal drug resistance.

Comparison of toxin-antitoxin expression among drug-susceptible and drug-resistant clinical isolates of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), is a significant global public health threat. Besides extensive multidrug resistance, MTB possesses several properties for long-term viability in the host as well as stress adaptation and resistance in harsh conditions. The role of toxin-antitoxin (TA) systems in disseminating and maintaining antimicrobial resistance in bacterial populations has also been demonstrated. This study aimed to evaluate differences in expression of MazEF (a well-known TA system) related genes (mazE3, mazF3, mazE6, and mazF6) amongst drug-susceptible and resistant MTB isolates in Iran. A total of 20 confirmed clinical isolates of MTB including 10 drug-susceptible and 10 drug-resistant (nine MDR, and one XDR) species were included in this study. M. tuberculosis H37Rv was used as the standard strain. RNA extraction, cDNA synthesis, and relative quantitative real-time PCR were performed according to the standard procedures. Our analysis indicated significant enhanced expression of the mazE6 antitoxin gene in drug-susceptible isolates compared to drug-resistant isolates and the standard strain. The expression of the mazF6 toxin gene was also increased in drug-susceptible isolates compared with the standard strain. In drug-resistant isolates, the expression levels of mazF3 and mazF6 genes were significantly higher than that in the susceptible isolates and the standard strain. In this study, there was significant overexpression of mazE6 in drug-susceptible isolates. As well, mazF3 and F6 were overexpressed in drug-resistant isolates when compared with the standard strain. The changes in expression levels of MazEF6 associated genes were greater than that of MazEF3 in both groups of isolates.

N-Thio-β-lactams targeting L,D-transpeptidase-2, with activity against drug-resistant strains of Mycobacterium tuberculosis

Effective treatment of tuberculosis is frequently hindered by the emerging antimicrobial resistance of Mycobacterium tuberculosis. The present study evaluates monocyclic β-lactam compounds targeting the mycobacterial cell wall remodeling. Novel N-thio-β-lactams were designed, synthesized, and characterized on the L,D-transpeptidase-2, a validated target in M. tuberculosis. The candidates were evaluated in biochemical assays identifying five compounds presenting target-specific kinetic constants equal or superior to meropenem, a carbapenem currently considered for tuberculosis therapy. Mass spectrometry in line with the crystal structures of five target-ligand complexes revealed that the N-thio-β-lactams act via an unconventional mode of adduct formation, transferring the thio-residues from the lactam ring to the active-site cysteine of LdtMt2. The resulting stable adducts lead to a long-term inactivation of the target protein. Finally, the candidates were evaluated invitro against a drug-susceptible and multidrug-resistant clinical isolates of M. tuberculosis, confirming the antimycobacterial effect of these novel compounds.

Proteomic Analyses of Acinetobacter baumannii Clinical Isolates to Identify Drug Resistant Mechanism.

Acinetobacter baumannii is one of the main causes of nosocomial infections. Increasing numbers of multidrug-resistant Acinetobacter baumannii cases have been reported in recent years, but its antibiotic resistance mechanism remains unclear. We studied 9 multidrug-resistant (MDR) and 10 drug-susceptible Acinetobacter baumannii clinical isolates using Label free, TMT labeling approach and glycoproteomics analysis to identify proteins related to drug resistance. Our results showed that 164 proteins exhibited different expressions between MDR and drug-susceptible isolates. These differential proteins can be classified into six groups: a. proteins related to antibiotic resistance, b. membrane proteins, membrane transporters and proteins related to membrane formation, c. Stress response-related proteins, d. proteins related to gene expression and protein translation, e. metabolism-related proteins, f. proteins with unknown function or other functions containing biofilm formation and virulence. In addition, we verified seven proteins at the transcription level in eight clinical isolates by using quantitative RT-PCR. Results showed that four of the selected proteins have positive correlations with the protein level. This study provided an insight into the mechanism of antibiotic resistance of multidrug-resistant Acinetobacter baumannii.

Potency of vancomycin against Mycobacterium tuberculosis in the hollow fiber system model

ObjectivesTo determine whether an inhaled vancomycin formulation resulting in high intrapulmonary 24-h area under the concentration–time curve (AUC0–24) could be optimised for tuberculosis treatment. We also explored vancomycin synergy and antagonism with d-cycloserine and benzylpenicillin. MethodsWe determined MICs of two Mycobacterium tuberculosis (Mtb) laboratory strains (H37Ra and H37Rv) and two drug-susceptible and nine multidrug resistant clinical strains. Second, in the hollow fiber system model of TB [HFS-TB] using Mtb H37Ra strain, we recapitulated vancomycin intrapulmonary pharmacokinetics of eight doses administered twice daily over 28 days, mimicking a 6-h half-life. Using the HFS-TB, vancomycin was tested in combination with d-cycloserine and benzylpenicillin to determine synergy or antagonism between drugs targeting the same pathway. ResultsVancomycin MICs were 12 and 48 mg/L in drug-susceptible clinical isolates but >96 mg/L in all MDR isolates.In the HFS-TB, vancomycin killed 3.9 ± 0.6 log10 CFU/mL Mtb. The EC50 was calculated as AUC0–24/MIC of 184.6 ± 106.5. Compared with day 0, 1.0 and 2.0 log10 CFU/mL kill was achieved by AUC0–24/MIC of 168 and 685, respectively. Acquired vancomycin resistance developed to all vancomycin doses tested in the HFS-TB. In the HFS-TB, vancomycin was antagonistic to benzylpenicillin, which works downstream to glycopeptides in peptidoglycan synthesis, but synergistic with d-cycloserine, which inhibits upstream d-Ala-d-Ala ligase and alanine racemase. ConclusionOur proof-of-concept studies show that vancomycin optimal exposure target for Mtb kill could be achieved via inhalational drug delivery. Addition of drugs synergistic with vancomycin, e.g. d-cycloserine, may lower the vancomycin concentrations required to kill Mtb.

Comparative sequences of the Wolbachia genomes of drug-sensitive and resistant isolates of Dirofilaria immitis

The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40–75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected.Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.

Molecular Mechanism of Multi-Drug Resistance in Mycobacterium tuberculosis

In this study, we demonstrate the clinical applicability of molecular assays for the differential identification of M. tuberculosis isolates by Double Repetitive Element-PCR (DRE-PCR), Duplex PCR (DPCR), Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and IS6110 flanking PCR and for the detection of specific codon mutations in antibiotic-resistant genes, rpoB and katG in 55 MDR-TB and 25 drug-susceptible clinical isolates by Multiplex PCR assays. The MAS-PCR assay was identified as the most prevalent rpoB gene mutations at codon 531 (83.6%), followed by codon 526 (12.7%), and no mutation was found in codon 516. Among the 55 MDR-TB isolates, 49 (89%) isolates had S315T, 5 (9%) had S315N mutations. DRE-PCR and RAPD-PCR generated similar banding of cluster III strains and suggested that MDR-TB strain genotype C may be responsible for the transmission of TB infection among the study population. PCR based differential identification of mtp40 and rpoB DPCR procedures identified two NTM strains among the isolates studied. Genotypic method DRE-PCR was found highly reproducible, followed by RAPD-PCR and mtp40, and rpoB DPCR methods effectively-identified NTM infection in this region. The presence of S315T mutation in katG gene and S531L, H526Y mutations in rpoB gene in MDR-TB isolates proved resistant phenotype. The simplicity of the MAS-PCR assay permits its implementation for the detection of resistance to INH and RIF in clinical laboratories in regions where this mutation is predominant among MDR-TB strains.

In Vitro Activity of a Novel Quinolone, UB-8902, Against Ofloxacin-Resistant Mycobacterium tuberculosis Isolates.

The main objective of this study was to compare in vitro activities of a novel fluoroquinolone (FQ), UB-8902, with ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MOX) against Mycobacterium tuberculosis isolates. Eleven OFX-resistant and 11 drug-susceptible clinical isolates were studied. Individual minimum inhibitory concentrations of OFX, LFX, MOX, and UB-8902 were determined using Middlebrook 7H11 agar. The concentrations studied ranged from 0.125 to 128 μg/mL in twofold dilutions. UB-8902 was more active than LFX and similar to MOX for OFX-resistant M. tuberculosis isolates. In addition, UB-8902 and MOX showed equal activity against drug-susceptible isolates, both being more active than OFX and LFX. In conclusion, the new FQ, UB-8902, showed good activity against OFX-resistant isolates. Moreover, it showed better activity than OFX and LFX and was equivalent to MOX against FQ-susceptible clinical isolates. UB-8902 can be considered as a drug with potential antituberculous activity, similar to MOX.

A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic

BackgroundAtypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations.MethodsWe conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants.ResultsPhylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role.ConclusionThe presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.

Enterotoxigenicity of Multidrug Resistant Staphylococcus aureus Isolated from Dry Catfish Sold in Some Open Markets in Zaria - Nigeria

Aim: The aim of this study was to determine the enterotoxigenic potential of MDR S. aureus isolated from dry catfish sold in some open markets in Zaria, Nigeria.&#x0D; Place and Duration of Study: Department of Pharmaceutical Microbiology, Ahmadu Bello University, Zaria and Central Laboratory, City Campus of the Usman Danfodio University, Sokoto, Nigeria, from December 2017 to March 2019.&#x0D; Methodology: We collected five, each of multidrug resistant and susceptible S. aureus isolates from the Pharmaceutical Microbiology Laboratory of the Ahmadu Bello University, Zaria and confirmed them as S. aureus. Cell concentrates were harvested from 24 hours old Luria and Bertani (LB) pure broth culture of S. aureus isolates by centrifugation at 4°C, 8000 rpm (6800 × g) in a micro-centrifuge for two minutes at room temperature. We used ZymoBIOMICS™ DNA Miniprep Kits to extract the genomic DNA from the harvested cells. We used PCR to amplify the 16S RNA, mecA and the enterotoxin genes after an external optimization of the reaction to ensure a better amplification. Finally, we used exactly two percent agarose gel to resolve the PCR genomic DNA fragments with their primers.&#x0D; Results: The results of the PCR and gel electrophoresis indicated that all the ten (10) strains of S. aureus tested for 16S rDNA gene where positive for the gene whereas only two strains (20%) out of the ten (10) were positive for mecA gene. Among the ten (10) strains of S. aureus, tested, four (40%) strains were positive for two or more staphylococcal enterotoxin genes. All but one phenotypic multidrug resistant isolates carried genes for enterotoxins whereas, phenotypically drug susceptible isolates showed no band for enterotoxins.&#x0D; Conclusion: This study showed that enterotoxin genes were common amongst the antibiotics resistant strains, which indicated a possibility of a relationship between antibiotics resistance and enterotoxogenicity.

In vitro bactericidal activity of 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone (CHP) against drug-susceptible, drug-resistant and drug-tolerant isolates of Mycobacterium tuberculosis

ObjectivesTuberculosis (TB) poses a serious global threat to humans. New bactericidal agents that can shorten treatment duration and target drug resistance still remain a top priority in the discovery of anti-TB drugs. The objective of this study was to investigate the bactericidal potential of 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone (CHP) against drug-susceptible, drug-resistant clinical isolates and drug-tolerant Mycobacterium tuberculosis. MethodsThe minimum bactericidal concentration (MBC) was determined by colony-forming unit (CFU) enumeration. The kill curve analysis was done at different concentrations spanning over 16 days. Drug combination studies with antituberculosis drugs were done to investigate possible synergy. The potential against drug- resistant isolates of M. tuberculosis was done by broth dilution assay. CFU enumeration was done to determine its activity against nutrient-starved drug tolerants, and its feasibility for oral administration was tested by serum inhibitory titre. ResultsCHP displayed bactericidal activity with an MBC of 4 μg/mL against M. tuberculosis H37Rv. The kill curve analysis exhibited a biphasic pattern of killing. CHP showed synergy with rifampicin, isoniazid and amikacin but was indifferent towards ethambutol and levofloxacin. CHP retained its full activity against drug-susceptible, monoresistant and multidrug-resistant (MDR) clinical isolates. CHP showed very strong bactericidal activity against nondividing, drug-tolerant M. tuberculosis that on comparison was highly superior to rifampicin. Furthermore, CHP significantly improved the bactericidal activity of rifampicin and isoniazid in a combination study. The serum inhibitory titre in mice indicated its high oral bioavailability. ConclusionOur results show strong bactericidal potential of CHP against M. tuberculosis that warrant its immediate mechanistic, pharmacokinetic and pharmacodynamic studies.

The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis.

Fluoroquinolones (FQ) form the backbone in experimental treatment regimens against drug-susceptible tuberculosis. However, little is known on whether the genetic variation present in natural populations of Mycobacterium tuberculosis (Mtb) affects the evolution of FQ-resistance (FQ-R). To investigate this question, we used nine genetically distinct drug-susceptible clinical isolates of Mtb and measured their frequency of resistance to the FQ ofloxacin (OFX) in vitro. We found that the Mtb genetic background led to differences in the frequency of OFX-resistance (OFX-R) that spanned two orders of magnitude and substantially modulated the observed mutational profiles for OFX-R. Further, in vitro assays showed that the genetic background also influenced the minimum inhibitory concentration and the fitness effect conferred by a given OFX-R mutation. To test the clinical relevance of our in vitro work, we surveyed the mutational profile for FQ-R in publicly available genomic sequences from clinical Mtb isolates, and found substantial Mtb lineage-dependent variability. Comparison of the clinical and the in vitro mutational profiles for FQ-R showed that 51% and 39% of the variability in the clinical frequency of FQ-R gyrA mutation events in Lineage 2 and Lineage 4 strains, respectively, can be attributed to how Mtb evolves FQ-R in vitro. As the Mtb genetic background strongly influenced the evolution of FQ-R in vitro, we conclude that the genetic background of Mtb also impacts the evolution of FQ-R in the clinic.

Resistance To First-Line Antituberculosis Drugs And Prevalence Of pncA Mutations In Clinical Isolates Of Mycobacterium tuberculosis From Zunyi, Guizhou Province Of China.

BackgroundChina is one of the high-burden countries for multidrug-resistant tuberculosis (MDR-TB), and pyrazinamide is one of the anti-TB drugs used for the shorter MDR-TB treatment regimen. The aim of this study was to determine the correlation between pncA gene mutations and resistance to four first-line anti-TB drugs as well as treatment history in clinical isolates of Mycobacterium tuberculosis.Patients and methodsM. tuberculosis clinical isolates were collected from 318 in-patients with smear-positive TB between October 2008 and September 2016 at a major hospital in Zunyi, Guizhou Province of China, and used for drug susceptibility testing against four first-line anti-TB drugs. Genomic DNA extracted from clinical isolates was used for PCR amplification and DNA sequencing of the pncA gene.ResultsAmong 318 clinical isolates, 129 (40.6%), 170 (53.5%), 66 (20.8%) and 109 (34.3%) were resistant to rifampicin, isoniazid, ethambutol and streptomycin respectively. In addition, 124 clinical isolates were MDR-TB and 71.8% of them were previously treated cases. Sequencing results showed that 46.8% of MDR-TB and 2.2% of drug susceptible isolates harbored a pncA mutation, and 52 types of pncA mutations were detected from 64 isolates. The prevalence of pncA mutations in isolates resistant to first-line anti-TB drugs and previously treated TB cases was significantly higher than that in drug-susceptible isolates and new cases of TB.ConclusionHigh prevalence of pncA mutations in clinical isolates of M. tuberculosis from Zunyi, Guizhou Province of China, is correlated with resistance to four first-line anti-TB drugs, MDR-TB and previously treated TB cases.

The association of acetylacetate (Acr AB-Tol C) and QepA genes with multiple antibiotic resistance among Escherichia coli clinical isolates

The role of efflux pumps in antibiotic resistance development among Escherichia coli may have been underappreciated. The objective of this study was assessment the association of AcrAB-TolC efflux pumps and qepA genes with resistance to common antibiotics among E. coli isolates. A total of 200 E. coli isolates were obtained from diverse samples of inpatients. Minimum inhibitory concentrations and Kirby–Bauer disk diffusion tests were determined for ceftazidime, cefotaxime, imipenem, gentamicin, and tetracycline. The Acr-AB-TolC and qepA genes were amplified using PCR technique and their association with antibiotics was also evaluated using Chi-square test. A majority of isolates (64%) were retrieved from gastrointestinal samples, followed by urinary tract infections (33%), and bloodstream (3%). All the isolates were resistant to ampicillin (100%), followed by cefazolin (59%), and cefoxitin (58%). However, 100% of the isolates showed susceptibility to fosfomycin. The prevalence of acrA, acrB, and qepA genes was 94% (n = 188), 86% (n = 172), and 8% (n = 16), respectively. The acrA and acrB were significantly associated with resistance to cefoxitin and cefazolin (P < 0.01), ceftazidime (P < 0.01), carbapenems (P = 0.022), and tetracycline (P = 0.0112). In addition, qepA gene was significantly associated with tetracycline resistance (P = 0.032). None of the patients had death outcome. A majority of E. coli isolates harbored the AcrAB genes, but qepA was observed among lower number of the isolates. It is notable that three strains lacked the extended spectrum beta-lactamase and carbapenemases and none of multidrug resistant strains carried tet and aminoglycoside modifying enzymes genes. Over-expression of efflux pumps has been increasingly is associated with clinically relevant antibiotic resistance. For this reason, the expression and functionality of efflux pumps should be more investigated profoundly and be compared between drug-resistant and drug-susceptible isolates.

The antitrypanosomal diarylamidines, diminazene and pentamidine, show anthelmintic activity against Haemonchus contortus in vitro

Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC50 4.9 μM and 16.1 μM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the spider-venom peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC50 of 22.9 ± 1.9 μM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target.

Contribution of Pretomanid to Novel Regimens Containing Bedaquiline with either Linezolid or Moxifloxacin and Pyrazinamide in Murine Models of Tuberculosis.

Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure tuberculosis (TB) in BALB/c mice compared to that of the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined, and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to bedaquiline, moxifloxacin, and pyrazinamide (BMZ) resulted in a 1-log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to that for BMZ treatment alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival (≥60 versus 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to the level for BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.

Expression analysis of 10 efflux pump genes in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis clinical isolates.

Active extrusion of antituberculosis drugs via efflux pumps (EPs) has been suggested as contributing to drug resistance in Mycobacterium tuberculosis. This study was conducted to determine the role of 10 drug efflux transporters in the development of drug resistance in a series of clinical M. tuberculosis isolates. A total of 31 clinical M. tuberculosis isolates without drug exposure [21 multi/extensively drug-resistant (M/XDR-TB) and 10 drug-susceptible isolates] were studied. The expression profile of 10 EP genes, including efpA, mmr, stp, drrA, drrB, mmpL7, Rv1250, Rv1634, Rv2994 and Rv1258c, was investigated against the H37Rv standard strain by quantitative reverse transcription PCR (RT-qPCR). Among the 21M/XDR-TB isolates, 10 showed significantly increased levels of gene expression (>4-fold) for at least one of the studied EPs. Moreover, of the isolates with overexpressed genes, three and seven lacked genetic alterations in the surveyed regions of the rpoB+katG+inhA and katG+inhA genes, respectively. Whilst no elevation was observed in the expression of mmr, Rv1250, Rv1634 and Rv1258c genes in any of the isolates, drrA, stp and drrB were found to be the most commonly overexpressed, being overexpressed in seven, five and three isolates, respectively. Decreased minimum inhibitory concentrations (MICs) of rifampicin, but not isoniazid, were observed in the presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Overexpression of EP genes can contribute to the emergence of a MDR phenotype in M. tuberculosis. Inhibition of EPs may provide a promising strategy for improving tuberculosis treatment outcomes in patients infected with M/XDR-TB isolates.

Molecular epidemiology of tuberculosis in Tasmania and genomic characterisation of its first known multi-drug resistant case.

BackgroundThe origin and spread of tuberculosis (TB) in Tasmania and the types of strains of Mycobacterium tuberculosis complex (MTBC) present in the population are largely unknown.ObjectiveThe aim of this study was to perform the first genomic analysis of MTBC isolates from Tasmania to better understand the epidemiology of TB in the state.MethodsWhole-genome sequencing was performed on cultured isolates of MTBC collected from 2014–2016. Single-locus variant analysis was applied to determine the phylogeny of the isolates and the presence of drug-resistance mutations. The genomic data were then cross-referenced against public health surveillance records on each of the cases.ResultsWe determined that 83.3% of TB cases in Tasmania from 2014–2016 occurred in non-Australian born individuals. Two possible TB clusters were identified based on single locus variant analysis, one from November-December 2014 (n = 2), with the second from May-August 2015 (n = 4). We report here the first known isolate of multi-drug resistant (MDR) M. tuberculosis in Tasmania from 2016 for which we established its drug resistance mutations and potential overseas origin. In addition, we characterised a case of M. bovis TB in a Tasmanian-born person who presented in 2014, approximately 40 years after the last confirmed case in the state’s bovids.ConclusionsTB in Tasmania is predominantly of overseas origin with genotypically-unique drug-susceptible isolates of M. tuberculosis. However, the state also exhibits features of TB that are observed in other jurisdictions, namely, the clustering of cases, and drug resistance. Early detection of TB and contact tracing, particularly of overseas-born cases, coordinated with rapid laboratory drug-susceptibility testing and molecular typing, will be essential for Tasmania to reach the World Health Organisation’s TB eradication goals for low-incidence settings.

Automated broth-based systems versus the MYCOTB plate for antimicrobial susceptibility testing of the Mycobacterium tuberculosis complex: challenges in interpretation

We examined categorical agreement between automated mycobacterial susceptibility testing methods (Mycobacterial Growth Indicator Tube [MGIT] 960 System and the VersaTREK Mycobacteria Detection and Susceptibility System) which are based on single critical concentration (CC) “breakpoints” and a commercial microbroth dilution method (Sensititre Mycobacterium tuberculosis MIC Plate [MYCOTB]) which provides an MIC value. Mycobacterium tuberculosis isolates (n=355) were tested against three first-line antimycobacterial agents (ethambutol [EMB], isoniazid [INH], rifampin [RIF]) using the MYCOTB plate and either the MGIT 960 (site 1, n=142) or VersaTREK (site 2, n=213) systems. Overall categorical agreement was 96.8%. When stratified by drug and CC-defined susceptible and resistant isolates, concordance ranged from 75% to 100%. Interpretation of MIC-based results versus established CC-based results was challenging for drugs whose CC was not represented by an exactly equivalent concentration in the manufacturer-defined dilutions on the MYCOTB plate (EMB, INH). We propose interpretations of MYCOTB plate MICs using the currently available plate configuration.