Minimum Inhibitory Concentration Levels Research Articles

Antimicrobial and Antibiofilm Effects of Combinatorial Treatment Formulations of Anti-Inflammatory Drugs-Common Antibiotics against Pathogenic Bacteria.

With the spread of multi-drug-resistant (MDR) bacteria and the lack of effective antibiotics to treat them, developing new therapeutic methods and strategies is essential. In this study, we evaluated the antibacterial and antibiofilm activity of different formulations composed of ibuprofen (IBP), acetylsalicylic acid (ASA), and dexamethasone sodium phosphate (DXP) in combination with ciprofloxacin (CIP), gentamicin (GEN), cefepime (FEP), imipenem (IPM), and meropenem (MEM) on clinical isolates of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) as well as the transcription levels of biofilm-associated genes in the presence of sub-MICs of IBP, ASA, and DXP. The minimal inhibitory concentrations (MICs), minimal biofilm inhibitory concentrations (MBICs), and minimum biofilm eradication concentrations (MBECs) of CIP, GEN, FEP, IPM, and MEM with/without sub-MICs of IBP (200 µg/mL), ASA (200 µg/mL), and DXP (500 µg/mL) for the clinical isolates were determined by the microbroth dilution method. Quantitative real-time-PCR (qPCR) was used to determine the expression levels of biofilm-related genes, including icaA in S. aureus and algD in P. aeruginosa at sub-MICs of IBP, ASA, and DXP. All S. aureus isolates were methicillin-resistant S. aureus (MRSA), and all P. aeruginosa were resistant to carbapenems. IBP decreased the levels of MIC, MBIC, and MBEC for all antibiotic agents in both clinical isolates, except for FEP among P. aeruginosa isolates. In MRSA isolates, ASA decreased the MICs of GEN, FEP, and IPM and the MBICs of IPM and MEM. In P. aeruginosa, ASA decreased the MICs of FEP, IPM, and MEM, the MBICs of FEP and MEM, and the MBEC of FEP. DXP increased the MICs of CIP, GEN, and FEP, and the MBICs of CIP, GEN, and FEP among both clinical isolates. The MBECs of CIP and FEP for MRSA isolates and the MBECs of CIP, GEN, and MEM among P. aeruginosa isolates increased in the presence of DXP. IBP and ASA at 200 µg/mL significantly decreased the transcription level of algD in P. aeruginosa, and IBP significantly decreased the transcription level of icaA in S. aureus. DXP at 500 µg/mL significantly increased the expression levels of algD and icaA genes in S. aureus and P. aeruginosa isolates, respectively. Our findings showed that the formulations containing ASA and IBP have significant effects on decreasing the MIC, MBIC, and MBEC levels of some antibiotics and can down-regulate the expression of biofilm-related genes such as icaA and algD. Therefore, NSAIDs represent appropriate candidates for the design of new antibacterial and antibiofilm therapeutic formulations.

Characterization of antibiotic resistance and genotyping of Helicobacter pylori isolates in patients with gastroduodenal disease

Background: In this study, we assessed the status of antibiotic resistance of H. pylori isolates to antimicrobial agents at Hospital of Hue University of Medicine and Pharmacy in Central Vietnam and identified the underlying molecular mechanisms related to tetracycline and amoxicillin resistance. In addition, the cagA, cagE, cagT, vacA, iceA genotypes of H. pylori strains were investigated to predict clinical outcomes. Materials and methods: H. pylori was successfully cultured in 52 patients with different gastrointestinal disorders at the Hospital of Hue University of Medicine and Pharmacy in Central Vietnam. The minimum inhibitory concentrations (MICs) of five antimicrobials; clarithromycin (CLR), metronidazole (MTZ), levofloxacin (LE), amoxicillin (AMX) and tetracycline (TE) were determined by the E-test method. Genetic determinants of AMX and TE resistance were identified with the polymerase chain reaction (PCR), followed by sequencing and gene analysis. Allelic variants of cagA, cagE, cagT, vacA, iceA were identified by the PCR. Results: The resistance to CLR, MTZ, LE, AMX, and TE were 90.4%, 86.5%, 65.4%, 40.4% and 0%, respectively. Multidrug resistance was observed in 88.5% of the isolates investigated. Several known AMX resistance mutations were identified in PBP1A (A369T, V374L, S543R, T556S, N562Y), whereas a known mutation in 16S rRNA (A926G) was detected in strains with higher MIC level (TE MICs of 0.25 and 0.5 mg/L). The cagA, cagE and cagT genotypes were found together in 46 isolates (88.5%), vacAs- region genotype in 51 (98.1%, predominantly vacAs1), vacAmregion genotype in all strains studied (vacAm1 - 51.9%, vacAm2 - 40.4%, vacAm1 and vacAm2 - 7.7%), iceA1 in 22 (42.3%) and iceA2 in 20 (38.5%) of strains. The allelic variant vacAs1m1 was prominent (57.4%), and vacAs1m2 (42.6%). Conclusion: Overall, resistance rates to CLR, MTZ, LE and AMX were high in H. pylori from Central Vietnam, except for TE, which serves as a foundation for developing local guidelines for more effective therapeutic strategies. Neither the single genes nor the combination of genes was significantly helpful in predicting the clinical outcome of H. pylori infection in patients in our study. Key words: H. pylori, antibiotic resistance, genotype.

Role of Vancomycin Area Under the Curve Trapezoidal Dosing in Prevention of Nephrotoxicity as Compared with Traditional Trough-based Dosing

Published on: October 2022Journal of Young Pharmacists, 2022; 14(4):408-411Original Article | doi:10.5530/jyp.2022.14.82 Authors:Robert J Kranz*, Erin M LockardDepartment of Pharmacy, MercyOne Medical Center, 3421 W 9th Street, Waterloo, IA 50702, USA. Abstract:Background: Vancomycin is a fundamental treatment for resistant grampositive infections and is implicated in vancomycin-induced-nephrotoxicity (VIN) at high concentrations. Troughs greater than 18 mcg/ml are associated with a 4-fold increase in nephrotoxicity. Additionally, Area Under the Curve/ Minimum Inhibitory Concentration (AUC/MIC) levels over 600 mg*h/L are implicated in VIN. It is proposed that trapezoidal AUC/MIC dosing strategies reduce VIN instances. Materials and Methods: Patients greater than 18 years of age who received 72 hr of vancomycin therapy for a non-skin or soft-tissue or urinary indication were included in this retrospective cohort study. The primary endpoint was rate of nephrotoxicity based on the Acute Kidney Injury Network (AKIN) criteria for those patients who suffered an increase in serum creatinine of 0.3 mg/dl or greater after 48 hr of vancomycin therapy. The secondary endpoints were mean 24-hr vancomycin daily doses and instances of trough excursions above 18 mcg/ mL between groups. Forty patients met inclusion criteria for the AUC/MIC arm while seventy-nine met inclusion to the trough arm. Results: Rates of nephrotoxicity in the trough versus AUC/MIC were 36 patients (30.3%) and 3 patients (7.5%) respectively with a p<0.001. AUC/MIC based dosing was associated with decreased rates of nephrotoxicity and decreased rates of trough excursions above 18 mcg/ml. Conclusion: The AUC/MIC dosing strategy was associated with lower rates of VIN compared with trough based dosing.Keywords: Acute Kidney Injury, Creatinine Clearance, Vancomycin.

Antibacterial Effect and Possible Mechanism of Salicylic Acid Microcapsules against Escherichia coli and Staphylococcus aureus.

Microcapsules serve as a feasible formulation to load phenolic substances such as salicylic acid, a natural and safe antimicrobial agent. However, the antibacterial efficacy of salicylic acid microcapsules (SAMs) remains to be elucidated. Here, salicylic acid/β-cyclodextrin inclusion microcapsules were subjected to systematic antibacterial assays and preliminary antibacterial mechanism tests using Escherichia coli and Staphylococcus aureus as target organisms. It was found that the core-shell rhomboid-shaped SAMs had a smooth surface. SAMs exhibited a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 4 mg/mL against both bacteria. In the growth inhibition assay, 1/4 × MIC, 1/2 × MIC, and 1 × MIC of SAMs effectively retarded bacterial growth, and this effect was more prominent with the rise in the level of SAMs. Practically, SAMs possessed a rapid bactericidal effect at the 1 × MIC level with a reduction of more than 99.9% bacterial population within 10 min. A pronounced sterilization activity against E. coli and S. aureus was also observed when SAMs were embedded into hand sanitizers as antimicrobial agents. Moreover, exposure of both bacteria to SAMs resulted in the leakage of intracellular alkaline phosphatases and macromolecular substances (nucleic acids and proteins), which indicated the disruption of bacterial cell walls and cell membranes. In conclusion, SAMs were able to inactivate E. coli and S. aureus both in vitro and in situ, highlighting the promising utilization of this formulation for antimicrobial purposes in the area of food safety and public health.

Phenotypic detection of methicillin resistance, biofilm production, and inducible clindamycin resistance in Staphylococcus aureus clinical isolates in Kathmandu, Nepal

IntroductionMethicillin resistance, inducible clindamycin resistance (ICR), biofilm production, and increased minimum inhibitory concentration (MIC) of vancomycin in Staphylococcus aureus are major causes of antibiotic treatment failure and increased morbidity and mortality. The surveillance of such isolates and the study of their antimicrobial pattern are essential in managing the infections caused by these isolates. This study aimed to determine methicillin resistance, biofilm production, and ICR in S. aureus isolates from a tertiary care hospital in Kathmandu, Nepal.Materials and methodsA total of 217 S. aureus isolated from different samples were processed following standard laboratory procedures. Antibiotic susceptibility testing was performed by the Kirby–Bauer disk diffusion technique. Methicillin-resistant S. aureus (MRSA) were identified by the cefoxitin disk diffusion test, and biofilm producers were examined using the microtiter plate technique. D-test and E-test were performed to determine inducible clindamycin resistance and minimum inhibitory concentration of vancomycin, respectively.ResultsAmong the 217 S. aureus isolates, 78.3% were multidrug-resistant (MDR), 47.0% were MRSA, 62.2% were biofilm producers, and 50.7% showed ICR. All MRSA isolates exhibited MIC levels of vancomycin within the susceptible range. Biofilm producers and MRSA isolates showed elevated antimicrobial resistance. MRSA was significantly associated with MDR. Biofilm-producing and multidrug-resistant MRSA isolates showed significantly higher MIC levels of vancomycin (p = 0.0013 and < 0.0001, respectively), while ICR was significantly higher in MDR (p = 0.0001) isolates.ConclusionHigh multidrug resistance, MRSA, and ICR in this study call for routine evaluation of antibiotic susceptibility patterns of S. aureus. Vancomycin can be used to treat serious staphylococcal infections. Clindamycin should be prescribed only after performing the D-test. Drugs like teicoplanin, chloramphenicol, doxycycline, amikacin, and levofloxacin can treat MRSA infections.

Antifungal effects of Zataria multiflora Boisss (Shirazi Thyme) extract against Aspergillus fumigatus Afs35 genes: crza and rpda

Aspergillus fumigatus causes a fatal infection particularly among immunocompromised individuals. Nowadays herbal and medicinal essential oils play a vital role as an alternative safe medication. Zataria multiflora Boiss essential oils have antifungal activities mainly on the expression of some gene required for fungal survival. The main objective of the study was evaluation of antifungal effects of Z. multiflora Boiss extract against A. fumigatus AfS35 strain. The strain was obtained from department of biology, Baghdad University. Various concentrations (10-300mg/mL) of Z. multiflora Boiss extract were prepared and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined using micro-broth dilution method. The expression of crzA and rpdA genes was evaluated using the quantitative real-time PCR (RT-qPCR) technique. The concentrations ≥100mg/mL of extract inhibited the fungal growth which was non-toxic to normal cell line (MIC=100mg/mL and MFC=150mg/mL). The combination of 50mg/mL of extract plus each of amphotericin B (10mg/ml) and nystatin (10mg/mL) significantly decreased the MIC level (p&lt;0.001). The concentration of 10mg/mL of the extract decreased the expression of crzA and rpdA genes 2.4 (p&lt;0.001) and 3.6 (p&lt;0.0001) fold, respectively. The same concentration of amphotericin B non- significantly decreased the crzA and rpdA genes expression by 0.6 (p=0.224) and 1.1 (p=0.033) fold, respectively. The study concluded that Z. multiflora Boiss extract exerted antifungal effect against A. fumigatus by inhibiting the cell wall biosynthesis (down-regulation of crzA), sustainability and virulence expression (rpdA). The combination therapy with Z. multifora Boiss is promising to eradicate A. fumigatus infections.

Identification of a novel aminoglycoside O-nucleotidyltransferase AadA33 in Providencia vermicola.

A novel chromosome-encoded aminoglycoside O-nucleotidyltransferase AadA33 was identified in Providencia vermicola strain P13. The AadA33 shares the highest amino acid identity of 51.28% with the function characterized AadA31. Antibiotic susceptibility testing and enzyme kinetics analysis revealed that the function of AadA33 is to mediate spectinomycin and streptomycin resistance. The recombinant strain harboring aadA33 (pUCP20-aadA33/Escherichia coli DH5α) displayed >256- and 128-fold increases in the minimum inhibitory concentration levels to spectinomycin and streptomycin, respectively, compared with the control strains pUCP20/DH5α. Enzyme kinetic parameters manifested the substrate of AadA33 including spectinomycin and streptomycin, with kcat/Km of 3.28 × 104 (M−1 s−1) and 3.37 × 104 (M−1 s−1), respectively. Bioinformatics analysis revealed its structural mechanism of antimicrobial resistance, genetic context, and phylogenetic relationship with other aminoglycoside O-nucleotidyltransferases. This study of AadA33 contributed to understanding the function and resistance mechanism of aminoglycoside O-nucleotidyltransferase.

Whole-Genome Sequencing and Drug-Susceptibility Analysis of Serial Mycobacterium abscessus Isolates from Thai Patients.

Simple SummaryWe undertook genetic diversity analysis between subspecies, mutation-rate estimation and identification of drug-resistant mutations analysis of Mycobacterium abscessus. We differentiated between re-infection and persistent infection of M. abscessus based on genetic distance using WGS analysis. We found a high mutation rate of M. abscessus during the initial infection process, followed by a dramatic reduction in this rate in serial isolates separated by more than 180 days. Mutation rate also differed between the two subspecies that we included: subsp. abscessus (MAB) and subsp. massilense (MMAS). MMAS strains show more susceptibility to clarithromycin, amikacin and linezolid than MAB. This can inform choice of an appropriate regimen for patient management and reduce the speed of development of drug resistance for M. abscessus patients.Mycobacterium abscessus is an important pathogen that can cause serious human diseases and is difficult to treat due to antibiotic resistance. In this study, we analyzed, using whole-genome sequence (WGS) data, M. abscessus strains serially isolated from patients at various time intervals. We undertook genetic diversity analysis between subspecies, mutation-rate estimation and identification of drug-resistant mutations with minimum inhibitory concentration (MIC) analysis. Clonal isolates of M. abscessus:—subsp. abscessus (MAB) and subsp. massiliense (MMAS)—causing persistent infection through time, differed by 0–7 and 0–14 SNPs, respectively, despite being isolated 1 to 659 days apart. Two cases caused by MMAS differed by ≥102 SNPs at 350 days apart and were regarded as examples of reinfection. Isolates collected ≤7 days apart exhibited a high mutation rate (133.83 ± 0.00 SNPs/genome (5 Mb)/year for MMAS and 127.75 SNPs/genome (5 Mb)/year for MAB). Mutation rates declined in a time-dependent manner in both subspecies. Based on isolates collected > 180 days apart, MMAS had a significantly higher average mutation rate than MAB (2.89 ± 1.02 versus 0.82 ± 0.83 SNPs/genome (5 Mb)/year, (p = 0.01), respectively). All well-known drug-resistance mutations were found to be strongly associated with high MIC levels for clarithromycin and ciprofloxacin. No known mutations were identified for strains resistant to linezolid and amikacin. MAB strains in the study were susceptible to amikacin, while most MMAS strains were susceptible to clarithromycin, amikacin and linezolid. No hetero-resistance was found in the strains analyzed. Our study reports the genetic diversity and mutation rate of M. abscessus between the two major subspecies and confirms the drug resistance-associated mutations. Information about drug-resistance and associated mutations can be applied in diagnosis and patient management.

Terpinen-4-ol, An Active Constituent of Kewda Essential Oil, Mitigates Biofilm Forming Ability of Multidrug Resistant Staphylococcus aureus and Klebsiella pneumoniae

The increased incidence of microbial resistance to traditional antibiotics has urged the scientific community to look for alternative therapeutic regimens. In this context, mitigation of biofilm formation is considered as viable alternative. Since, plant derived essential oils are rich heritage of bioactive phytochemicals with widespread pharmacological values, in the present study Terpinen-4-ol, bioactive constituent of Kewda essential oil (KEO) extracted from Pandanus odorifer male flower was evaluated for its antimicrobial and antibiofilm activities against Staphylococcus aureus and Klebsiella pneumoniae and their reference strains, MTCC-740 and MTCC-109. The minimum inhibitory concentration (MIC) of Terpinen-4-ol against S. aureus and K. pneumoniae was 50 and 25 mM, respectively. At MIC level, Terpinen-4-ol exhibited antibacterial activities against both the reference strains i.e. MTCC-740 and MTCC-109 with a zone of 16 and 14 mm, respectively. On treatment with sub-MIC of Terpinen-4-ol, a significant reduction in exopolysaccharides (EPS) production was observed as evident from qualitative Congo red agar (CRA) assay. Further, the reduction in EPS production was quantified with a reduction of 67.51±1.29% against S. aureus. The light and fluorescence microscopic analysis also corroborated the antibiofilm potential of Terpinen-4-ol as a significant reduction in the thickness of biofilm formation was observed. In silico studies provided an insight into the action of Terpinen-4-ol in binding to target proteins associated with biofilm formation and drug resistance. Thus, Terpinen-4-ol could be considered as putative drug candidate in the fight against biofilm associated chronic infections and drug resistance.

Novel Antimicrobial Peptide "Octoprohibitin" against Multidrug Resistant Acinetobacter baumannii.

Octoprohibitin is a synthetic antimicrobial peptide (AMP), derived from the prohibitin-2 gene of Octopus minor. It showed substantial activity against multidrug resistant (MDR) Acinetobacter baumannii with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 200 and 400 µg/mL, respectively. Time-kill kinetics and bacterial viability assays confirmed the concentration-dependent antibacterial activity of octoprohibitin against A. baumannii. The morphology and ultrastructure of A. baumannii were altered by treatment with octoprohibitin at the MIC and MBC levels. Furthermore, propidium iodide-fluorescein diacetate (PI-FDA) staining and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining of octoprohibitin-treated A. baumannii revealed membrane permeability alterations and reactive oxygen species (ROS) generation, respectively. Agarose gel retardation results confirmed the DNA-binding ability of octoprohibitin to the genomic DNA of A. baumannii. Furthermore, octoprohibitin showed concentration-dependent inhibition of biofilm formation and eradication. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of octoprohibitin were 1000 and 1460 µg/mL, respectively. Octoprohibitin produced no significant cytotoxicity up to 800 µg/mL, and no hemolysis was observed up to 400 µg/mL. Furthermore, in vivo analysis in an A. baumannii-infected zebrafish model confirmed the effective bactericidal activity of octoprohibitin with higher cumulative survival percent (46.6%) and fewer pathological signs. Histological analysis showed reduced alterations in the gut, kidney, and gill tissues in the octoprohibitin-treated group compared with those in the phosphate-buffered saline (PBS)-treated group. In conclusion, our results suggest that octoprohibitin is a potential antibacterial and antibiofilm agent against MDR A. baumannii.

Molecular characterization of florfenicol and oxazolidinone resistance in Enterococcus isolates from animals in China.

Florfenicol is widely used for the treatment of bacterial infections in domestic animals. The aim of this study was to analyze the molecular mechanisms of florfenicol and oxazolidinone resistance in Enterococcus isolates from anal feces of domestic animals. The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method. Polymerase chain reaction (PCR) was performed to analyze the distribution of the resistance genes. Whole-genome sequencing and comparative plasmid analysis was conducted to analyze the resistance gene environment. A total of 351 non-duplicated enteric strains were obtained. Among these isolates, 22 Enterococcus isolates, including 19 Enterococcus. faecium and 3 Enterococcus. faecalis, were further studied. 31 florfenicol resistance genes (13 fexA, 3 fexB, 12 optrA, and 3 poxtA genes) were identified in 15 of the 19 E. faecium isolates, and no florfenicol or oxazolidinone resistance genes were identified in 3 E. faecalis isolates. Whole-genome sequencing of E. faecium P47, which had all four florfenicol and oxazolidinone resistance genes and high MIC levels for both florfenicol (256 mg/L) and linezolid (8 mg/L), revealed that it contained a chromosome and 3 plasmids (pP47-27, pP47-61, and pP47-180). The four florfenicol and oxazolidinone resistance genes were all related to the insertion sequences IS1216 and located on two smaller plasmids. The genes fexB and poxtA encoded in pP47-27, while fexA and optrA encoded in the conjugative plasmid pP47-61. Comparative analysis of homologous plasmids revealed that the sequences with high identities were plasmid sequences from various Enterococcus species except for the Tn6349 sequence from a Staphylococcus aureus chromosome (MH746818.1). The current study revealed that florfenicol and oxazolidinone resistance genes (fexA, fexB, poxtA, and optrA) were widely distributed in Enterococcus isolates from animal in China. The mobile genetic elements, including the insertion sequences and conjugative plasmid, played an important role in the horizontal transfer of florfenicol and oxazolidinone resistance.

Role of some proteins in resistance of clinical Acinetobacter baumannii isolates to imipenem

Acinetobacter baumannii is one of the ESKAPE pathogens which are the leading cause of nosocomial infections throughout the world. The aim of this study is to detect the role of Some Proteins in Resistance of Acinetobacter baumannii to imipenem. The research included the collection of 100 different clinical specimens of( urine, burns, and wounds) isolated from patients in Al-Diwaniyah Teaching Hospital for the period from September to December 2021. 20 isolates out of 100 isolates belonging to A.baumannii were obtained. The samples were collected from different clinical specimens distributed as follows:7(35%) Swabs of burns,8(40%) swabs of wounds, and 5(25%)from urine, an examination was conducted for (8)Antibiotics by ( Antibiotic Susceptibility Test-AST ) on 20 isolates. The results showed that all isolates are resistant to antibiotics except for imipenem showed a sensitivity of 20% and resistance of 80% to imipenem. The results of the Minimum Inhibitory Concentration level of imipenem that were conducted for five isolates showed that all isolates are resistant to imipenem at concentrations of (128 mg/ml and 256 mg/ml ), while 2(40%) isolates out of 5 isolates were resistant to imipenem in the concentration of 64 mg /ml.

Antibacterial activity and mechanism of essential oils in combination with medium-chain fatty acids against predominant bovine mastitis pathogens.

Bovine mastitis has become a significant economic importance for the dairy industry. Concerns regarding poor milk quality and emergence of bacterial resistance have necessitated to develop an alternative therapeutic approach to antibiotics for the treatment of mastitis. Saturated medium-chain fatty acids (MCFAs) and essential oils (EOs) are known natural antimicrobials, but their combined effect has not been investigated extensively. The objective of the present investigation was to examine the bactericidal effect of various combined treatments of eight EOs and three saturated MCFAs to inactivate predominant mastitis pathogens, including Staphylococcus aureus ATCC 29213; Escherichia coli ATCC 25922; Klebsiella pneumoniae ATCC 27736 and Streptococcus agalactiae ATCC 27956. The minimum inhibitory concentration (MIC) values confirmed that all the tested pathogens were variably susceptible to both EOs and saturated MCFAs. Among essential oils, carvacrol (CAR), trans-cinnamaldehyde (TC) and thymol (TM) showed the highest inhibitory activity at concentration 0·38-1·32 mg/mL. Carvacrol exhibited effective additive antibacterial activity in combined treatment with octanoic acid (OA) in terms of its fractional inhibitory index (0·63-0·88) and time-kill effect in reducing about 6 log CFU/mL bacterial cells in less than 5 min. The effort was also made to elucidate the mechanism of antibacterial action of CAR and OA against selected mastitis pathogens by observing changes in cell microstructure, permeability and integrity of cell membrane and their membrane potential. After adding CAR and OA at MIC level, there were obvious changes in cell morphology, leakage of small electrolytes and macromolecules at the initial few hours of treatment i.e. within 1-2 h were observed. Our results indicated that CAR and OA could be evaluated as alternatives or adjuncts to antibiotics as intramammary infusion or topical application to treat bovine mastitis, significantly improving the microbiological safety of milk.

Antibacterial Activity of Bioactive Compound in Salvadora persica (Chewing Stick) Against Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans

This study aims to determine the phytochemical properties of Salvadora persica extracts and their antibacterial activities against Gram-negative oral anaerobes which are responsible of periodontitis such as Porphorymonas gingivalis and Aggregatibacter actinomycetemcomitans. Aqueous and ethanolic extracts were prepared using the root powdered stem of S. persica. The phytochemical compounds were determined with Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS). The antibacterial activities were assessed according to the levels of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The S. persica ethanolic and aqueous extracts contained antibacterial compounds such as Octadecenoic acid, methyl ester, n-Hexadecanoic acid and Pentadecanoic acid, 2,6,10,14-tetramethyl-, methylester, 1-(2,2-Dimethylpropanoyl)-L-prolyl-L-prolyl-N,2-dimethylalaninamide and 2-({4-[(Difluoromethyl)sulfanyl]phenyl} amino) benzoic acid, respectively. Both P. gingivalis (0.025 mg/mL) and A. actinomycetemcomitans (0.125 mg/mL) were susceptible to S. persica ethanolic extract. Ethanolic extract preparation has equal antibacterial activity with 0.2% chlorhexidine gluconate solution (p&lt;0.05). Ethanolic extract (0.025-0.125 mg/mL) preparation has comparable require biocompatible testing.

Effects of four efflux pump inhibitors on the activities of clarithromycin against Mycobacterium abscessus

Objective: To detect the effects of four efflux pump inhibitors on the minimum inhibitory concentration of clarithromycin (CLA) against Mycobacterium abscessus (M. abscessus) in vitro, and to explore the role of efflux pump in CLA resistance of M. abscessus. Methods: Four frequently-used efflux pump inhibitors (Carbonyl Cyanide 3-chlorophenylhydrazone, CCCP, N, N'-dicyclohexylcarbodiimide, DCC, Verapamil, VP, Reserpine, RSP) were evaluated in this study. The minimum inhibitory concentration (MIC) values of clarithromycin against M. abscessus reference strain and 60 clinical strains with or without efflux pump inhibitors were detected by Alamar Blue method. Sequence analysis of erm(41) and rrl genes known to be associated with CLA resistance in M. abscessus was performed to analyze the correlation between the effect of efflux pump inhibitors on MIC and mutation of resistance-related genes. Results: CCCP, DCC, VP and RSP could reduce the MIC of M. abscessus to CLA, and the effect of RSP was weaker than the other three efflux pump inhibitors. Among the sixty M. abscessus clinical strains, ten strains were resistant to clarithromycin, seven of which had rrl gene mutation. The CLA resistance rate of smooth phenotype isolates was higher than that of rough phenotype isolates. At 3 day of clarithromycin incubation, the MICs of resistant strains were all reduced by efflux pump inhibitors. Compared with the strains with rrl gene mutation, efflux pump inhibitors had a greater effect on the strains without rrl gene mutation. At 14 day of clarithromycin incubation, 83% of M. abscessus subsp. abscessus, were induced to be resistant, and all of them were T28 sequence type of erm(41). With the occurrence of induced drug resistance, the effect of efflux pump inhibitor on CLA MIC decreased. Efflux pump inhibitors had no statistically significant diffence in the effect of effcux pump inhibitors on CLA MIC levels in different phenotypes of isolates. Conclusions: Efflux pump is involved in the resistance process of M. abscessus to CLA. Efflux pump inhibitors reduce the drug resistance to clarithromycin against M. abscessus in different degrees. The use of efflux pump inhibitors may provide a new way to alleviate the drug resistance of M. abscessus.

Colistin Resistance and Molecular Characterization of the Genomes of mcr-1-Positive Escherichia coli Clinical Isolates.

Research on resistance against polymyxins induced by the mcr-1 gene is gaining interest. In this study, using agar dilution method, polymerase chain reaction, and comparative genomic analysis, we investigated the colistin resistance mechanism of clinical E. coli isolates. The minimum inhibitory concentration (MIC) analysis results revealed that of the 515 isolates tested, bacteria with significantly increased MIC levels against colistin were isolated in 2019. Approximately one-fifth (17.14% to 19.65%) of the isolates showed MIC values ≥1 mg/L against colistin in 2015, 2016, and 2017. However, in 2019, up to three-quarters (74.11%, 146/197) of the isolates showed MIC values ≥1 mg/L against colistin indicating an increase in colistin resistance. Six isolates (EC7518, EC4968, EC3769, EC16, EC117, EC195, 1.13%, 6/515) were found to carry the mcr-1 gene and a novel mcr-1 variant with Met2Ile mutation was identified in EC3769. All six strains showed higher MIC levels (MIC=4 mg/L) than any mcr-1-negative strains (MIC ≤ 2 mg/L). Whole-genome sequencing of the six mcr-1-positive isolates revealed that EC195 carried the highest number of resistance genes (n = 28), nearly a half more than those of the following EC117 (n = 19). Thus, EC195 showed a wider resistance spectrum and higher MIC levels against the antimicrobials tested than the other five isolates. Multi-locus sequence typing demonstrated that these mcr-1-positive strains belonged to six different sequence types. The six mcr-1 genes were located in three different incompatibility group plasmids (IncI2, IncHI2 and IncX4). The genetic context of mcr-1 was related to a sequence derived from Tn6330 (ISApl1-mcr-1-pap2-ISApl1). Investigations into the colistin resistance mechanism and characterization of the molecular background of the mcr genes may help trace the development and spread of colistin resistance in clinical settings.

Effect of Ciprofloxacin on Planktonic Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial, acute, and chronic infections in patients with conditions such as cystic fibrosis, severe burns, and cancer. P. aeruginosa is known for its persistence in infections due largely to its numerous means of antimicrobic resistance, one of which is its ability to form biofilms. This organism has been placed on the World Health Organization list of Priority Pathogens for Research and Development of New Antibiotics at the critical level for many reasons, that includes its high mortality, healthcare burden, prevalence of resistance, and treatability. P. aeruginosa is resistant to many antibiotics; however, &gt;70% of P. aeruginosa are susceptible to ciprofloxacin. For these reasons, the purpose of the current study was to establish antimicrobic levels of ciprofloxacin effective against P. aeruginosa. To accomplish this purpose, antimicrobic levels of bacteria in biofilms and those not associated with a biofilm (planktonic) need separate methods of testing. For planktonic bacteria, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are performed, whereas other methods are used for biofilm‐associated bacteria. To initiate inhibition studies, a model system was identified of ciprofloxacin effect on standardized strains of P. aeruginosa (ATCC strains 10145, 27853, 1744, CRM‐9027, 9721). These strains were used to establish ciprofloxacin effect, in terms of MIC and MBC levels, on planktonic bacteria. Results indicate that MIC and MBC levels of these strains are in the same range as reported values of P. aeruginosa. These results establish ciprofloxacin efficacy of growth inhibition for each ATCC strain of planktonic P. aeruginosa and will serve as baseline results for future studies, such as effect on biofilm‐associated bacteria and combination antimicrobic treatments.

Colistin Action Against Planktonic Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial, acute, and chronic infections in patients with conditions such as cystic fibrosis, severe burns, and cancer. P. aeruginosa is associated with increased patient morbidity, mortality, and healthcare costs and is known for its persistence of infections. P. aeruginosa is resistant to many antibiotics; however, most strains are susceptible to colistin. The World Health Organization has identified antimicrobial resistance as a major threat and Antimicrobial Stewardship Programs (ASP) have been initiated. ASP goals are to improve patient outcomes, optimize patient safety, reduce antimicrobial resistance, and control healthcare costs. Recommendations to reduce antimicrobial resistance include optimizing the use of antimicrobials and preventing the transmission of drug‐resistant organisms. Using antimicrobic agents wisely leads to optimization of their use. For these reasons, we sought to adopt a model system for testing minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) levels for standardized strains of P. aeruginosa (ATCC strains 10145, 27853, 1744, CRM‐9027, 9721) using standardized methods. This study sought to compare results of standardized analysis that used Mueller‐Hinton Broth (MHB), the recommended and standardized growth medium, or tryptic soy broth (TSB), the growth medium used in many labs with published MIC and MBC reports. Given the ASP recommendations of optimizing use of antimicrobial agents, it is important to know the impact of variations in and deviations from standardized MIC and MBC methodologies in reporting of research results relevant to antimicrobic agents. This comparison study tested the assumption that MIC and MBC results are the same regardless of whether MHB or TSB is used as the growth medium. Preliminary results suggest that MIC and MBC values obtained by using TSB or MHB may display some degree of variability.

In Vitro Activity of Ceftazidime-avibactam and Colistin Against Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates

Infections caused by multi drug-resistant gram-negative bacilli are increasingly reported worldwide. Colistin, tigecycline and aminoglycosides are almost the only and last choice antibiotics in the treatment of infections caused by carbapenem-resistant Enterobacterales members. Ceftazidime-avibactam is a novel antibiotic combination consisting of a broad-spectrum cephalosporin and avibactam with good antimicrobial activity against carbapenem-resistant Enterobacterales members. The aim of this study was to assess the in vitro activity of ceftazidime-avibactam and colistin against carbapenem-resistant Klebsiella pneumoniae isolates and to obtain local antimicrobial surveillance data. A total of 150 carbapenem-resistant K.pneumoniae isolates obtained from various clinical samples of the patients hospitalized in our hospital between 2018-2021 were included in the study. Duplicate isolates were excluded from the study. The isolates were recovered from blood (n= 72), tracheal aspirate (n= 40), wound (n= 20), biopsy and abscess (n= 10), steril body fluid (n= 5), and peripheral venous catheter (n= 3) samples. Isolates were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, Germany). The minimum inhibitory concentration (MIC) values of the isolates for meropenem, colistin, ceftazidime, and ceftazidime-avibactam were determined by broth microdilution method. Susceptibility of the isolates to the tested antibiotics was evaluated by the European Committee of Antimicrobial Susceptibility Testing (EUCAST) criteria. The presence of carbapenemases (VIM, IMP, NDM, KPC, and OXA-48) was investigated by polymerase chain reaction (PCR) using specific primers. The mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were evaluated by PCR for plasmid-mediated colistin resistance. All K.pneumoniae isolates were found to be positive for at least one of the carbapenemase genes evaluated in the study. The blaOXA-48 gene was detected in 107 (71.3%), blaKPC gene in 25 (16.7%); blaNDM gene in 7 (4.7%), co-production of blaOXA-48 and blaKPC genes in 10 (6.7%), co-production of blaOXA-48 and blaNDM genes in 1 (0.6%) isolate. None of the isolates harbored the blaVIM and blaIMP genes. None of the mcr genes screened in the study were detected among the isolates. The susceptibility of the isolates to ceftazidime-avibactam and colistin was 92.7% (139/150) and 48% (72/150), respectively. The MIC50 and MIC90 values for meropenem, ceftazidime, ceftazidime-avibactam, and colistin of the isolates were determined as 32/256, > 128/> 128, 1/8, and 4/16 µg/ml, respectively. Of the ceftazidimeavibactam resistant isolates, seven were positive for blaNDM, three for blaKPC, and one for both blaOXA-48 and blaNDM genes. High ceftazidime-avibactam MIC levels (> 128 µg/ml) were detected in metallo-betalactamase producing isolates. Consequently, our data suggested that ceftazidime-avibactam exhibited as a good alternative therapeutic choice for carbapenem-resistant K.pneumoniae isolates. It is noteworthy that high rate of colistin resistance was detected in K.pneumoniae isolates. Another notable finding of this study is the increase in K.pneumoniae isolates producing blaKPC for our country. To prevent the development of resistance which is observed even in last-choice therapeutic antibiotics, the principles of rational antibiotic use should be followed. The appropriate antimicrobial susceptibility testing should be routinely performed for surveillance of ceftazidime-avibactam and colistin.

The Antibacterial Analysis of Alcohol-Free and Alcohol-Based Chlorhexidine Mouthwashes Against Oral Bacteria

Background: Chlorhexidine (CHX) is the gold standard chemical agent against oral pathogenic bacteria and is widely used for plaque/gingivitis control. The aim of the present study was to compare the effect of alcohol-based and alcohol-free CHX mouthwashes on oral microorganisms. Methods: In the present in vitro study, the standard strains of four microorganisms present in the oral cavity were prepared, including Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, and Lactobacillus casei. The serial dilutions of CHX antimicrobial agents were obtained, and the level of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was determined using the broth dilution method. Finally, data were analyzed using the Kruskal-Wallis test, the Mann-Whitney U test, and SPSS-16. Results: The MIC values of 0.12% and 0.2% alcohol-free CHX and 0.2% alcohol-based CHX for S. mutans were 1.17, 0.48, and 0.24 µg/mL, respectively. The MBC values of 0.12% and 0.2% alcohol-free CHX and 0.2% alcohol-based CHX for S. mutans were 18.78, 7.81, and 7.81 µg/mL, respectively. The MIC and MBC values of the tested CHX mouthwashes for S. mutans were significant (P≤0.05). Conclusions: Overall, the 0.2% alcohol-based CHX mouthwash had the highest antibacterial activity against gram-positive bacteria.