Determination Of Minimum Inhibitory Concentration Research Articles

Genotypic to Phenotypic Resistance Discrepancies Identified Involving β-Lactamase Genes, blaKPC, blaIMP, blaNDM-1, and blaVIM in Uropathogenic Klebsiella pneumoniae.

IntroductionKlebsiella pneumoniae carbapenemase (KPC) belongs to the Group-A β-lactamases that incorporate serine at their active site and hydrolyze various penicillins, cephalosporins, and carbapenems. Metallo-beta-lactamases (MBLs) are group-B enzymes that contain one or two essential zinc ions in the active sites and hydrolyze almost all clinically available β-lactam antibiotics. Klebsiella pneumoniae remains the pathogen with the most antimicrobial resistance to KPC and MBLs.MethodsThis research investigated the blaKPC, and MBL genes, namely, blaIMP, blaVIM, and blaNDM-1 and their phenotypic resistance to K. pneumoniae isolated from urinary tract infections (UTI) in Bangladesh. Isolated UTI K. pneumoniae were identified by API-20E and 16s rDNA gene analysis. Their phenotypic antimicrobial resistance was examined by the Kirby-Bauer disc diffusion method, followed by minimal inhibitory concentration (MIC) determination. blaKPC, blaIMP, blaNDM-1, and blaVIM genes were evaluated by polymerase chain reactions (PCR) and confirmed by sequencing.ResultsFifty-eight K. pneumoniae were identified from 142 acute UTI cases. Their phenotypic resistance to amoxycillin-clavulanic acid, cephalexin, cefuroxime, ceftriaxone, and imipenem were 98.3%, 100%, 96.5%, 91.4%, 75.1%, respectively. Over half (31/58) of the isolates contained either blaKPC or one of the MBL genes. Individual prevalence of blaKPC, blaIMP, blaNDM-1, and blaVIM were 15.5% (9), 10.3% (6), 22.4% (13), and 19% (11), respectively. Of these, eight isolates (25.8%, 8/31) were found to have two genes in four different combinations. The co-existence of the ESBL genes generated more resistance than each one individually. Some isolates appeared phenotypically susceptible to imipenem in the presence of blaKPC, blaIMP, blaVIM, and blaNDM-1 genes, singly or in combination.ConclusionThe discrepancy of genotype and phenotype resistance has significant consequences for clinical bacteriology, precision in diagnosis, the prudent selection of antimicrobials, and rational prescribing. Heterogeneous phenotypes of antimicrobial susceptibility testing should be taken seriously to avoid inappropriate diagnostic and therapeutic decisions.

QUANTITATIVE ANALYSIS OF GYRA AND GYRB KNOWN MUTATIONS IN DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS TREATED WITH OFLOXACIN

Objective: The aim of the study is to measure the minimum inhibitory concentrations (MICs) of ofloxacin antibiotic from gyrA and gyrB mutations present in fluoroquinolones (FQs) resistant strains of Mycobacterium tuberculosis (MTB) and to further concentrate the potential association between gene mutations and phenotypic resistance based on their MICs.
 Methods: Different levels of ofloxacin MICs levels were detected in 31 archived multi drug-resistant MTB isolates showing gyrA mutations in codon at A90V, S91P, D94A, D94N/Y, D94 G, and D94H and two gyrB probes (N538D and E540V). The MIC determinations were made using the 1% proportion method.
 Results: Genotypic assay detected 32 mutations in the gyrA (n=29) and gyrB (n=3) genes among the 31 FQs resistant isolates. Most frequently seen in gyrA mutations at codon D94G (n=16; 50%), these mutations had a clearly elevated MIC level from 2 to 16 μg/ml, that was phenotypically resistant. The A90V mutation region consistently exhibited the lowest levels of ofloxacin resistance, with three out of eight (37.50%) of these isolates had a MIC of <2 μg/ml. In addition, a further strain of S91P mutations for MIC was determined to be less than the critical concentration (CC). These low levels of resistance have been detected in a phenotypic manner which is noticeable in the study. Furthermore, fewer mutations in codons at D94A, D94N/Y were identified. Three wild-type absent isolates from gyrB QRDR were identified and the MIC of those strains for ofloxacin was lower than the critical cutoff value.
 Conclusion: Based on the results, it is shown that different resistance mutations were associated with different levels of MICs and the current concentration for MGIT will be lowered from 2 μg/ml to 1 μg/ml for the ofloxacin drug.

Encapsulation-based a novel antibiotic selection technique for Agrobacterium-mediated genetic transformation of Dendrobium Broga Giant orchid

Dendrobium Broga Giant (Dendrobium Bobby Messina × Dendrobium superbiens) is a new hybrid orchid in Malaysia. This study has established encapsulation based a novel antibiotic selection technique to determine the minimal inhibitory concentration of potential antibiotic for Agrobacterium-mediated transformation of Dendrobium Broga Giant (DBG) orchid. For determination of minimal inhibitory concentration, two steps of antibiotic selection techniques were used in this experiment such as potential antibiotic was selected before encapsulation and minimal inhibitory concentration was determined after encapsulation. The effectiveness of three antibiotics (kanamycin, geneticin and neomycin) with their different concentrations were evaluated as a selection agent for transformation studies. The effectiveness was determined by identifying the minimal concentration of the selection agent required to fully inhibit the growth of Dendrobium Broga Giant orchid PLBs. Non transformed PLBs were cultured on hormone-free ½MS selection medium containing varying concentrations (0–300 mg/l) of the antibiotics. The growth of PLBs were recorded every week up to four weeks. Among the selection agents evaluated, geneticin proved to be the most effective antibiotic which could inhibit the growth of PLBs at 50 mg/l. Neomycin requires 250 mg/l for inhibition of all PLBs after four weeks of cultured but Kanamycin is the least effective as it only inhibits 80% of the PLBs grown at 300 mg/l. For stringent minimal inhibitory concentration of antibiotic selection, encapsulated PLBs were cultured on similar concentrations of geneticin containing ½MS selection media and growth of PLBs evaluated every week up to four weeks of cultured. It was observed that 50 mg/l geneticin fully inhibited PLBs after two weeks. Hence, encapsulation based novel antibiotic selection techniques revealed that geneticin is the potential antibiotics and 50 mg/l concentration is the minimal inhibitory concentration for Dendrobium Broga Giant orchid PLBs.

Detecting In-Vitro Colistin Resistance- A Comparative Study Between Broth Microdilution Versus Vitek-2 For Colistin Susceptibility Testing

Background: Susceptibility testing for polymyxins is a great challenge for a Clinical Microbiology laboratory. There are several methodological issues associated with MIC (Minimum Inhibitory Concentration) determination of colistin.
 Methods: In our study, we have compared the results of colistin susceptibility testing by Automated system (Vitek-2, Biomerieux, France) with the reference Broth Microdilution method (BMD) to identify the type of discrepancies by Vitek-2 method and thus develop a practical and accurate approach for colistin susceptibility testing in a Clinical Microbiology laboratory. A total of 730 strains of Gram negative bacteria [Escherichia coli (325), Klebsiella sp.(346), Acinetobacter baumanii complex (37) and Pseudomonas aeruginosa (22)] from 485 patents were tested simultaneously by BMD and Vitek-2 method for colistin susceptibility testing.
 Results: The Essential agreement (EA), Categorical agreement (CA), Very major error (VME) and Major error (ME) rates for Klebsiella sp. were 87.3%, 89.3%, 8% and 2.3% respectively, for Escherichia coli were 88.3%, 89.5%, 9.2% and 1.2% respectively, for Acinetobacter baumannii complex were 89.1%, 91.8%, 8.1% and 0% respectively, for Pseudomonas aeruginosa were 68.1%, 72.7%, 0% and 27.2% respectively.
 Conclusions: Colistin susceptibility testing by Vitek-2 method is an easily adoptable method and the results of Vitek-2 with reference to BMD are acceptable to a great extent in Klebsiella sp., Escherichia coli and Acinetobacter baumanii complex. So, we believe that Vitek-2 method may be used for colistin susceptibility testing in low risk patients. However, BMD should be used in high risk immunosupressed and immunocompromised patients who are admitted in critical care units. For Pseudomonas aeruginosa, BMD should be routinely used.

Antibacterial Activity Evaluation of Psidium guajava L. (Myrtaceae) Crude Extracts Against Selected Bacterial Pathogens

Methanol, ethanol, acetone, ethyl acetate and hot water extracts of different parts (leaves, seeds, fruits and twigs fractions) of Psidium guajava L. were investigated for their antibacterial activities against 8 bacterial isolates. Inhibitory effects of P. guajava L. extracts have been screened by disc- diffusion method (Zone of inhibition, ZI), activity index (AI) and minimum inhibitory concentration (MIC) determination. Ciprofloxacin antibiotic was used as standard for P. guajava L. antimicrobial activity comparison. From the ZI, AI and MIC values, methanolic and hot water extracts of twigs < 1cm diameter were the most potent against all tested micro-organisms by showing the highest ZI and AI value and the lowest MIC values. Whereas, no inhibitory activity was recorded for both seeds and fruits extracts using all tested solvents. These observations make this plant a potential source that can be used in management of bacterial infections. Moreover, methanolic and hot water extracts of twigs < 1cm required further in depth study.
 
 

Natural antioxidant pterostilbene as an effective antibiofilm agent, particularly for gram-positive cocci.

Pterostilbene (PTE), a dimethylated analogue of resveratrol, mostly contained in Vitis vinifera leaves or in other plant sources is well-known for its antioxidant activity. Due to its bioavailability, low hydrophilicity and thus ability to penetrate hydrophobic biological membranes it was found to be an antimicrobial agent. These properties of PTE offer the possibility of its use in the treatment of microbial infections. The emergence of antibiotic resistance of microorganisms is often caused by their ability to form biofilm; new substances with antibiofilm activity are therefore sought. The representatives of opportunistic pathogenic gram-positive and gram-negative bacteria as well as fungi were used for the determination of minimum inhibitory concentrations (MIC50 and MIC80), minimum biofilm inhibitory concentrations (MBIC50 and MBIC80) and minimum biofilm eradication concentrations (MBEC50 and MBEC80) of PTE and commonly used antibiotics erythromycin, polymyxin B or antimycotic amphotericin B. Total biofilm biomass was investigated by crystal violet staining, and the results were confirmed using microscopic techniques. The most significant antibiofilm action was proved for gram-positive cocci, e.g., MBEC50 of PTE for all strains of Staphylococcus epidermidis tested was 25mg/L. By contrast, the antibiotic ERM did not exhibit antibiofilm activity in most cases. The permeabilization of cell membranes of gram-positive cocci biofilm by MBIC50 and MBEC50 of PTE was confirmed by LIVE/DEAD staining using spinning disc confocal microscopy. PTE significantly influenced the ability of gram-positive cocci to form biofilm and it effectively eradicated pre-formed biofilm in vitro; its potential for the treatment of biofilm-associated infections of Staphylococcus spp. or Enterococcus faecalis is thus apparent.

Specific gyrA Gene Mutations Correlate with High Prevalence of Discordant Levofloxacin Resistance in Mycobacterium tuberculosis Isolates from Beijing, China

Although rapid, highly sensitive molecular diagnostics tests are useful for diagnosing fluoroquinolone-resistant tuberculosis (TB), results of molecular testing versus conventional sequential phenotypic drug susceptibility testing (pDST) are frequently discordant. This article determined the discordance rate of levofloxacin (LFX) resistance results, obtained via MeltPro TB molecular testing, versus pDST of clinical TB isolates collected in Beijing, China, between January and December 2018. Isolates with discordant results were further subjected to LFX minimal inhibitory concentration (MIC) determinations and DNA sequence analysis to explore causes of discordance. Of 571 total TB cases, 126 (22.1%) were identified as LFX resistant using the MeltPro TB assay. However, 34 of these 126 LFX-resistant isolates yielded LFX-susceptible test results using pDST, for an overall discordance rate of 27.0%. LFX MICs mainly clustered around the critical LFX concentration, with 7 (21.2%) and 13 (39.4%) of isolates exhibiting MICs of 2.0 and 4.0 mg/L, respectively. The most prevalent LFX resistance mutations associated with discordant results were involved in DNA gyrase subunit A amino acid substitutions Ala90Val (13, 39.4%) and Asp94Ala (11, 33.3%). Notably, more than one-quarter of isolates deemed LFX resistant via the MeltPro assay were scored as LFX susceptible on the basis of pDST results. Ultimately, highly discordant LFX-resistance test results were associated with specific gyrA mutations in isolates with MICs approaching the critical LFX concentration.

The gastroprotective potential of silibinin against Helicobacter pylori infection and gastric tumor cells

AimsSilibinin is the major component of flavonolignans complex mixture (Silymarin), which is obtained from Silybum marianum (L.) Gaertn. Despite several reports about silibinin, little is known about its effects on gastric diseases. Then, the present study aims to evaluate the silibinin effect against Helicobacter pylori infection, gastric tumor cells and immunomodulation. Main methodsThe anti-H. pylori effect was performed on 43504 and 43629 strains by minimum inhibitory concentration (MIC) determination, observing morphological alterations by scanning electron microscopy and in silico evaluation by molecular docking. Immunomodulatory activity (Interleukins-6 and 10, TNF-α and NO inhibition) was determined in H. pylori-stimulated macrophages and the cytotoxic activity on gastric adenocarcinoma cells prior and after metabolization by S9 fraction. Key findingsSilibinin showed anti-H. pylori activity with MIC of 256 μg/mL, promoted important morphological changes in the bacterial cell wall, as blebs and clusters, suggesting interaction with Penicillin Binding Protein (PBP) subunits. Immunomodulatory potential was observed at 50 μg/mL with the inhibition of produced cytokines and NO by H. pylori-stimulated macrophages of 100% for TNF-ɑ, 56.83% for IL-6, and 70.29% for IL-10 and 73.33% for NO. Moreover, silibinin demonstrated significant cytotoxic activity on adenocarcinoma cells (CI50: 60.17 ± 0.95 μg/mL) with a higher selectivity index (SI: 1.52) compared to cisplatin. After metabolization silibinin showed an increase of cytotoxicity with a CI50 six-fold decrease (10.46 ± 0.25). SignificanceThe use of silibinin may become an important alternative tool in the prevention and treatment of H. pylori infection and, consequently, in gastric cancer.

Anti-Bacterial Effects of Essential Oils against Uropathogenic Bacteria.

Given the increasing antimicrobial resistance in urinary tract infections (UTI), alternative strategies need to be investigated. Determination of minimal inhibitory and bactericidal concentrations of essential oils from cajeput, lemongrass, tea tree, and thyme in artificial urine, revealed bactericidal activity of all four tested essential oils against seven uropathogenic species with values ranging between 0.78–50 mg/mL. Tea tree and thyme essential oils were more efficient than lemongrass and cajeput. In addition, antibiotic-resistant strains showed similar susceptibility as antibiotic-sensitive strains, suggesting no cross-resistance between antibiotics and these essential oils. Checkerboard assays revealed a synergistic activity of the combination of thyme and tea tree. Furthermore, the combination with thyme and tea tree essential oils increased the activity of fosfomycin and pivmecillinam, but not nitrofurantoin, against Escherichia coli. This study provides a basis for further investigation of the potential of thyme and tea tree oil as an alternative or additional treatment of UTI.

Chemical composition and antibacterial effect of Smyrnium olusatrum L. Fruit Essential Oil

The plants of the <em>Apiaceae</em> family are mainly used for food purposes and their multiple therapeutic properties and biological activities. This study aims to determine the chemical composition of Essential Oil (EO) of the fruits of <em>Smyrnium olusatrum</em> L. obtained by hydrodistillation in Clevenger-type apparatus. GC/MS analyzed the EO and the antibacterial activity was evaluated against clinical bacterial strains by two complementary methods (Disc diffusion and micro-atmosphere) and determination of Minimal Inhibitory Concentration (MIC). The results of chemical composition show that the EO is dominated by hydrocarbon monoterpenes (55,95%). The antibacterial effect shows that Gram-positive bacteria are the most sensitive to the inhibitory action of EO with inhibition zone diameters (DZI) ranging from 18 to 20 mm for <em>Staphylococcus aureus</em> and <em>Streptococcus faecium</em> strains. The same results were reported with the micro-atmosphere

Combined effects of Allium sativum and Cuminum cyminum essential oils on planktonic and biofilm forms of Salmonella typhimurium isolates.

Sa lmonella typhimurium (S. typhimurium) represents an important global public health problem and has the ability to survive under desiccation conditions in foods and food processing facilities for years. The aim of this study was to investigate the effects of Allium sativum (A. sativum) and Cuminum cyminum (C. cyminum) essential oils (EOs) against planktonic growth, biofilm formation and quorum sensing (QS) of S. Typhimurium isolates, the strong biofilm producers. The major components of EOs were determined by gas chromatography-mass spectrometry (GC-MS). Biofilm formation of S. Typhimurium isolates was measured by crystal violet staining. Then, the effects of the EOs on the planktonic cell growth (using determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)), measurement of the synergistic effects of EOs (using checkerboard method), biofilm formation (using microtiter-plate test and scanning electron microscope (SEM)), and expression of QS and cellulose synthesis genes (using quantitative real-time PCR) were assessed. Finally, tetrazolium-based colorimetric (MTT) assay was used to examine EOs cytotoxicity on the Vero cell line. GC-MS analysis showed that terpineol, carene and pinene in C. cyminum EO and sulfur compounds in A. sativum EO were the major components of the plant extract. The Geometric mean of MIC values of the A. sativum and C. cyminum were 0.66 and 2.62 μLmL-1, respectively. The geometric means of the fractional inhibitory concentration index (FICi) for both EOs were calculated as 1.05. The qPCR results showed that MIC/2 concentrations of both EOs significantly down-regulated of QS (sdiA and luxS) and cellulose synthesis (csgD and adrA) genes. Scanning electron microscopy showed the EOs reduced the amount of S. Typhimurium mature biofilm. In general, we showed that C. cyminum and A. sativum EOs can be considered as the potential agents against planktonic and biofilm form of S. Typhimurium without any concern of cytotoxic effect at 4 MIC concentrations on the eukaryotic Vero cells.

Activity of Specialized Biomolecules against Gram-Positive and Gram-Negative Bacteria.

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 μg/mL) to induce the same effects as the AMPs (500–7.8 μg/mL) or EOs (365.2–19.7 μg/mL). Pexiganan and CLO were the most effective biomolecules, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5–7.8 μg/mL and 39.3–19.7 μg/mL, respectively), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

Antimicrobial Activity of Some Novel 1-(3-(4-Chlorophenylimino)-3,4- dihydroquinoxalin-2-yl)-3-substituted phenyl-1H-pyrazole-4-carbaldehyde

Background: In this study, synthesis of some novel 1-(3-(4-chlorophenylimino)-3,4- dihydroquinoxalin-2-yl)-3-substituted phenyl-1H-pyrazole-4-carbaldehyde were done by cyclization of dehydrated hemiketal using VilsmeierHaack reaction. The structures of synthesized compounds were in accordance of the basis of IR, 1HNMR, Mass spectral data and elemental analyses. The synthesized compounds had given good yields and high purity. Methods: The antimicrobial activity of the synthesized compounds was preliminarily screened by paper disc diffusion technique. The determination of minimum inhibitory concentrations (MIC) of the synthesized compounds were done by using tube dilution method. Results and Conclusion: All the screened compounds showed antibacterial and antifungal activity against selected strains of Gram positive and Gram negative bacteria and two strains of fungus, respectively but exhibited considerably less activity when compared to the reference drug ciprofloxacin and Fluconazole for antibacterial and antifungal activity, respectively. Among the synthesized compounds VV5 exhibited significant activity.

Cyclo(L-Pro-L-Tyr) from Streptomyces sp. 150: Exploiting in vitro Potential in Controlling Foodborne Pathogens and Phytopathogens

Background: In the prokaryotic unicellular bacteria, Streptomyces species are the most frequent producers of bioactive secondary metabolites. Our continuous quest for new antibiotics from Actinomycetes genera put us forward for isolation of a strain Streptomyces sp. 150 from the soil samples collected at the Daegu University premises in Korea. Objective: The aims of this study was to isolate and identify bioactive compounds from the isolated microorganism and assess the efficacy of the compounds in controlling foodborne pathogens and phytopathogens. Methods: The isolated bacterium was characterized by the taxonomic analyses and a compound was isolated from the fermentation broth by applying different chromatographic techniques e.g. column chromatography, TLC and PTLC. The structure of the compound was established by UV, IR, 1H-NMR and 13C-NMR spectral data analyses. The antibacterial and antifungal efficacy of the compound was assessed by disc diffusion assay, poisoned food technique, MIC determination and SEM analysis. Results: Different chromatographic techniques resulted in isolation and purification of a secondary metabolite from the fermentation broth of Streptomyces sp. 150. The analyses of the spectroscopic data identified the compound as cyclo(L-Pro-L-Tyr). The compound exhibited potential efficacy in controlling all the seven foodborne pathogenic bacteria with corresponding inhibition zone and minimum inhibitory concentration (MIC) ranging from 15.1 to 20.1 mm and 15.6 to 62.5 μg/mL respectively, and tested phytopathogenic fungi with mycelium growth inhibition varying from 57.1 to 68.5% and MIC from 125 to 250 μg/mL. Moreover, in scanning electron microscopy, morphological changes in Listeria monocytogenes ATCC 19166 at MIC dose of compound was observed. Conclusion: This study demonstrated the possibility to use the compound cyclo(L-Pro-L-Tyr) in food and agrochemical industries to control foodborne pathogens and phytopathogens.

Detection of Colistin Resistance in Salmonella enterica Using MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer.

Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 h) and challenging to perform in clinical and veterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.

Study of Plasmid-Mediated Quinolone Resistance in Escherichia coli from Nosocomial Urinary Tract Infections.

The aim of the present study was to study the prevalence of plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac) in Escherichia coli (E. coli) isolated from patients with nosocomial urinary tract infections (UTIs) and its relation to the extended-spectrum β-lactamase (ESBL) production. ; Methods: A cross-sectional study was carried out on 200 non-duplicated isolates of E. coli isolated from patients with nosocomial UTIs. E.coli isolates were subjected to antibiotic susceptibility testing by disc diffusion method, determination of minimum inhibitory concentrations (MICs) of ciprofloxacin by Epsillometer (E) test strips, detection of ESBL production by double disc synergy method and detection of qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac genes by polymerase chain reaction (PCR). ; Results: The antimicrobial susceptibility testing of the isolated E. coli revealed a high frequency of resistance to ampicillin (73.5%), ceftazidime (72%) and imipenem (71.5%). The less frequent resistance was for aztreonam (21.5%), amikacin (36.5%) and gentamicin (38.5%). ESBL production was found in 131 isolates (65.5%) and phenotypic quinolone resistance was detected by MIC in 65 isolates (32.5%), with 52.3% of them showed high resistance to ciprofloxacin with an MIC more than 32 μg/ml. PMQR genes were found in 40 isolates. The frequency of the detected genes was 40%, 37.5%, 35%, 20% and 5% for qnrA, qnrS, qepA, qnrB and oqxA, respectively. Significant association was found between the presence of PMQR genes and ESBL production (P=0.0001). ; Conclusion: The study highlights the prevalence of PMQR genes in E. coli with high association with the ESBL phenotype. This finding is a sign of limited therapeutic options for E. coli.

Chemical Separation and Antibacterial Activity of Nutmeg seed Essential Oil against Shigella sp. and Escherichia coli ATCC 25922

The essential oil of the nutmeg seeds has antibacterial activity and contains some major compounds: sabinene, α-pinene, 2-β-pinene, myristicin, safrole, and 1-4-terpineol. This essential oil was extracted from the nutmeg seeds by separation process using the distillation method. The purpose of this study is to separate compounds in essential oils of nutmeg seed and to evaluate the antibacterial activity against two bacterial species. Nutmeg essential oils compound separation was conducted by distillation at 95 mmHg with different temperature 408, 428, 438, and 503 K. The separation results analyzed physical properties, chemical compounds with GC-MS, and the antibacterial activity test of Shigella sp. and Escherichia coli ATCC 25922 using dilution and diffusion method. The samples antibacterial activity were evaluated by the determination of minimum inhibitory concentration. The GC-MS analysis of nutmeg essential oils shows the presence of 25 compounds. Four fractions of fractional distillation under reduced pressure have 57.12%; 17.00%; 1.40%; and 19.68% of yield with different physical properties as well as the number of different compounds in each fraction. Fraction 1 has 14 compounds with α-pinene (27, 44%), sabinene (23,78%), dan 2-β-pinene ( 18,29%) as major compound. Fraction 2 has 15 compounds with sabinene (16,18%), 2-β-pinene (14,27%), β-phellandrene (12,99%), dan γ-terpinene (14,48%) as major compound. Fraction 3 has 12 compounds with α-terpinene (11,24%), para-cymene (23,74%), limonene (13,23%), γ-terpinene (23,46%), dan α-terpinolene (15,01%) as major compound. Fraction 4 has 12 compounds with 1-4-terpineol (40,65%), safrol (20,34%) danmyristicin (16,06%) as major compound. Results showed that MIC and MBC on Shigella sp. nutmeg essential oils had 12.11mm inhibitory effects at 1.25%; fraction 2 has 5.88mm inhibitory effects at 2.5%, and fraction 4 has 9.67mm inhibitory effects at 1.25%. MIC and MBC in Escherichia coli ATCC 25922 nutmeg essential oils have 10.89mm inhibitory effects at 2.5%; fraction one has 10.22mm inhibitory effects at 10%, and fraction 4 has 10.11mm inhibitory effects at 2.5%.

ANTIFUNGAL ACTIVITY SCREENING OF ANTARCTIC ACTINOBACTERIA AGAINST PHYTOPATHOGENIC FUNGI

The extreme weather conditions in the Antarctic have exerted selective pressures favoring differential features in bacteria to survive this untapped environment (i.e., antibiotic molecules). Notably, higher chances of antibiotic discovery from extremophiles have been proposed recently. Althoughnew organic and environmentally friendly sources for helping in the control of plant pathogenic fungi are necessary, the information about anti-phytopathogenic applications of extremophile microorganisms from untapped environments is limited. In this study, we determined the antifungal effect of actinobacterial strains isolated from Antarctic soils and sediments. Co-culture inhibition assays and Minimum Inhibitory Concentration (MIC) determination revealed that all Antarctic strains (x28) can inhibit the growth of at least one phytopathogenic fungi including Fusarium oxysporum,Rhizoctonia solani,Botrytissp. and Phytophthora infestans. Additionally, new novel antagonistic relationships are reported. Our work establishes a precedent on Antarctic actinobacteria strains with the capacity to produce antifungal compounds, and its potential for developing new fungicides or biocontrol agents solving current agriculture problems.

AKTIVITAS ANTIBAKTERI EKSTRAK KULTUR JAMUR ENDOFIT Fusarium sp. CSP-4 YANG DIISOLASI DARI Curcuma sumatrana Miq.

Endophytic fungus Fusarium sp. CSP-4 was isolated from the rhizome of Curcuma sumatrana Miq. The ethyl acetate extract of CSP-4 was fractionated by chromatographic column with silica as a stationary phase. The fractions were tested for antibacterial activity against S.aureus and E.coli by Thin Layer Chromatography (TLC)- bioautography. Determination of Minimum Inhibitory Concentration (MIC) was carried out by the microdilution method on a 96-microwell plate with the concentrations from 2 to 256 µg/ml. The results showed that fraction 15 and 11 were able to inhibit bacterial growth with the MIC values of 256 and> 256 μg / mL against S.aureus and>256 μg / mL against E.coli, respectively.The antibacterial activity of fractions 15 and 11 was weaker compared to the positive control of chloramphenicol with the MIC value of 4 µg/mL against S.aureus and E.coli. Based on the results obtained showed that the fractions of the methanol extract of endophytic fungus Fusarium sp. CSP-4 have weak antibacterial activity.

In Vitro and In Vivo Antifungal Activity of Clove (Eugenia caryophyllata) and Pepper (Piper nigrum L.) Essential Oils and Functional Extracts Against Fusarium oxysporum and Aspergillus niger in Tomato (Solanum lycopersicum L.).

In this study, hydrodistillation was used to obtain essential oils (EOs) from pepper (Piper nigrum L.) and clove (Eugenia caryophyllata) and co-hydrodistillation (addition of fatty acid ethyl esters as extraction co-solvents) was used to obtain functional extracts (FEs). Antifungal activity of EOs and FEs was evaluated by determination of minimum inhibitory concentration (MIC) against Fusarium oxysporum and Aspergillus niger. The results showed that pepper (Piper nigrum) and clove (Eugenia caryophyllata) essential oils and their functional extracts are effective in vitro at concentrations from 400 to 500 ppm after 10 days of culturing. The essential oils and functional extracts were used on tomato fruit samples at three different concentrations: 350, 400, and 450 ppm5. Clove essential oil reduced the growth of Aspergillus niger from 50% to 70% and Fusarium oxysporum to 40%. The functional extracts (FEs) of clove and pepper, mixed with ethyl decanoate (FEs-C10), were the best combination for protecting the tomato fruit in vivo against both phytopathogenic fungi. Gas chromatography-mass spectrometry (GC-MS) was used to identify eugenol as the principal compound in clove oil and limonene, sabinene, and β-caryophyllene in pepper oil.