Characterization of Tigecycline Resistance Among Tigecycline Non-susceptible Klebsiella pneumoniae Isolates From Humans, Food-Producing Animals, and in vitro Selection Assay

Emergence of extensively drug-resistant isolates of Klebsiella pneumoniae has prompted increased reliance on the last-resort antibiotics such as tigecycline (TGC) for treating infections caused by these pathogens. Consumption of human antibiotics in the food production industry has been found to contribute to the current antibiotic resistance crisis. In the current study, we aimed to investigate the mechanisms of TGC resistance among 18 TGC-non-susceptible (resistant or intermediate) K. pneumoniae (TGC-NSKP) isolates obtained from human (n = 5), food animals (n = 7), and in vitro selection experiment (n = 6). Isolates were genotyped by multilocus sequence typing (MLST). ramR, acrR, rpsJ, tetA, and mgrB (for colistin resistance) genes were sequenced. The presence of tetX, tetX1, and carbapenemase genes was examined by PCR. Susceptibility to different classes of antibiotics was evaluated by disc diffusion and broth macrodilution methods. The expression level of acrB was quantified by RT-qPCR assay. The 12 TGC-NSKP isolates [minimum inhibitory concentrations (MICs) = 4–32 mg/l] belonged to 10 distinct sequence types including ST37 (n = 2), ST11, ST15, ST45, ST1326 (animal isolates); ST147 (n = 2, human and animal isolates); and ST16, ST377, ST893, and ST2935 (human isolates). Co-resistance to TGC and colistin was identified among 57 and 40% of animal and human isolates, respectively. All human TGC-NSKP isolates carried carbapenemase genes (blaOXA–48, blaNDM–1, and blaNDM–5). tetX/X1 genes were not detected in any isolates. About 83% of TGC-NSKP isolates (n = 15) carried ramR and/or acrR alterations including missense/nonsense mutations (A19V, L44Q, I141T, G180D, A28T, R114L, T119S, Y59stop, and Q122stop), insertions (positions +205 and +343), or deletions (position +205) for ramR, and R90G substitution or frameshift mutations for acrR. In one isolate ramR amplicon was not detected using all primers used in this study. Among seven colistin-resistant isolates, five harbored inactivated/mutated MgrB due to premature termination by nonsense mutations, insertion of IS elements, and frameshift mutations. All isolates revealed wild-type RpsJ and TetA (if present). Increased expression of acrB gene was detected among all resistant isolates, with the in vitro selected mutants showing the highest values. A combination of RamR and AcrR alterations was involved in TGC non-susceptibility in the majority of studied isolates.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (β-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Antifungal Effects of Essential Oils of Zataria multiflora, Mentha pulegium, and Mentha piperita

Background: Among important fungi associated with foods are Aspergillus spp., Penicillium spp., and Geotrichum spp. In this study, we evaluated antifungal effects of Essential Oils (EOs) of Zataria multiflora, Mentha pulegium, and Mentha piperita. Methods: Antifungal properties of EOs of M. piperita, M. pulegium, and Z. multiflora against Aspergillus spp., Penicillium spp., and Geotrichum candidum were determined by agar well diffusion and broth macrodilution method. Data were analyzed by SPSS 20. Results: Among three studied plant EOs, Z. multiflora EO had the strongest antifungal activity (p<0.05) on tested fungi; so that the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were 0.01 and 0.3% for G. candidum, 0.005 and 0.3% for Penicillium spp., and 0.1 and 0.3% for Aspergillus spp. Conclusion: All three studied plant EOs showed antifungal activities. However, as Z. multiflora EO showed the most antifungal effect, it could be specially suggested as natural powerful antifungal preservatives in the food industry.

Inhibitory effect and disinfectant activity of Syzygium aromaticum L. and Ocimum gratissimum L. essential oils against Escherichia coli and Staphylococcus aureus isolated from sheep carcasses

Syzygium aromaticum L. and Ocimum gratissimum L. essential oils were tested for their effectiveness in reduction the counts of inoculated Staphylococcus aureus and Escherichia coli in sheepmeat. The inhibitory effect was analysed by disk diffusion and broth macrodilution method with four strains and seven concentrations. Disinfectant activity of the oils was assessed using the suspension test with two strains and two concentrations. The inhibition was observed at concentrations 400, 200 and 100 μL/mL. Syzygium aromaticum L. oil was better than Ocimum gratissimum L. against bacteria isolated from sheep carcasses. The minimum bactericidal concentration of the essential oils aginst inocula ranged from 50 to 200 μL/mL. The suspension test showed that after 5 minutes of contact, Syzygium aromaticum L. disinfectant at 400 μL/mL deactivated the inoculants completely. The results demonstrated that essential oils exerted a significant bactericidal and bacteriostatic action against pathogens.

Application of Lemongrass Essential Oil as a Natural Preservative Agent for Pineapple Juice

Pineapple juice is frequently consumed for its extensive health benefits, but is prone to microbiological degradation. Pasteurization – as a current preservation technique – have been degrading the nutritional qualities of fruit juices, and the use of synthetic preservatives have been perceived negatively by consumers. In this study, lemongrass essential oil was applied for the preservation of pineapple juice. Agar Well Diffusion Method was used for antimicrobial activity screening of lemongrass essential oil against E. coli, S. cerevisiae, and A. niger, while Broth Macrodilution Method was used in determining Minimum Inhibitory Concentration (MIC) of lemongrass essential oil, which was evaluated to be 0.1%. Furthermore, the addition of 0.1% lemongrass essential oil into pineapple juice had a negative impact regarding the sensorial acceptability, but was able to significantly suppress the microbial count for bacteria, yeast, and mould during 5 days of storage time in chilling temperature (4oC). Vitamin C content degradation and pH changes during storage were not significantly affected by the addition oflemongrass essential oil. Furthermore, the addition of 0.1% lemongrass essential oil was observed to be not synergistic with the enzyme activity of pineapple juice, and has negatively impacted the enzyme stability of the pineapple juice.

In Vitro Inhibition of Staphylococcus aureus subsp. aureus (ATCC® 6538™) by Artemether-Lumefantrine Tablets: A Comparative Study of Three Dosage Strengths

Purpose: Antibiotics are progressively failing in the fight against infections due to S. aureus because the bacterium has an outstanding ability to acquire multi-antibiotic resistance and become resistant to most antibiotics. Multi-drug resistant S. aureus poses a major threat to the foundation upon which standard antibacterial chemotherapy stands, hence the need to consider non-antibiotic solutions to manage invasive bacterial infections. This study investigated the inhibitory activities of three dosage strengths of artemether-lumefantrine tablets against Staphylococcus aureus subsp. aureus (ATCC® 6538™) and determined the minimum concentrations of the tablets that are able to completely inhibit growth of the bacterium in vitro. Methods: The agar dilution and broth macrodilution techniques were used to determine the susceptibility of the Staphylococcus aureus subsp. aureus (ATCC® 6538™) strain to artemether-lumefantrine 20/120mg, 40/240mg and 80/480mg tablets. Results: The most active inhibitor was artemether-lumefantrine 80/480mg tablet with a minimum inhibitory concentration value of 2.5mg/mL while artemether-lumefantrine 20/120mg and 40/240mg tablets exhibited moderate but equal activities against the test strain. Conclusions: The study has revealed that artemether-lumefantrine, an antimalarial drug, also has anti-staphylococcal properties and inhibits S. aureus in vitro. This study presents the first report on the in vitro activity of artemether-lumefantrine tablet against S. aureus and suggests the need to consider it as an alternative in the treatment of staphylococcus infections.

Two-Site Evaluation of the Colistin Broth Disk Elution Test To Determine Colistin In Vitro Activity against Gram-Negative Bacilli

ABSTRACT Limited methods for colistin MIC determination are available to clinical microbiology laboratories. The purpose of this study was to evaluate the accuracy of the colistin broth disk elution (CBDE) test compared to that of broth microdilution (BMD) for identifying colistin MICs. CBDE was compared to colistin BMD using a collection of Gram-negative bacilli tested at two U.S. microbiology laboratories. The isolates tested included 121 retrospective clinical isolates, 45 prospective clinical isolates, and 6 mcr-1-positive Escherichia coli isolates. CBDE was performed with four 10-ml cation-adjusted Mueller-Hinton broth tubes per isolate, to which 0, 1, 2, and 4 colistin 10-µg disks were added, generating final concentrations in the tubes of 0 (growth control), 1, 2, and 4 µg/ml, respectively. MICs were evaluated visually and interpreted using Clinical and Laboratory Standards Institute breakpoints. Site 2 also compared CBDE to the reference broth macrodilution (BMAD) method (n = 110 isolates). Overall, CBDE yielded a categorical agreement (CA) and essential agreement (EA) of 98% and 99%, respectively, compared to the results of colistin BMD. Very major errors occurred for mcr-1-producing strains, with MICs fluctuating from 2 to 4 µg/ml on repeat testing. The results for all other isolates were in CA with those of BMD. CBDE versus BMAD had an EA of 100% and a CA of 100%. Compared to currently used techniques, CBDE is an easy and practical method to perform colistin MIC testing. Some mcr-1-producing isolates yielded MICs of 2 µg/ml by CBDE and 4 µg/ml by BMD. As such, the results for isolates with colistin MICs of 2 µg/ml by CBDE should be confirmed by the reference BMD method, and isolates with MICs of ≥2 µg/ml should be evaluated for the presence of mcr genes.

Reduced susceptibility in leptospiral strains of bovine origin might impair antibiotic therapy

Leptospirosis is a worldwide zoonotic disease determined by pathogenic spirochetes of the genus Leptospira. The control of bovine leptospirosis involves several measures including antibiotic treatment of carriers. Despite its importance, few studies regarding antimicrobial susceptibility of strains from bovine origin have been conducted. The aim of this study was to determine the in vitro susceptibility of Leptospira strains obtained from cattle in Rio de Janeiro, Brazil, against the main antibiotics used in bovine veterinary practice. A total of 23 Leptospira spp. strains were investigated for minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) using broth macrodilution. At the species level, there were not differences in MIC susceptibility except for tetracycline (P < 0.05). Nevertheless, at the serogroup level, differences in MIC were observed among Sejroe strains, mainly for ceftiofur, doxycycline and in MBC for streptomycin (P < 0.05). One strain presented MBC values above maximum plasmatic concentration described for streptomycin and was classified as presenting reduced susceptibility. Efficacy of antimicrobial therapy on bovine leptospirosis could be compromised due to occurrence of infection by Leptospira strains presenting reduced susceptibility.

In vitro susceptibility testing of four antifungal drugs against fungal isolates in onychomycosis

Background: Onychomycosis is chronic fungal infection of fingernails and toenails. Variety of fungi cause onychomycosis. Due to importance of high prevalence rate of onychomycosis this study was conducted.Methods: In this study 100 patients suspected of onychomycosis were examined. Diagnosis of onychomycosis was based on the patient’s history, physical examination, microscopy and culture of nail specimens.Results: Direct microscopy of the nail clippings in 20% KOH solution was positive in 61% and culture was positive in 54% cases. The common etiological agent was dermatophytes (79.6% cases) followed by non dermatophyte moulds (11.1% cases) and yeasts (9.2% cases). Amongst dermatophytes, T. rubrum was found to be commonest etiological agent (57.6%) followed by T. mentagrophytes. We had performed the in vitro antifungal susceptibility testing of isolated fungal species against Amphotericin B, Fluconazole, Itraconazole and Terbinafine according to standard guidelines recommended by the CLSI. Antifungal susceptibility testing of dermatophytes and non-dermatophyte moulds was performed by broth macrodilution method. For Candida species we used broth macrodilution method as well as disk diffusion method. All three Candida albicans isolates were sensitive to amphotericin B, fluconazole and itraconazole. Two strains of Candida krusei were sensitive to amphotericin B and resistant to fluconazole and itraconazole. Two isolates of T. rubrum had MIC >64µg/ml and one T. Mentagrophytes isolate had MIC 32µg/ml for fluconazole. Among non dermatophyte moulds, Aspergillus niger and one isolate of Fusarium oxysporum showed high MICs against fluconazole.Conclusions: Terbinafine exhibited the lowest MICs among all the tested antifungal drugs.