Fractional Inhibitory Concentration Index Research Articles

In vitro antibacterial effect of enrofloxacin combined with lavender essential oil on selected Salmonella serotypes isolated most commonly poultry

The present study investigated the antimicrobial activity of enrofloxacin in combination with lavender (Lavandula angustifolia) essential oil against three serotypes of Salmonella: Salmonella Enteritidis ATCC 13076, Salmonella Gallinarum biovar Pullorum ATCC 13036, Salmonella Typhimurium ATCC 14028 and Salmonella Typhimurium – a moderately sensitive monophasic strain. The analysis of the interaction of enrofloxacin with lavender essential oil was carried out using the checkerboard method. The obtained FICI values were interpreted as follows: synergistic when ≤ 0.5; additive when > 0.5 but ≤ 1; non-interactive when > 1 but ≤ 4; antagonistic when > 4. When analysing individual combinations of enrofloxacin with lavender oil, a positive additive effect was found in all cases (S. Enteritidis ATCC 13076 FICI – 0.85; S. Gallinarum biovar Pullorum ATCC 13036 FICI – 1.0; S. Typhimurium ATCC 14028 and S. Typhimurium – a moderately sensitive monophasic strain FICI – 0.7). Antagonistic interaction was not recorded in any case. When analysing individual combinations of enrofloxacin with lavender oil against the serotypes tested, an additive effect was demonstrated in all cases (FIC indices: 0.7-1.0). There was no antagonistic effect in any of the analysed combinations.

New synergistic antibacterial mechanism of bulky mixed Ti/w hetero-polyoxometalates composed of multi lacunary Keggin structure with oxacillin against vancomycin intermediate-resistant Staphylococcus aureus.

Considering the lack of detailed research on the antibacterial mechanism of polyoxometalates, we examined the synergistic effect of novel bulky mixed Ti/W hetero-polyoxometalates (K9.5H2.5 ［α-Ge2Ti4W20O78］・ 29H2O; αTi4, K9H5 ［α-Ge2Ti6W18O77］・16H2O; αTi6, K23H5［α-Ge4Ti12W36O154］・39H2O; αTi12, K9H5 ［β-Ge2Ti6W18O77］・ 45H2O; βTi6) with the antibiotic oxacillin against vancomycin intermediate-resistant Staphylococcus aureus (VISA) using fractional inhibitory concentration (FIC) index and growth curve in this study. All polyoxometalates used in this study showed remarkable synergistic effects with oxacillin. Its synergistic antibacterial mechanism was examined using reverse transcription PCR (RT-PCR) and penicillin binding protein-2' (PBP2') latex agglutination test. The results suggested that these polyoxometalates did not inhibit mecA gene transcription but resulted in PBP2' protein malfunction. From these results, we concluded that the substances producing resistance in VISA were affected by polyoxometalates depending on their molecular size, facilitating a synergistic antibacterial effect with oxacillin.

Siderophore Production and its Role as Therapeutic Agent

Siderophores are iron chelators, which are produced by bacteria under iron-deficient conditions required for their growth. Therefore, siderophores can be used as a carrier to direct drugs into the bacteria and kill them. The present study was designed to screen siderophore production using different bacteria using an iron-deficient medium and its synergistic capability to kill drug-resistant bacteria. Siderophore under iron-deprived condition was evaluated by chrome azurol S (CAS) assay. Whereas, broth micro-dilution method and checkerboard assay were used to determine the antimicrobial properties of selected drugs or epigallocatechin gallate (EGCG) individually or in combination with synthetic siderophore. Results demonstrated that the entire tested microorganisms produced siderophore under the iron-deprived condition as evidenced by orange halo zones in CAS agar plates. Gram-negative bacteria produced more siderophores as reflected by orange color with bacterial zone inhibition of 17-22mm as compared to Gram-positive bacteria (13-15mm). As compared to antibiotics and EGCG, acetohydroxamic acid (aHa; synthetic siderophore) showed no antibacterial properties (1500 - 6500 µg/ml). The synergism of aHa with tetracycline, ceftriaxone, and EGCG (FIC index <0.5) against S. typhi, methicillin-resistant and sensitive Staphylococcus aureus, and E. coli were evident. In conclusion, siderophore may be considered a potential candidate to design different combination therapy against emerging antimicrobial-resistant pathogens.

Synergistic Effects of Capric Acid and Colistin against Colistin-Susceptible and Colistin-Resistant Enterobacterales.

Colistin is a last-line antibiotic against Gram-negative pathogens. However, the emergence of colistin resistance has substantially reduced the clinical effectiveness of colistin. In this study, synergy between colistin and capric acid was examined against twenty-one Gram-negative bacterial isolates (four colistin-susceptible and seventeen colistin-resistant). Checkerboard assays showed a synergistic effect against all colistin-resistant strains [(FICI, fractional inhibitory concentration index) = 0.02-0.38] and two colistin-susceptible strains. Time-kill assays confirmed the combination was synergistic. We suggest that the combination of colistin and capric acid is a promising therapeutic strategy against Gram-negative colistin-resistant strains.

Investigation of the synergistic effect of glycolipid biosurfactant produced by Shewanella algae with some antibiotics against planktonic and biofilm forms of MRSA and antibiotic resistant Acinetobacter baumannii.

To tackling antibiotic resistance and the appearance of multidrug-resistant (MDR) strains, one current approach is the combined use of biosurfactants with antibiotics to increase their efficacy. The antimicrobial ability of biosurfactant produced by Shewanella algae strain B12 was examined using the agar well diffusion method versus some resistant Gram-negative and Gram-positive bacteria. The Minimum Inhibitory Concentration (MIC) of Glycolipid-Biosurfactant of B12 (GBB12) was performed by the broth dilution technique. The inhibition of biofilm formation, disruption of biofilm, and reducing the population of viable cells in biofilm were evaluated by the microtiter plate method. Finally, Scanning Electron Microscopy (SEM) analysis was used to confirm the disruption of the cell membrane by GBB12. In all experiments, when GBB12 was added to antibiotics (except Amikacin), the antimicrobial activity was increased. The synergistic effects of GBB12 and antibiotics (Ciprofloxacin and Gentamycin) against Methicillin-Resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii were confirmed by the Fractional Inhibitory Concentration Index (FICI). GBB12-Gentamycin mixture almost completely inhibits the formation of A. baumannii biofilm, reaching 99.8% inhibition. Also, the rate of MRSA biofilm inhibition treated with GBB12-Ciprofloxacin mixture was found to be 99.4%. biosurfactant-antibiotic mixture could be adequate replacements for traditional antibiotics in the near future. This study shows the potential of GBB12 as antimicrobial and antibiofilm agent.

1432. Exploring Cell Wall Targets to Overcome Mycobacterium tuberculosis (Mtb): Ceftriaxone (CRO) Inhibits LdtMt2, a Major Peptidoglycan (PG) Synthase

Abstract Background Drug-resistant tuberculosis (DR TB) is a deadly, difficult-to-treat infection, and new treatment strategies are needed. Despite the wide success of β-lactams (BLs), DR TB guidelines only include meropenem (MEM) and imipenem (IPM), given with clavulanate (CLA). BlaC, the Mtb β-lactamase, hydrolyzes CRO less efficiently than other cephems and β-lactamase inhibitors improve the in vitro susceptibility of Mtb to CRO. Surprisingly, CRO has not been evaluated in DR TB clinical studies. Moreover, the mechanisms by which CRO disrupts Mtb PG synthesis are not well characterized. CRO inhibits LdtMt1­, but activity against LdtMt2, an important Mtb PG synthase, is unknown. To explore this knowledge gap, we examined CRO inhibition of LdtMt2. In addition, we investigated if combining CRO with MEM or IPM would lower minimum inhibitory concentrations (MICs) more than each agent alone. Methods A panel of Mtb isolates was selected for susceptibility testing with a broth microdilution method. Timed electrospray ionization-mass spectrometry (ESI-MS) and inhibition kinetic assays were performed. Results CRO MICs ranged 0.25 to 16 µg/mL and lowered ≤ 0.06 to 2 µg/mL with CLA (Table 1). Fractional inhibitory concentration indices for CRO + MEM or IPM was < 0.5 for six isolates, suggesting synergy. ESI-MS captured CRO-LdtMt2­ acyl-complexes at timepoints 5 to 120 min, and a 158 Da fragment loss was observed; MEM and IPM were unchanged (Table 2, Figure 1). When LdtMt2 was co-incubated with MEM and CRO together, only MEM complexes were captured. Interestingly, Kiapp­ with CRO (0.07 ± 0.01 µM) was comparable to that with MEM (0.09 ± 0.01 µM). Figure 1Mass spectrometry chromatograms with LdtMt2 (A) alone incubated with ceftriaxone at (B) 5 min, (C) 15 min, (D) 30 min, (E) 120 min. [Ldt¬Mt2] = 13.2 µM and [ceftriaxone] = 264 µM. Changes in MW are also listed in Table 1. (F) shows the structure of ceftriaxone with the proposed leaving group in red, leaving the remaining bound structure accounting for the observed change in MW. Conclusion CRO was effective in lowering MICs with MEM, IPM, and CLA for our Mtb isolates. Based upon ESI-MS, we found that CRO forms a stable complex with LdtMt2 and the R2 side chain is eliminated, while kinetic observations support inhibition of LdtMt2 by CRO (Figure 2). Previous work found MEM and IPM also inhibit multiple other PG synthases (e.g., PonA1, LdtMt1, LdtMt3). We hypothesize that CRO + MEM/IPM inhibits the growth of Mtb by the combined inactivation of multiple cell wall enzymes. Our observations support the further exploration of the notion of “target redundancy” as an approach to treat multidrug-resistant mycobacteria with BLs. Figure 2.Ceftriaxone-LdtMt2 adduct formation leading to release of the R2 side group Disclosures Krisztina Papp-Wallace, Ph.D, Merck: Grant/Research Support|Venatorx: Grant/Research Support|Wockhardt: Advisor/Consultant Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.

1691. Bacteriophage and Protein Synthesis Pathway Inhibitor Combinations: Antibiotic mechanism of action drives antagonistic interactions

Abstract Background Bacteriophage (phage) to augment antibiotic efficacy is a possible therapeutic option in the era of antimicrobial resistance. Studies to date have assessed phage-antibiotic synergy (PAS), however, its efficacy may be dependent upon antibiotic mechanism of action. Here, we report our in-vitro evaluation of phage-antibiotic antagonism (PAA) among phage and protein synthesis inhibitor combinations in multidrug-resistant clinical strains of P. aeruginosa, S. aureus, and E. faecium. Methods The following bacteria (phage) regimens were evaluated: 10266 (EM) and R9010 (14207) (P. aeruginosa) against gentamicin (GEN), azithromycin (AZM), and ciprofloxacin (CIP); N315 and 494 (Intesti) (S. aureus), and R497 and HOU503 (NV-497) (E. faecium) against linezolid (LNZ), minocycline (MIN), and daptomycin (DAP). Modified checkerboard (CB) MIC assays were used for preliminary screening followed by 24h time kill analyses (TKA). For CB, synergy, additive activity, and antagonism were defined as an FIC index of ≤0.5, 1–4, and >4, respectively. In 24h TKA, synergy and additivity were defined as a ≥2 and ≥1 log10 CFU/mL reduction from baseline, while antagonism was defined as phage-antibiotic combinations with CFU/mL higher than the most effective single treatment at 24h. Data were compared by one-way ANOVA and Tukey (HSD) test (P< 0.05). Results In CB analyses and 24h TKA of S. aureus and E. faecium isolates, phage-LZD and phage-MIN combinations were antagonistic (FIC >4) while phage-DAP was synergistic (FIC 0.5) (ANOVA range of mean differences 0.52 to 2.59 log10 CFU/mL; P< 0.001). For P. aeruginosa, phage-AZM and phage-GEN were antagonistic (FIC >4) and additive (FIC=1), respectively (ANOVA range of mean differences 1.04 to 1.95 log10 CFU/mL; P< 0.001). Conclusion Our results suggest that antibiotics that act on the protein synthesis pathway may lead to PAA, however, PAA interactions may be highly dependent upon antibiotic mechanism of bacterial inhibition (i.e., location of ribosomal protein synthesis inhibition, bactericidal vs. bacteriostatic) protein synthesis inhibit. Studies assessing PAA in a wider array of phage-antibiotic combinations are warranted. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

Repurposing streptomycin and chloramphenicol against bacterial pathogens by combination with diminazene aceturate.

Bacterial resistance is a threat to health worldwide, mainly due to reduced effective treatment. In this context, the search for strategies to control such infections and suppress antimicrobial resistance is necessary. One of the strategies that has been used is combination therapy. In the present work, we investigated the in vitro efficacy of the antimicrobials diminazene aceturate (DA), chloramphenicol (CHL), and streptomycin (STP) alone and in combination against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus clinical isolates. DA was capable of inhibiting all strains with MIC of 25-400 μg mL-1, while STP and CHL showed antibacterial activity with minimum inhibitory concentration (MICs) of≤3.12-400 μg mL-1. The combination of aceturate with STP showed synergism toward almost all Gram-negative bacteria, with fractional inhibitory concentration index (FICIs) of 0.09-0.37. In addition, for CHL and aceturate, synergisms for Gram-negative and -positive strains were observed. A time-kill assay against E. coli revealed that the aceturate and STP combination can inhibit bacterial growth in a shorter time when compared with single antibiotics. In addition, antimicrobials did not show hemolytic activity even at the highest concentrations used. Therefore, the antimicrobial combinations presented in this work showed important results, demonstrating that combined therapy can be used as an alternative strategy for pathogen control.

Towards Green Strategies of Food Security: Antibacterial Synergy of Essential Oils from Thymus vulgaris and Syzygium aromaticum to Inhibit Escherichia coli and Staphylococcus aureus Pathogenic Food Isolates.

Foodborne diseases continue to represent an important public health issue. The control of food spoilage and pathogenic microorganisms is achieved mainly by synthetic chemicals, unfortunately associated to several undesirable aspects. The growing requirement for new and safe alternative strategies has resulted in the research of agents from natural sources with antimicrobial properties, such as essential oils (EOs). This study's purpose was to define the antibacterial profile of thyme (Thymus vulgaris) and cloves (Syzygium aromaticum) essential oils against both Gram-positive and Gram-negative important foodborne pathogenic bacteria. Gas chromatography mass spectrometry analysis was performed for EOs' chemical composition. Qualitative in vitro antimicrobial assays (i.e., agar well diffusion method and disk-volatilization method) allowed for verification of the efficacy of EOs, used individually and in binary combination and both in liquid and vapor phase, against Staphylococcus aureus and Escherichia coli food isolates. Minimal inhibitory concentrations and minimal bactericidal concentration values have been used to quantitatively measure the antibacterial activity of EOs, while the fractional inhibitory concentration index has been considered as a predictor of in vitro antibacterial synergistic effects. The microbiological tests suggest that thyme and cloves EOs, rich in bioactive compounds, are able to inhibit the growth of tested foodborne bacteria, especially in vapor phase, also with synergistic effects. Results provide evidence to consider the tested essential oils as promising sources for development of new, broad-spectrum, green food preservatives.

In Vitro Antimycobacterial Activity of Human Lactoferrin-Derived Peptide, D-hLF 1-11, against Susceptible and Drug-Resistant Mycobacterium tuberculosis and Its Synergistic Effect with Rifampicin.

Tuberculosis is a highly contagious disease caused by the Mycobacterium tuberculosis complex (MTBC). Although TB is treatable, multidrug-resistant, extensively drug-resistant, and totally drug-resistant forms of M. tuberculosis have become a new life-threatening concern. New anti-TB drugs that are capable of curing these drug-resistant strains are urgently needed. The purpose of this study is to determine the antimycobacterial activity of D-enantiomer human lactoferricin 1-11 (D-hLF 1-11) against mycobacteria in vitro using a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide colorimetric assay, resazurin microplate assay, and microscopic observation drug susceptibility assay. Three previously described antimicrobial peptides, protegrin-1, AK 15-6, and melittin, with potent anti-TB activity, were included in this study. The findings suggest that D-hLF 1-11 can inhibit the growth of M. tuberculosis with a minimum inhibitory concentration of 100-200 µg/mL in susceptible, isoniazid (INH)-monoresistant, rifampicin (RF)-monoresistant, and MDR strains. The peptide can also inhibit some nontuberculous mycobacteria and other MTBC in similar concentrations. The antibiofilm activity of D-hLF 1-11 against the biofilm-forming M. abscessus was determined by crystal violet staining, and no significant difference is observed between the treated and untreated biofilm control. The checkerboard assay was subsequently carried out with M. tuberculosis H37Rv and the results indicate that D-hLF 1-11 displays an additive effect when combined with INH and a synergistic effect when combined with RF, with fractional inhibitory concentration indices of 0.730 and 0.312, respectively. The red blood cell hemolytic assay was initially applied for the toxicity determination of D-hLF 1-11, and negligible hemolysis (<1%) was observed, despite a concentration of up to 4 mg/mL being evaluated. Overall, D-hLF 1-11 has potential as a novel antimycobacterial agent for the future treatment of drug-sensitive and drug-resistant M. tuberculosis infections.

Antibacterial Activity of Syzygium aromaticum (Clove) Bud Oil and Its Interaction with Imipenem in Controlling Wound Infections in Rats Caused by Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infection worldwide. Clove oil's ability to inhibit the growth of MRSA was studied through in vitro and in vivo studies. The phytochemical components of clove oil were determined through gas chromatography-mass spectrometry (GC-MS) analysis. The antibacterial effects of clove oil and its interaction with imipenem were determined by studying MIC, MBC, and FIC indices in vitro. The in vivo wound-healing effect of the clove oil and infection control were determined using excision wound model rats. The GC-MS analysis of clove oil revealed the presence of 16 volatile compounds. Clove oil showed a good antibacterial effect in vitro but no interaction was observed with imipenem. Clove bud oil alone or in combination with imipenem healed wounds faster and reduced the microbial load in wounds. The findings of this study confirmed the antibacterial activity of clove oil in vitro and in vivo and demonstrated its interaction with imipenem.

Methanolic extract of Hemidesmus indicus root augments the antibacterial and antibiofilm activity of amoxicillin and clindamycin against methicillin-resistant Staphylococcus aureus of bovine origin.

The present study evaluated the antibacterial and antibiofilm activity of methanolic extract of Hemidesmus indicus root (MHIR) in combination with amoxicillin and clindamycin against biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk samples of mastitic cows. Microdilution susceptibility testing and microtitre plate assays were used to evaluate the invitro efficacy of MHIR and antibiotic combinations against MRSA (n = 12). Furthermore, invitro findings were validated in a murine model. Minimum inhibitory concentration and minimum biofilm inhibitory concentration of amoxicillin and clindamycin in combination with MHIR were significantly (P < 0·05) lower than when used alone against MRSA. In terms of antibacterial activity, MHIR showed additive interaction (fractional inhibitory concentrationindex >0·5-4) with amoxicillin and clindamycin against all the MRSA isolates, whereas MHIR synergizes (fractional biofilm inhibitory concentrationindex ≤0·5) the antibiofilm activity of amoxicillin and clindamycin against 58·33% and 83·33% of the MRSA isolates respectively. Amoxicillin/clindamycin in combination with MHIR significantly (P < 0·05) reduced disease activity score, and bacterial load and Gram-positive spots in kidney and liver of MRSA-infected mice. The combined efficacy of MHIR and amoxicillin/clindamycin was comparable to clindamycin alone but superior to amoxicillin alone. Hence, the combination of MHIR with amoxicillin/clindamycin is advocated in the treatment of MRSA-associated infections.

SINERGISTIC COMBINATION OF ALOE VERA AND KERSEN LEAF EXTRACTS FOR INHIBITION OF THE GROWTH OF Staphylococcus aureus 25923

Aloe vera leaf (Aloe vera L.) and cherry leaf (Muntingia calabura L.) contain bioactive compounds that serve as the potential antibacterial agent. The combination treatment of multiple extracts can produce a stronger effect than a single compound. This study aims to determine the synergistic combination of aloe vera leaf extract (EDLB) and cherry leaf extract (EDK) as an antibacterial agent against Staphylococcus aureus ATCC 25923. The extract was obtained by maceration using 96% ethanol. Chemical content in the extract was analyzed by tube test and thin layer chromatography (TLC). Antibacterial activity against S. aureus ATCC 25923 was measured by the checkerboard microdilution method on a 96 well microplate. The effect of the combination of extracts on antibacterial activity was based on the fractional inhibitory concentration index (FICI) value. EDLB and EDK contain flavonoids, tannins, alkaloids, and saponins. The second combination showed a synergistic effect with a FICI value of 0.5 in inhibiting the growth of S. aureus with minimal inhibitory levels in a mixture of 0.625 mg/mL EDLB and 2.5 mg/mL EDK.

Characterization and Isolation of the Major Biologically Active Metabolites Isolated from Ficus retusa and Their Synergistic Effect with Tetracycline against Certain Pathogenic-Resistant Bacteria.

Globally, pathogenic microbes have reached a worrisome level of antibiotic resistance. Our work aims to identify and isolate the active components from the bioactive Ficus retusa bark extract and assess the potential synergistic activity of the most major compounds' constituents with the antibiotic tetracycline against certain pathogenic bacterial strains. The phytochemical screening of an acetone extract of F. retusa bark using column chromatography led to the identification of 10 phenolic components. The synergistic interaction of catechin and chlorogenic acid as the most major compounds with tetracycline was evaluated by checkerboard assay followed by time-kill assay, against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, and Salmonella typhi with fraction inhibitory concentration index values (FICI) of 0.38, 0.43, 0.38, 0.38, 0.38, and 0.75 for catechin and 0.38, 0.65, 0.38, 0.63, 0.38, and 0.75 for chlorogenic acid. The combination of catechin and chlorogenic acid with tetracycline significantly enhanced antibacterial action against gram-positive and gram-negative microorganisms; therefore, catechin and chlorogenic acid combinations with tetracycline could be employed as innovative and safe antibiotics to combat microbial resistance.

Prevalence of classes 1 and 2 integrons in multidrug-resistant Acinetobacter baumanni isolates recovered from some aquatic environment in South Africa

The emergence of antibiotic-resistance genes (ARGs) by means of integrons in multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) has become a significant challenge in the management of infections from this pathogen. In this paper, we report on the variable region of class 1 and 2 integrons observed in MDR A. baumanni isolates recovered from rivers in the Eastern Cape Province, South Africa. Class 1 and 2 integrons with their variable regions were evaluated with polymerase chain reaction techniques followed by sequencing. Antibiotic sensitivity testing, checkerboard assay, time-kill independent assay, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) were carried out using standard microbiological techniques. A total of fifty-six (56) isolates were examined, among which 45 (79%) tested positive for class 1 integron, and 7 (12.3%) had class 2 integron. None was found to be class 3 integron positive among the isolates. The variable region contained aadA1, aadA5, and aadA2 genes, which confer resistance against streptomycin and spectinomycin, aac(6′)-Ib against amikacin/ tobramycin and dfrA17 genes against trimethoprim. The minimum inhibitory concentrations of the antimicrobials for one of the tested organisms were resistant against meropenem, colistin sodium methanesulfonate, tetracycline, ceftazidime, and ciprofloxacin (16, > 16, > 8, > 256, and 128 ug/mL respectively). The impact of colistin combined with quinolones (ciprofloxacin), with the FICIs (0.31) indicated synergistic effects against MDR A baumanni. However, when colistin was combined with meropenem and ceftazidime, additive effects with fractional inhibitory concentration (FIC) index ranging from 0.52 to 1 were observed. No antagonistic effect was evaluated among the examined isolates. ERIC-PCR analyses of A. baumanni isolates revealed significant genetic diversity, suggesting various sources of environmental contamination. We conclude that A. baumanni harbouring class 1 integrons in aquatic milieus are a significant source of ARGs and can transmit these elements to other organisms and consequently to man with significant public health implications.

The synergy of thanatin and cathelicidin-BF-15a3 combats Escherichia coli O157:H7

Escherichia coli O157:H7 is a pathogen that commonly causes foodborne illness and represents a health hazard to consumers. The combined use of synergistic antimicrobial peptides (AMPs) is a promising way to improve the microbiological safety of foods. In this study, we detected the synergistic interactions between thanatin and BF-15a3 to reduce their usage and obtain more efficient antibacterial activity. The minimal inhibitory concentrations (MICs) of thanatin and BF-15a3 against 49 E. coli O157:H7 strains were ranged from 2 to 8 μg/mL and 4–32 μg/mL, showed a general inhibitory effect on E. coli O157:H7 strains, respectively, even multidrug-resistant strains. Their fractional inhibitory concentration index (FICI) was 0.375, which suggested that their combination presented synergistic antibacterial effect against E. coli O157:H7. The killing kinetic curves indicated that the 0.25 × MIC combination had equivalent bactericidal effects to 1 × MIC thanatin or BF-15a3. When AMP combinations were used to treat eukaryotic cells to evaluate the hemolytic characteristics against rabbit erythrocytes and cytotoxicity against human embryonic kidney 293T (HEK-293T) cells and intestinal porcine enterocyte J2 (IPEC-J2) cells, no magnified adverse effects were observed, exhibiting higher specificity to bacteria and lower toxicity to eukaryotic cells. Compared with bacteriostasis of thanatin or BF-15a3 alone, the proportion of membrane-damaged bacteria treated with the synergetic combination did not appear a significant rise, interestingly the Zeta potential of them greatly decreased and their cell membrane permeability significantly increased. Besides, more release of ions and cytoplasm were detected, confirming a more severe loss of membrane integrity. These results suggested that the synergistic action mode of thanatin and BF-15a3 is likely attributed to damage aggravation to E. coli membrane. When applying in fresh-cut lettuce and cucumber, their combination allowed for 2.5 log CFU/piece reductions of E. coli O157:H7 in 24 h. In conclusion, the combination of thanatin and BF-15a3 showed excellent synthetic efficacy to kill E. coli O157:H7 in vitro under lower MICs than single use of them.

Characterization of Wild Turmeric and Holy Basil Combination Herbal Extracts and Their Synergistic Effect on the Antibacterial Properties of Knitted Fabrics

ABSTRACT In this work, the characterization of combination herbal extracts of wild turmeric (WT) and holy basil (HB) and their synergistic effect on the antibacterial properties of finished single jersey knitted fabrics of cotton, lyocell and micro-denier polyester were analyzed. The main objective of this work is to find out the influence of combination herbal extract proportion, type of material and their interactive effect on the antibacterial properties. The quantitative antibacterial test (AATCC 100) results of the combination herbal extracts-treated single jersey knitted fabrics show that the 50%WT: 50%HB proportion showed consistent bacterial reduction performance against both Staphylococcus aureus and Escherichia coli bacterial strains in all the materials as supported from the test results of phytochemical screening, Minimum Inhibitory Concentration, Fractional Inhibitory Concentration index for synergy, Antioxidant, Cytotoxicity and GC-MS analysis. In terms of material, lyocell and micro-denier polyester fabrics showed good results in 12 hr and 24 hr contact time against S. aureus. Against E. coli, cotton and micro-denier polyester showed good results in 12 hr and 24 hr contact time, respectively.

Candida albicans Reactive Oxygen Species (ROS)-Dependent Lethality and ROS-Independent Hyphal and Biofilm Inhibition by Eugenol and Citral.

Candida albicans is part of the normal human flora but is most frequently isolated as the causative opportunistic pathogen of candidiasis. Plant-based essential oils and their components have been extensively studied as antimicrobials, but their antimicrobial impacts are poorly understood. Phenylpropenoids and monoterpenes, for example, eugenol from clove and citral from lemon grass, are potent antifungals against a wide range of pathogens. We report the cellular response of C. albicans to eugenol and citral, alone and combined, using biochemical and microscopic assays. The MICs of eugenol and citral were 1,000 and 256 μg/mL, respectively, with the two exhibiting additive effects based on a fractional inhibitory concentration index of 0.83 ± 0.14. High concentrations of eugenol caused membrane damage, oxidative stress, vacuole segregation, microtubule dysfunction and cell cycle arrest at the G1/S phase, and while citral had similar impacts, they were reactive oxygen species (ROS) independent. At sublethal concentrations (1/2 to 1/4 MIC), both oils disrupted microtubules and hyphal and biofilm formation in an ROS-independent manner. While both compounds disrupt the cell membrane, eugenol had a greater impact on membrane dysfunction. This study shows that eugenol and citral can induce vacuole and microtubule dysfunction, along with the inhibition of hyphal and biofilm formation. IMPORTANCE Candida albicans is a normal resident on and in the human body that can cause relatively benign infections. However, when our immune system is severely compromised (e.g., cancer chemotherapy patients) or underdeveloped (e.g., newborns), this fungus can become a deadly pathogen, infecting the bloodstream and organs. Since there are only a few effective antifungal agents that can be used to combat fungal infections, these fungi have been exposed to them over and over again, allowing the fungi to develop resistance. Instead of developing antifungal agents that kill the fungi, some of which have undesirable side effects on the human host, researchers have proposed to target the fungal traits that make the fungus more virulent. Here, we show how two components of plant-based essential oils, eugenol and citral, are effective inhibitors of C. albicans virulence traits.

Synergistic Effects of Some Methoxyflavones Extracted from Rhizome of Kaempferia parviflora Combined with Gentamicin against Carbapenem-Resistant Strains of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

The present study aimed to investigate the antibacterial activity of ethanolic Kaempferia parviflora extracts and the combined effects of the plant's specific compounds with gentamicin against clinical strains of carbapenem-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The minimal inhibitory concentrations (MIC) of gentamicin and Kaempferia parviflora extracts against the tested bacterial strains were determined by using broth microdilution. The combined effects of Kaempferia parviflora extract and gentamicin were investigated by using a checkerboard assay and expressed as a fractional inhibitory concentration index (FICI). Crude ethanolic extract of Kaempferia parviflora showed the lowest median values of MIC towards the tested isolates (n = 10) of these tested bacteria at doses of 64 µg/mL, compared to those of other Kaempferia extracts. Among the isolated compounds, only three compounds, namely 3,5,7-trimethoxyflavone, 3,5,7,3'4'-pentamethoxyflavone, and 5,7,4'-trimethoxyflavone, were identified by NMR structural analysis. According to their FICIs, the synergistic effects of gentamicin combined with 3,5,7,3'4'-pentamethoxyflavone were approximately 90%, 90%, and 80% of tested carbapenem-resistant Klebsiella pneumoniae (CRKP), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB), respectively. The present study concluded that 3,5,7,3'4'-pentamethoxyflavone extracted from Kaempferia parviflora potentiated the antibacterial action of gentamicin to combat bacterial resistance against the tested bacteria.

In vitro synergistic interaction between Melaleuca armillaris essential oil and erythromycin against Staphylococcus aureus isolated from dairy cows.

Staphylococcus aureus frequently causes subclinical mastitis around the world with a high impact on the milk industry and public health. Essential oils (EO) are recognized antimicrobials that can be synergistic with antibiotics. The main objective of this study was to evaluate the essential oil (EO) of Melaleuca armillaris as an adjuvant of erythromycin (ERY) for the alternative treatment of bovine mastitis caused by S. aureus. The Minimum Inhibitory and Bactericidal Concentrations (MIC and MBC) of EO, ERY, and its combinations were established against S. aureus at different pHs (7.4, 6.5 and 5.0), emulating extra and intracellular conditions. Sensitive (N = 3) and resistant (N = 3) strains to ERY and S. aureus ATCC 29213 as control were used. Math models were applied to describe the antibacterial activity of EO and combinations EO-ERY. The EO was bactericidal against all the strains independently of the pH with a slight improvement in acid conditions. The synergism between EO and ERY was estimated by the Fractional Inhibitory Concentration Index (FIC) and by mathematical modeling of the bacterial killing data. Synergism was observed with ERY, where combinations had bactericidal activity also even with pH modification. M. armillaris EO is an interesting adjuvant for ERY, being a promissory option for further analysis of intracellular efficacy against S. aureus.