Antimicrobial Susceptibility of Clinical Oral Isolates of Actinomyces spp.

Actinomyces species play an important role in the pathogenesis of oral diseases and infections. Susceptibility testing is not always routinely performed, and one may oversee a shift in resistance patterns. The aim of the study was to analyze the antimicrobial susceptibility of 100 well-identified clinical oral isolates of Actinomyces spp. against eight selected antimicrobial agents using the agar dilution (AD) and E-Test (ET) methods. We observed no to low resistance against penicillin, ampicillin-sulbactam, meropenem, clindamycin, linezolid and tigecycline (0–2% ET, 0% AD) but high levels of resistance to moxifloxacin (93% ET, 87% AD) and daptomycin (83% ET, 95% AD). The essential agreement of the two methods was very good for benzylpenicillin (EA 95%) and meropenem (EA 92%). The ET method was reliable for correctly categorizing susceptibility, in comparison with the reference method agar dilution, except for daptomycin (categorical agreement 87%). Penicillin is still the first-choice antibiotic for therapy of diseases caused by Actinomyces spp.

ANTIFUNGAL ACTIVITY OF CHITOSAN AGAINST Rhizopus stolonifer

These days instead of chemical pesticides, various natural alternatives have been used for the management of post-harvest diseases. This research was carried out to evaluate the antifungal potential of different chitosan concentrations viz., 5, 10, and 15 mg/µl against Rhizopus stolonifer by the agar dilution and well diffusion methods. The evaluation was carried out after seven days of incubation by measuring the inhibition of R. stolonifer mycelial growth. Results of the study revealed that the chitosan had a significant inhibitory effect on mycelial growth and maximum mycelial growth inhibition was reported at the 10 and 15 mg/µl concentrations and these two treatments are not significantly different. Based on these results, the best concentration (15 mg/µl) was further evaluated by the well diffusion technique. The average inhibition zones formed by the higher concentration was 25mm. Results of the study suggested that chitosan can be used for the management of post-harvesting diseases of tomatoes.

AcrB-TolC efflux system is essential for macrolide resistance in Helicobacter pylori

The prevalence of Helicobacter pylori strains resistant to macrolide is increasing worldwide. Macrolide molecules can be generally extruded by the AcrB-TolC system in bacteria. The H. pylori 26695 genome was assessed for putative translocases and the outer membrane efflux of AcrB (HP607) and TolC (HP605) proteins. We investigated the role of the AcrB-TolC efflux system in macrolide resistant (M-R) H. pylori. Both acrB- and tolC-mutant M-R strains were constructed from M-R strains by insertional inactivation of the acrB and tolC genes. The minimal inhibition concentrations (MICs) of erythromycin (EM) and clarithromycin (CLR) were determined by an agar dilution assay. To investigate the efflux ability of macrolides, intracellular accumulation of radiolabeled EM in the H. pylori 26695 strain, M-R strain, and acrB- and tolC-mutant M-R strains was measured by a liquid scintillation counter. For Post antibiotic effect (PAE), EM-treated H. pylori was diluted 1000-fold to remove antimicrobial activity. After additional 24 hours incubation, the CFU was measured. The decrease in the levels of resistance to EM and CLR was 32-fold higher for the acrB- and tolC-mutant M-R strains than the M-R strains. The intracellular EM concentration significantly increased in the acrB- and tolC-mutant M-R strains than the H. pylori 26695 and M-R strains. Diluted acrB, and tolC M-R mutant H. pylori after EM treatment was markedly reduced compared to M-R H. pylori. Our result showed that the M-R mechanism of H. pylori is significantly associated with AcrB-TolC efflux system.

In Vitro Activity of Gepotidacin Against Gram-negative and Gram-positive Anaerobes

Gepotidacin (formerly GSK2140944) is a first in class triazaacenaphthylene antibacterial currently in Phase III clinical trials. When tested against Gram-negative ( n =333) and Gram-positive ( n =225) anaerobes by agar dilution, gepotidacin inhibited 90% of isolates (MIC 90 ) at concentrations of 4 and 2 μg/ml, respectively. Given gepotidacin’s in vitro activity against the anaerobic isolates tested, further study is warranted to better understand gepotidacin’s utility in the treatment of infections caused by clinically relevant anaerobic organisms.

Klebsiella pneumoniae and Colistin Susceptibility Testing: Performance Evaluation for Broth Microdilution, Agar Dilution and Minimum Inhibitory Concentration Test Strips and Impact of the “Skipped Well” Phenomenon

The emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the “skipped well” (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains (“very major errors”), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.

Antibacterial Activity and Optimal Treatment of Ceftazidime-Avibactam and Aztreonam-Avibactam Against Bloodstream Infections Caused by Carbapenem-Resistant Klebsiella pneumoniae

Objectives: This work was to investigate the activity and optimal treatments of ceftazidime-avibactam (CZA) and aztreonam-avibactam (AZA) against bloodstream infections caused by carbapenem resistant Klebsiella pneumoniae (BSIs-CRKP).Methods: A total of 318 nonduplicate BSIs-CRKP isolates were collected from Blood Bacterial Resistant Investigation Collaborative System (BRICS) program. The minimum inhibitory concentration (MIC) of CZA and AZA were determined by agar dilution method. Carbapenemase genes and multilocus sequence typing were amplified by PCR. Monte Carlo simulation (MCS) was conducted to calculate cumulative fraction of response (CFR) of different CZA or AZA administrations.Results: The MIC90 of CZA and AZA were 128/4 and 1/4 mg/L, respectively. There are 87.4 and 3.5% isolates carried blaKPC-2 and blaNDM-1. A total of 68 ST types were identified and 29 novel ST types. ST11 accounted for 66.6%. Further MCS showed CFR of CZA using two-step infusion therapy (rapid first-step 0.5 h infusion and slow second-step 3 h infusion, TSIT) (2.5 g 0.5 h, 3.75 g every 8 h with 3 h infusion and 3.75 g 0.5 h, 2.5 g every 8 h with 3 h infusion) was above 89%. The CFR of AZA with TSIT was above 96%.Conclusion: TSIT with sufficient pharmacokinetic conditions could be useful for enhancing the therapeutic efficacy of CZA and AZA against BSIs-CRKP.

Utility of bile esculin azide agar for screening of stool samples for vancomycin resistant enterococci from patients with gut colonization

Background: Due to increased prevalence of vancomycin resistant enterococci (VRE) in hospital settings as an important nosocomial pathogen, microbiology laboratories should be prepared with test protocol for prompt detection and reporting of these resistant organisms. This helps in appropriate treatment of patients without delay and implementation of infection control measures in order to prevent spread of such infections. With this background present study was conducted to demonstrate utility of bile esculin azide agar with vancomycin (BEAV) for screening of enterococci for vancomycin drug resistance.Methods: Over a period of one year 200 stool samples were collected from hospitalized patients in a tertiary care hospital. Samples were inoculated on bile esculin azide agar with vancomycin (6ug/ml) to screen for vancomycin drug resistance in enterococci isolated from stool samples. Vancomycin drug resistance was confirmed by agar dilution method.Results: Out of 200 stool samples collected from hospitalized patients, 13 (6.5%) samples showed growth on bile esculin azide agar with vancomycin (6 µg/ml). Of these 13 isolates, 12 (92.3%) isolates were confirmed as VRE by agar dilution method and demonstrated minimum inhibitory concentration (MIC) of ≥32 µg/ml and all 12 isolates were identified as E. faecium. One (7.7%) isolate grown on BEAV was identified as E. gallinarum and showed MIC value of 8 µg/ml.Conclusions: Present study recommends use of bile esculin azide agar with vancomycin (6 µg/ml) as a screening medium for isolation of VRE from stool samples which usually carries mixed commensal flora of gastrointestinal tract.

In vitro Inhibitory Action of the Essential Oils of Origanum Vulgare and Rosmarinus Officinalis against Aspergillus Fumigatus

Aspergillus fumigatus is the main etiological agent of aspergillosis. Considering azole antifungal drug resistance in A. fumigatus, which compromises treatment, new alternatives are needed. Among them, essential oils (EOs) can be an alternative treatment, having shown positive results in inhibiting phytopathogenic fungi in vitro. We aimed to determine the in vitro antifungal activity of Origanum vulgare L. subsp. hirtum (Link) (oregano) and Rosmarinus officinalis L. (rosemary) EOs alone and in association (O. vulgare+R. officinalis) against A. fumigatus. EOs were analyzed by gas chromatography (GC-FID and GC/MS systems), and analyses showed that the major components of O. vulgare EO were carvacrol (67.8%), p-cymene (14.8%), and thymol (3.9%); for R. officinalis, they were the monoterpenes 1,8-cineole (49.1%), camphor (18.1%) and α-pinene (8.1). For biological assays, five EO concentrations, 0.2; 0.4; 0.6; 0.8 and 1.0%, were used in disk diffusion and agar dilution tests for 21 days. In disk diffusion, O. vulgare EO alone and in association (O. vulgare+R. officinalis) showed fungicidal activity at all concentrations. In agar dilution, inhibitory action was demonstrated from 0.6% for O. vulgare EO and in association (O. vulgare+R. officinalis). R. officinalis EO at 1.0% showed no fungal growth, determining the minimum inhibitory concentration (MIC). The present study demonstrated inhibitory actions of O. vulgare and R. officinalis EOs in A. fumigatus. GC analyses corroborated the literature regarding their antibacterial and antifungal effects. However, further in vitro and in vivo studies are needed to evaluate EOs as alternative antifungals for treating aspergillosis.

Ammoides pusilla Essential Oil: A Potent Inhibitor of the Growth of Fusarium avenaceum and Its Enniatin Production

Enniatins are mycotoxins produced by Fusarium species contaminating cereals and various agricultural commodities. The co-occurrence of these mycotoxins in large quantities with other mycotoxins such as trichothecenes and the possible synergies in toxicity could lead to serious food safety problems. Using the agar dilution method, Ammoides pusilla was selected among eight Tunisian plants for the antifungal potential of its essential oil (EO) on Fusarium avenaceum mycelial growth and its production of enniatins. Two EO batches were produced and analyzed by GC/MS-MS. Their activities were measured using both contact assays and fumigant tests (estimated IC50 were 0.1 µL·mL−1 and 7.6 µL·L−1, respectively). The A. pusilla EOs and their volatiles inhibited the germination of spores and the mycelial growth, showing a fungistatic but not fungicidal activity. The accumulation of enniatins was also significantly reduced (estimated IC50 were 0.05 µL·mL−1 for the contact assays and 4.2 µL·L−1 for the fumigation assays). The most active batch of EO was richer in thymol, the main volatile compound found. Thymol used as fumigant showed a potent fungistatic activity but not a significant antimycotoxigenic activity. Overall, our data demonstrated the bioactivity of A. pusilla EO and its high potential to control F. avenaceum and its enniatins production in agricultural commodities.