Agar Dilution Test Research Articles

Influence of Plant Phenology on Chemical Composition of Monarda fistulosa L. Organs and their Bioactive Properties

Monarda fistulosa L. above-ground organs, collected at three phases of plant phenology, were investigated as potential raw materials for application in the food industry. They were evaluated regarding essential oil (EO) content, composition, and antimicrobial activity, as well as characteristics of phenolic fractions and antioxidant properties, which may determine health benefits and potential use in food preservation. The dominant constituent of leaf EO was carvacrol. In the inflorescence EO carvacrol content was especially high at the full flowering phase (45.12%), while during the fruit setting phase its content was lower than that of p-cymene (39.75%) and thymoquinone (25.04%). In the agar dilution test, leaf and inflorescence EOs inhibited the growth of the six tested microorganisms at the concentration range of 0.156–0.625 µL/mL. Leaves collected at the vegetative phase of plant growth were characterised by the highest content of rosmarinic acid and didymin. Inflorescences harvested during the flowering of plants were rich in linarin. Flavonoid content was highly correlated with antioxidant activity of extracts. Due to these properties, M. fistulosa extracts and essential oils could be used in the food industry as natural preservatives or antioxidants, thereby contributing to the development of safer and more sustainable food products.

Mistletoe (Loranthus europaeus Jacq.): antioxidant, antimicrobial and anticholinesterase activities

Plants are natural resources responsible for different biological activities. Determining the biological activities of plants is very important. In our study, some biological activities and total phenolic and flavonoid contents of Loranthus europaeus Jacq. were determined. In this context, ethanol and methanol extracts of the plant were obtained using the soxhlet device. Total antioxidant, total oxidant, and oxidative stress statuses were determined using Rel Assay kits. Antimicrobial activity was tested against standard bacterial and fungal strains using the agar dilution test. Anticholinesterase activity was determined by detecting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. Determination of total phenolic content was determined using the Folin-Ciocalteu reagent. Total flavonoid quantification was performed using an aluminum chloride assay. As a result of the study, TAS values (Total antioxidant status) of ethanol and methanol extracts of the plant were determined as 5.620±0.134 and 6.384±0.134 mmol/L, respectively. TOS values (Total oxidant status) were determined as 10.997±0.183 and 13.368±0.222 µmol/L, respectively. OSI values (Oxidative stress index) were measured as 0.196±0.004 and 0.209±0.003, respectively. TPC (Total phenolic content) value was determined as 34.49±1.78 and 55.82±2.34 mg/g, respectively, and TFC (Total flavonoid content) value was determined as 46.10±2.40 and 55.49±1.15 mg/g, respectively. Ethanol and methanol extracts of the plant were found to be effective against bacterial and fungal strains at concentrations between 50-200 µg/mL. In addition, the anti-AChE values of ethanol and methanol extracts were determined as 13.51±0.81 and 22.79±1.86 µg/mL, respectively, and the anti-BChE values were determined as 27.84±0.62 and 33.08±1.63 µg/mL, respectively. According to the results obtained, it was determined that L. europaeus has antioxidant, antimicrobial, and anticholinesterase activity. In this context, it is thought that it can be used as a natural material in pharmacological designs.

Agar dilution test and its Merits and Drawbacks in evolving dynamics of colistin susceptibility

ObjectivesAutomated results for determining colistin susceptibility tests are unreliable, micro broth dilution requires expertise, and CBDE has limited dilutions. International Consensus Document of 2022 suggested that agar dilution was unacceptable due to varying results in the literature. MethodsThe study was designed to evaluate the agar dilution method for colistin susceptibility in CRE isolates compared to CBDE. In the study, 108 carbapenem-resistant isolates were tested for Colistin susceptibility by Microbroth Dilution, agar dilution, and colistin broth disc elution. The comparisons were made using various statistical parameters. ResultsThe results of the agar dilution method revealed an essential agreement of 75 % and a categorical agreement of 92.5 %. The method showed a sensitivity of 75 % and a specificity of 97.7 %. The positive and negative predictive values were 88.2 % and 94.5 %, respectively. Youden's index was 0.727, indicating a moderate level of accuracy. Meanwhile, CBDE diagnostic accuracy tests were better, with Youden's index at 0.939. ConclusionsWhile CBDE demonstrated better accuracy parameters, it did not offer a broader MIC range. In contrast, agar dilution showed reasonable specificity and reliability for isolates with high MIC. Therefore, we propose using CBDE for screening and agar dilution as a supplementary test in the approach to colistin susceptibility.

Antibacterial activity of silver and gold nanoparticles that have been synthesized by curcumin

The green synthesis of metal nanoparticles with curcumin which is a compound with antibiotic effects, was considered for the synthesis of metal nanoparticles. Gold and silver nanoparticles were synthesized with an average diameter of 19.8 ± 6.2 nm and 29.8 ± 6.4 nm respectively. Both particles represent stability in the physiological environment and after a series of washing procedures to remove unreacted precursors. The antimicrobial efficacy of the synthesized nanoparticles was investigated in different techniques such as disk diffusion, agar well diffusion, minimum inhibitory concentration, and minimum bactericidal concentration tests. The results of both curcumin-coated metal nanoparticles in the disk diffusion test were negative. However, in the agar dilution and MIC tests of antibacterial effect of both nanoparticles on the tested bacteria was observed. The silver nanoparticles had an inhibitory effect on all selected bacteria in the agar dilution (8-256 µg/ml). However, gold nanoparticles had a lower inhibitory effect on the growth of some of the selected bacteria (64-512 µg/ml). In conclusion, silver nanoparticles were more effective than gold nanoparticles in most of the investigated methods.

Determination of Vancomycin Minimum Inhibitory Concentration in Methicillin-resistant Staphylococcus Aureus by Various Phenotypic Methods: A Cross-sectional Study

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) has become a global threat, causing nosocomial and community-acquired infections, thus significantly contributing to morbidity and mortality. These organisms often exhibit Multidrug Resistance (MDR) to various groups of antimicrobials. The emergence of reduced susceptibility to vancomycin and resistance further exacerbates the problem, leaving clinicians with few therapeutic options to treat serious, life-threatening infections. Aim: To determine the vancomycin susceptibility of MRSA using various phenotypic methods. Materials and Methods: The present cross-sectional study was conducted in the Department of Microbiology, SHKM Government Medical College, Nalhar, Haryana, India, from February 2019 to January 2020. All MRSA isolates were included, totaling 66 MRSA isolates. MRSA isolates were screened using the cefoxitin disc (30 μg) according to the Clinical and Laboratory Standards Institute (CLSI) recommended disc diffusion method. The Minimum Inhibitory Concentration (MIC) of vancomycin was determined using phenotypic methods such as broth dilution, agar dilution and Epsilometer test (E-test). Demographic details such as age, sex, site of the sample and diagnosis were recorded. Statistical analysis was performed using the Pearson’s product-moment correlation. The significance level was set at a probability of <0.05 with a confidence interval of 95%. Results: A total of 66 (30.3%) isolates of Staphylococcus aureus (S. aureus) were identified as MRSA using standard laboratory tests and were studied for vancomycin MIC using phenotypic methods: Broth dilution, agar dilution and E-test. Vancomycin- intermediate S. aureus (VISA) was observed in 54.5%, 42.4% and 51.5% of MRSA by E-test, agar dilution and broth dilution, respectively. The presence of Vancomycin-resistant S. aureus (VRSA) was not detected in the region. When compared with broth dilution, E-test results demonstrated higher accuracy (97%) than agar dilution (90.9%). Conclusion: The present study highlights the emergence of VISA in more than 40% of the MRSA in the region. The higher MIC of vancomycin emphasises the need for strict adherence to antibiotic prescription guidelines to prevent further emergence of vancomycin resistance.

Characterization of Bacillus velezensis 32a metabolites and their synergistic bioactivity against crown gall disease

Crown gall disease caused by Agrobacterium tumefaciens is considered to be the main bacterial threat of stone fruit plants in Mediterranean countries. In a previous study, Bacillus velezensis strain 32a was isolated from Tunisian rhizosphere soil and revealed high antagonistic potential against A. tumefaciens strains. In order to better characterize the antagonistic activity of this strain against this important plant pathogen, the production of secondary metabolites was analyzed using liquid chromatography coupled with mass spectrometry. The results revealed the production of different compounds identified as surfactins, fengycins, iturins and bacillibactin belonging to the lipopeptide group, three polyketides (macrolactins, oxydifficidin and bacillaenes), bacilysin and its chlorinated derivative; chlorotetaine. The involvement of lipopeptides in this antagonistic activity was ruled out by performing agar and broth dilution tests with pure molecules. Thus, the construction of B. velezensis 32a mutants defective in polyketides and bacilysin biosynthesis and their antagonistic activity was performed and compared to a set of derivative mutants of a comparable strain, B. velezensis GA1. The defective difficidin mutants (△dfnA and △dfnD) were unable to inhibit the growth of A. tumefaciens, indicating the high-level contribution of difficidin in the antagonism process. While the macrolactin deficient mutant (∆mlnA) slightly decreased the activity, suggesting a synergetic effect with difficidin. Remarkably, the mutant △dhbC only deficient in bacillibactin production showed significant reduction in its capacity to inhibit the growth of Agrobacterium.Taken collectively, our results showed the strong synergetic effect of difficidin and macrolactins and the significant implication of siderophore to manage crown gall disease.

Total phenolic, flavonoid, protein contents and biological activities of wild mustard

AbstractThe nutritional and therapeutic benefits of plants are outstanding. Wild mustard (Sinapis arvensis L.) collected from different regions was analysed for its phenolic, flavonoid, protein, and biological activity levels. In this case, the plant ethanol extract was obtained using a soxhlet apparatus. Rel assay kits and the DPPH test were used to assess the plant's antioxidant activity. The agar dilution test was used to determine antimicrobial efficacy. A549 lung cancer cells were used in an MTT assay to measure antiproliferative activity. The Folin−Ciocalteu reagent was used to quantify the total phenolic content of the sample. The amount of flavonoids was determined using an aluminium chloride test. The amount of protein was calculated using the AOAC's standard technique. Based on the research conducted, it was shown that the maximum total antioxidant status (TAS) value for the ethanol extract of wild mustard from various places was 5.232 ± 0.047 mmol L−1. At a concentration of 2 mg mL−1, DPPH activity was measured to be 82.06 ± 1.01. The maximum levels of total phenolic, flavonoid, and protein were 80.57 ± 2.19 mg g−1, 154.07 ± 2.79 mg g−1, and 7.75 ± 0.24%, respectively. Doses of 25–100 μg mL−1 of plant extracts were effective against fungal strains, whereas doses of 50–200 μg mL−1 were beneficial against bacterial strains. The plant extracts were shown to have potent antiproliferative properties. It was found that wild mustard's phenolic, flavonoid, protein, and biological activity levels varied according to the location from which it was gathered. It was also concluded that wild mustard had significant biological activity.

Lecithin-amended montmorillonite clays enhance the antibacterial effect of barrier creams

The objective of this study was to assess in vitro antibacterial activity of barrier cream (EVB) formulations containing either calcium montmorillonite (CM) or lecithin-amended montmorillonite (CML). All ingredients were generally recognized as safe (GRAS), and clay minerals were specifically studied due to their known ability to adsorb numerous toxins of human clinical relevance. Characterization of the EVB formulations showed good spreadability, pH, appearance, unity, viscosity, and no evidence of phase separation. Colony forming, disk diffusion susceptibility, and agar dilution assays were used to determine the minimal bactericidal concentration (MBC) of total EVB formulations, as well as respective individual ingredients, against E. coli. Active ingredients within the base EVB formulation were found to be essential oils and zinc oxide. EVB-CML at 0.5–25 mg/mL dose-dependently and significantly (p ≤ 0.01) enhanced the antibacterial activity of the base EVB formulation. MBC values for EVB-CML were 2.5 mg/mL in the colony forming assay and 0.75 mg/mL in the agar dilution test, with a zone of inhibition. Both EVB and EVB-CML displayed stronger antibacterial activity than four antimicrobial creams currently marketed in the United States. Moreover, this effect was rapid, favored by high temperature, and product stability testing suggested a shelf life of at least 10 months. Taken together, these findings demonstrate the ability of CML to enhance the antibacterial effect of the base EVB formulation against E. coli. This novel EVB-CML formulation represents a promising advancement toward improved antibacterial efficacy beyond current industry standards for commercial skin creams and sunscreens.

Antimicrobial susceptibility of western Canadian Brachyspira isolates: Development and standardization of an agar dilution susceptibility test method.

The re-emergence of Brachyspira-associated disease in pigs since the late 2000s has illuminated some of the diagnostic challenges associated with this genus; notably, the lack of standardized antimicrobial susceptibility testing (AST) methods and interpretive criteria. Consequently, laboratories have relied heavily on highly variable in-house developed methods. There are currently no published investigations describing the antimicrobial susceptibility of Brachyspira isolates collected from pigs in Canada. The first objective of this study was therefore to develop a standardized protocol for conducting agar dilution susceptibility testing of Brachyspira spp., including determining the optimal standardized inoculum density, a key test variable that impacts test performance. The second objective was to determine the susceptibility of a collection of western Canadian Brachyspira isolates using the standardized methodology. After assessing multiple media, an agar dilution test was standardized in terms of starting inoculum (1-2 × 108 CFU/ml), incubation temperature and time, and assessed for repeatability. The antimicrobial susceptibility of a collection of clinical porcine Brachyspira isolates (n = 87) collected between 2009-2016 was then determined. This method was highly reproducible; repeat susceptibility testing yielded identical results 92% of the time. Although most of the isolates had very low MICs to the commonly used antimicrobials to treat Brachyspira-associated infections, several isolates with elevated MICs (>32 μg/ml) for tiamulin, valnemulin, tylosin, tylvalosin, and lincomycin were identified. Overall, this study underscores the importance of establishing CLSI approved clinical breakpoints for Brachyspira to facilitate the interpretation of test results and support the evidence-based selection of antimicrobials in swine industry.

Total phenolic and flavonoid contents and antioxidant, antimicrobial and antiproliferative activities of Polycarpontetraphyllum

Plants are organic materials that support a variety of biological processes. The biological activity of the Polycarpon tetraphyllum (L.) L plant was identified in this study. In this case, the Soxhlet apparatus was used to obtain ethanol and methanol extracts of the plant's aerial portions. Total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) values for the plant were determined using Rel Assay kits. The antioxidant potential of the plant extract was determined by the DPPH test. Antimicrobial activity was determined using the agar dilution test against six bacterial and three fungal strains. The antiproliferative activity of the plant extract against A549 lung cancer cell line was determined by MTT assay. Phenolic compounds in the plant were screened with LC-MS/MS device. As a result of the study, the DPPH activity of the plant was found to be higher in the methanol extract (75.72 ​± ​0.32) at a concentration of 2 ​mg/mL. TAS value of the plant was determined as 8.449 ​± ​0.143 ​mmol/L, TOS value as 12.129 ​± ​0.200 ​μmol/L and OSI value as 0.144 ​± ​0.004. It was determined that the plant extracts showed the highest effect against Acinetobacter baumannii, Candida albicans and C. krusei at 50 ​μg/mL. The ethanol extract of the plant was found to have stronger antiproliferative activity. In addition, the presence of acetohydroxamic acid, catechin hydrate, resveratrol, fumaric acid, 4-hydroxycinnamic acid, 4-gydroxybenzoic acid, oleuropein, ellagic acid, quercetin, butein and luteolin were found in the plant. In this context, it was determined that the plant has important biological activity.

Molecular testing-guided therapy versus susceptibility testing-guided therapy in first-line and third-line Helicobacter pylori eradication: two multicentre, open-label, randomised controlled, non-inferiority trials

Helicobacter pylori infection is an important causal factor of gastric cancer and peptic ulcer disease and is associated with immune thrombocytopenic purpura and functional dyspepsia. In H pylori strains, point mutations in the 23S rRNA and gyrA genes are associated with clarithromycin resistance and levofloxacin resistance, respectively. Whether the efficacy of molecular testing-guided therapy is non-inferior to that of susceptibility testing-guided therapy for H pylori eradication is unclear. Therefore, we aimed to compare the efficacy and safety of molecular testing-guided therapy and traditional culture-based susceptibility testing-guided therapy in first-line and third-line treatment of H pylori infection. We did two multicentre, open-label randomised trials in Taiwan. In trial 1 (done at seven hospitals), treatment-naive individuals infected with H pylori who were aged 20 years or older were eligible for study inclusion. In trial 2 (done at six hospitals), individuals aged 20 years or older who failed treatment after two or more eradication therapies for H pylori infection were eligible for enrolment. Eligible patients were randomly assigned (1:1) to receive either molecular testing-guided therapy or susceptibility testing-guided therapy. The randomisation sequence was generated by computer using permuted block randomisation with a block size of 4. All investigators were masked to the randomisation sequence. Clarithromycin and levofloxacin resistance were determined by agar dilution test for measuring minimum inhibitory concentrations in the susceptibility testing-guided therapy group, and by PCR and direct sequencing for detection of 23S rRNA and gyrA mutations in the molecular testing-guided therapy group. Study participants received clarithromycin sequential therapy, levofloxacin sequential therapy, or bismuth quadruple therapy according to the resistance status to clarithromycin and levofloxacin. The 13C-urease breath test was used to determine the status of H pylori infection at least 6 weeks after eradication therapy. The primary outcome was the eradication rate by intention-to-treat analysis. The frequency of adverse effects was analysed in patients with available data. The prespecified margins for non-inferiority were 5% for trial 1 and 10% for trial 2. The trials are ongoing for post-eradication follow-up and registered with ClinicalTrials.gov, NCT03556254 for trial 1, and NCT03555526 for trial 2. Between March 28, 2018, and April 23, 2021, 560 eligible treatment-naive patients with H pylori infection were recruited and randomly assigned to the molecular testing-guided therapy group or the susceptibility testing-guided therapy group in trial 1. Between Dec 28, 2017, and Oct 27, 2020, 320 eligible patients with refractory H pylori infection were recruited and randomly assigned to the molecular testing-guided therapy group or the susceptibility testing-guided therapy group in trial 2. 272 men and 288 women were recruited for trial 1, and 98 men and 222 women were recruited for trial 2. In first-line H pylori treatment, infection was eradicated in 241 (86%, 95% CI 82-90) of 280 patients in the molecular testing-guided therapy group and 243 (87%, 83-91) of 280 patients in the susceptibility testing-guided therapy group by intention-to-treat analysis (p=0·81). In third-line H pylori treatment, infection was eradicated in 141 (88%, 83-93) of 160 patients in the molecular testing-guided therapy group and 139 (87%, 82-92) of 160 patients in the susceptibility testing-guided therapy group by intention-to-treat analysis (p=0·74). The difference in the eradication rate between the molecular testing-guided therapy group and the susceptibility testing-guided therapy group was -0·7% (95% CI -6·4 to 5·0; non-inferiority p=0·071) in trial 1 and 1·3% (-6·0 to 8·5; non-inferiority p=0·0018 in trial 2 by intention-to-treat analysis. We found no difference in adverse effects across both treatment groups in trial 1 and trial 2. Molecular testing-guided therapy was similar to susceptibility testing-guided therapy in first-line therapy and non-inferior to susceptibility testing guided therapy in third-line treatment of H pylori infection, supporting the use of molecular testing-guided therapy for H pylori eradication. Ministry of Science and Technology of Taiwan, and Centre of Precision Medicine of the Higher Education Sprout Project by the Ministry of Education of Taiwan.

Antibiograms of select anaerobic bacteria over 10 years

Abstract Antibiotics are critical complements to the immune response in combatting infection. Anaerobic bacteria are increasingly common causes of infection, and antibiotic resistance of anaerobes has been rising. Most laboratories do not perform antimicrobial susceptibility testing of anaerobes, and even fewer have compiled antibiogram data. Here we investigated antimicrobial susceptibility results of anaerobes from January 2012 to December 2021 obtained from inpatients at Mayo Clinic using reference agar dilution testing. Actinomyces (60–70 isolates annually), Bacteroides fragilis (30–80), Finegoldia magna (40–60), and Cutibacterium acnes (70–110) were included. Penicillin, clindamycin, metronidazole, ertapenem, piperacillin-tazobactam, and moxifloxacin were tested. Clinical and Laboratory Standards (CLSI) breakpoints were followed. B. fragilis showed ≥90% and Actinomyces 100% susceptibility to piperacillin-tazobactam and ertapenem. All F. magna and >95% B. fragilis were susceptible to metronidazole. The most striking fluctuations in percent susceptibility from year to year were observed for clindamycin, with Actinomyces ranging from 66–79%, B. fragilis 34–67%, and F. magna 47–85%. In the absence of local anaerobe antibiograms, providers may refer to such antibiogram data to guide empiric anti-anaerobic therapy at their institutions. In conclusion, anaerobe antibiotic susceptibility patterns must be monitored to ensure appropriate antibiotic empiric therapy and stewardship.

Comparison of in vitro fosfomycin susceptibility testing methods with agar dilution for carbapenem resistant Klebsiella pneumoniae and Escherichiacoli.

The emergence of Extensively drug resistant (XDR) pathogens like Carbapenem Resistant Klebsiella pneumoniae (CR Kpn) and Carbapenem Resistant Escherichia coli (CR Eco) has limited therapeutic options for treating them. Fosfomycin a broad-spectrum antibiotic, has emerged as a potential treatment option in combination with other agents. It is therefore important that accurate drug susceptibility testing (DST) results of fosfomycin should be available to all clinical microbiology laboratories. Agar dilution which is the recommended method for fosfomycin DST is not convenient to adopt in a routine set-up. This study aimed to determine the susceptibility pattern of CR Kpn and CR Eco to fosfomycin and to evaluate the discrepancies of the available manual MIC based alternative methods. Agar dilution (AD), broth microdilution (BMD), E-test and Ezy MIC test were performed on 235 CR-Kpn and Eco isolates respectively. Of 177 CR Kpn, 31.63% (n​=​56/177) of the isolates were susceptible by AD. Categorical Agreement (CA) by BMD, E-test and Ezy MIC were lower than the acceptable limit while Very Major Errors (VMEs) and Major Errors (MEs) were beyond the acceptable limits. In the case of CR Eco, 96.55% (n​=​56/58) were susceptible by AD. CA of 100% (n​=​58/58) was shown by both BMD and Ezy MIC while 86.20% (n​=​50/58) was shown by E-test, with no VME observed for CR Eco. ME was only observed for E-test method. The alternative methods were in poor agreement with AD method for CR Kpn and for CR Eco, BMD and Ezy MIC have shown reliable results.

A Coating Based on Bioactive Compounds from Streptomyces spp. and Chitosan Oligomers to Control Botrytis cinerea Preserves the Quality and Improves the Shelf Life of Table Grapes.

Botrytis cinerea is the most harmful postharvest disease of table grapes. Among the strategies that can be envisaged for its control, the use of coatings based on natural products is particularly promising. The study presented herein focuses on the assessment of the antagonistic capacity of two Streptomyces species and their culture filtrates against B. cinerea. Firstly, the secondary metabolites were characterized by gas chromatography-mass spectrometry, with N1-(4-hydroxybutyl)-N3-methylguanidine acetate and 2R,3S-9-[1,3,4-trihydroxy-2-butoxymethyl]guanine acetate as the main compounds produced by S. lavendofoliae DSM 40217; and cyclo(leucyloprolyl) and cyclo(phenylalanylprolyl) as the most abundant chemical species for S. rochei DSM 41729. Subsequently, the capacity of S. lavendofoliae DSM 40217 and S. rochei DSM 41729 to inhibit the growth of the pathogen was tested in dual culture plate assays, finding 85-90% inhibition. In agar dilution tests, their culture filtrates resulted in effective concentration values (EC90) in the 246-3013 μg·mL-1 range. Upon the formation of conjugate complexes with chitosan oligomers (COS) to improve solubility and bioavailability, a synergistic behavior was observed, resulting in lower EC90 values, ranging from 201 to 953 μg·mL-1. Ex situ tests carried out on 'Timpson' and 'Red Globe' table grapes using the conjugate complexes as coatings were found to maintain the turgor of the grapes and delay the appearance of the pathogen by 10-15 days at concentrations in the 750-1000 µg·mL-1 range. Hence, the conjugate complexes of COS and the selected Streptomyces spp. culture filtrates may be put forward as promising protection treatments for the sustainable control of gray mold.

Medium-chain fatty acid esters are effective even in azole-resistant Malassezia pachydermatis.

Malassezia (M.) pachydermatis as a frequent reason for dermatological consultation in dogs and cats was recently shown to be lipid-dependent, too. Lipolytic activity is a prerequisite for activating antimicrobial effectivity of fatty acid esters. It was therefore of interest whether it is possible to induce this mechanism in M. pachydermatis and to identify possible differences between minimal and strong lipid-dependent strains. In an agar dilution test, the minimal inhibitory concentrations of six fatty acid esters were determined for seventeen M. pachydermatis strains. GC analysis of parent compounds and liberated fatty acids was used to quantify ester cleavage. Hydrolysis was observed in all test strains in a homogenous manner but was dependent on the chemical structure. Lowest MICs (500 ppm after 14 days of incubation) were obtained applying glyceryl monocaprylate and 3-hydroxylpropyl caprylate, while the corresponding esters of undecylenic acid showed nearly twice the value. As shown by GC analysis with the reference strains CBS 1879 and CBS 1892 and 3-hydroxypropyl caprylate, hydrolysis and caprylic acid formation starts immediately and was dependent on yeast density. Furthermore, nine azole-resistant strains isolated from dogs with treatment failures showed MIC values comparable to the other strains and no resistance to monohydric fatty acid esters. Medium-chain fatty acid esters may represent a new therapeutic option for veterinary use even in azole-resistant strains. The in vivo verification in M. pachydermatis-associated dermatitis in dogs and cats will be the next step for the successful development of new therapeutics.

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.

Antimicrobial Resistance and Genetic Diversity of Campylobacter spp. Isolated from Broiler Chicken at Three Levels of the Poultry Production Chain in Costa Rica

Campylobacter spp. are considered the most common bacterial cause of human gastroenteritis, one of the four main causes of diarrheal disease worldwide, and they are one of the main foodborne pathogens causing hospitalizations and deaths. Here, 148 strains of Campylobacter spp. isolated from poultry at farms, processing plants, and retail stores in Costa Rica were examined for resistance to six antibiotics. An agar dilution test was used to determine the MIC and susceptibility profiles against doxycycline, ciprofloxacin, nalidixic acid, enrofloxacin, chloramphenicol, and erythromycin. In addition, a pulsed-field gel electrophoresis analysis was carried out to determine the genotype relatedness of a representative subset of the isolates. Approximately 136 (92%) of the 148 analyzed isolates showed resistance to the tested drugs. Nalidixic acid, ciprofloxacin, and enrofloxacin were the antibiotics for which resistance occurred most frequently (91.2, 85.8, and 85.8%, respectively), followed by doxycycline (25.0%), chloramphenicol (5.4%), and erythromycin (2.7%). The profile conferring only resistance to quinolones was the most frequently found, and only 2.0% of the isolates showed resistance to quinolones and macrolides simultaneously. Results showed a high frequency of resistant Campylobacter spp. strains and evidenced the distribution, selection, and circulation of resistant strains along the poultry chain from farms to consumers. Cross-contamination and resistance seem to play important roles in the dissemination of these strains at specific points of the poultry chain, even when control measures are being taken. The establishment of effective surveillance and control strategies represents an essential tool for foodborne diseases mitigation. The rational use of antibiotics, especially those still showing efficacy, should be a priority in both human and veterinary medicine to contain the progress of this phenomenon and its consequences.

Enterobacterales Üyelerinde Nadir Bir Plazmid Aracılı A Sınıfı Beta Laktamaz Olan IBC-1'in Araştırılması

Objective: The aim of the study was to investigate the presence of extended -spectrum beta-lactamases (ESBL) TEM, SHV, and CTX-M which are frequently observed all over the world, and the rare IBC-1 beta-lactamase enzyme. Material and Method: Thirty strains that isolated from various clinical samples from inpatient at Marmara University Hospital, which were phenotypically positive for ESBL, and IBC-1were included in the study. Antimicrobial susceptibility tests were performed both by disk diffusion and agar dilution tests. E-test and double-disc synergy method (DDS) were used for phenotypic detection of ESBLs. The presence of ESBL genes was detected by polymerase chain reaction (PCR). Results: All strains were susceptible to imipenem while ampicillin, amoxicillin-clavulanic acid, piperacillin, ceftazidime, and trimethoprim-sulfamethoxazole were resistant. While 4 strains were unidentified, 26 strains were detected as ESBL positive by E-test. Two strains were phenotypically positive for IBC-1 with DDS, while 5 strains were identified as doubtful. The rate of carrying the blaTEM, blaSHV, and blaCTX-M genes of strains was 73.3%, 60%, and 56.6%, respectively. The blaIBC gene was not detected in any of the strains. Conclusion: While the IBC-1 enzyme, which was first detected in Greece, have not caused a threat to our country yet, the rate of TEM, SHV, and CTX-M enzymes were found to be quite high.

Pattern of Esbls in Uro-Pathogens Obtained from a Nigerian Tertiary Hospital

Background: Multidrug resistance remains a challenge in the treatment of Urinary Tract Infections (UTI) in Nigeria, a key factor being the occurrence of ESBL producers. Earlier reports have emphasized the occurrence of major ESBLs, little is known about the minor subtypes' occurrence in this regard.Objective: This study sought to evaluate the occurrence of major and minor ESβL producers among a cohort of uropathogens collected from a Nigerian Teaching Hospital using molecular techniques.Material and Methods: Cultures from 1000 UTI positive urine specimens were collected from the hospital laboratory between May 2015 and December 2017. All samples were subjected to standard isolation culturing techniques and identified. They were further tested for susceptibility to 8 antibiotics. Of these, gram-negative isolates with presumptive ESBL production were evaluated for confirmatory ESBL production using Chromogenic ESBL agar (Oxoid) and Agar Dilution tests. One hundred and twenty-five of them were evaluated for the presence of six ESBL genes (TEM, SHV, CTX-M 15, PER, GES, VEB) using Multiplex PCR/Agarose Gel Electrophoresis.Results: The results revealed that 40 out of 97 (41%) detected ESBL genes were of the ESBL minor category (VEB, PER, and GES). These ESBL producers were also observed to be resistant to at least five of the 8 antibiotics tested.Conclusion: More attention should be paid to the emergence minor ESBL producers among uropathogens in this environment as they represent a potential underlying influence on the observed treatment failure in the treatment of UTI. 

In-vitro potential of crude extracts of selected garden herbs for mastitis management in Zambia

Introduction: The advent of bovine mastitis associated antimicrobial resistance has necessitated development of alternative herbal remedies. In this study, we tested, in-vitro, the antimicrobial efficacy of three crude extracts: (1) aqueous, (2) ethanol and (3) ethyl acetate extracts of Zambian cultivated Aloe barbadensis Miller (Aloe vera) and Curcuma longa (Turmeric) and also made comparisons to synthetic anti-mastitis formulations. Methods: The active compounds of these herbs were obtained as crude extracts using water, ethanol and ethyl acetate as solvents. The test microbes comprised of ten standard controls including Escherichia coli and Pseudomonas aeruginosa and 84 field isolates (all gram positive bacteria) from clinical and subclinical mastitis milk samples from selected districts of the Southern Province of Zambia belonging to the genera Staphylococcus, Streptococcus, Bacillus and Lactobacillus. The agar dilution method was used to determine the antimicrobial activity by observing the sensitivity which was graded as sensitive, intermediate or resistant. Commercial synthetic anti-mastitis preparations served as positive controls and their sensitivity was compared to that of the herbal preparations. The Minimum Inhibitory Concentrations (MIC) of the herbal formulation was also determined using two-fold serial dilutions. Results: The agar dilution test results of the field isolates indicated 18.8% resistance, 25.3% intermediate and 56% sensitivity for the herbal extracts, while the synthetic anti-mastitis preparation averaged 16.8% resistance, 4.75% intermediate and 78.5% sensitivity. The gram negative controls tested were generally resistant. On average, the agar dilution trials revealed; 41.8% resistance, 13% intermediate and 45.3% sensitivity for the herbal formulation while the synthetic antimicrobials produced 23.5% resistance, 5.2% intermediate and 71.3% sensitivity. Mean MIC was 11.5mg/ml at the 5% serial dilution for the herbal extract compared to the Cephalexin (semi-synthetic antimicrobial) MIC for S. aureus at 0.5mg/L or 0.0005mg/ml. Conclusion: The herbs Aloe vera and Curcuma longa from Zambia showed antimicrobial efficacy on bovine mastitis causing microbes and may be used as a cheaper alternative to synthetic anti-mastitis preparations and as an aid to fight antimicrobial resistance to the benefit of the most affected and resource limited small-scale dairy farmers.