Synergistic Interaction between Boesenbergia rotunda (L.) Mansf. Essential Oil and Cloxacillin on Methicillin-Resistant Staphylococcus aureus (MRSA) Inhibition.

Currently, antibiotic resistance is widespread among bacteria. This problem requires greater awareness because bacterial resistance increases, reducing antibiotic use effectiveness. Consequently, new alternative treatments are needed because the treatment options for these bacteria are limited. This work aims to determine the synergistic interaction and mechanism of action of Boesenbergia rotunda essential oil (BREO) against methicillin-resistant Staphylococcus aureus (MRSA). Gas chromatography-mass spectrometry identified 24 BREO chemicals (GC-MS). The main components of BREO were ß-ocimene (36.73%), trans-geraniol (25.29%), camphor (14.98%), and eucalyptol (8.99%). BREO and CLX inhibited MRSA DMST 20649, 20651, and 20652 with a minimum inhibitory concentration (MIC) of 4 mg/mL and 512 µg/mL, respectively. The checkerboard method and the time-kill assay revealed synergy between BREO and CLX with fractional inhibitory concentration (FIC) <0.5 and log reduction >2log10 CFU/mL at 24 hours compared to the most effective chemical. BREO inhibited biofilm formation and increased membrane permeability. Exposure alone to BREO or in combination with CLX inhibited biofilm formation and increased cytoplasmic membrane (CM) permeability. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results revealed that alterations in the cell walls, cytoplasmic membrane, and leakage of intracellular components of MRSA DMST 20651 after treatment with BREO alone and in combination with CLX were observed. These results indicate that BREO synergizes and could reverse the antibacterial activity of CLX against MRSA strains. The synergy of BREO may lead to novel drug combinations that increase the effectiveness of antibiotics against MRSA.

Identification of Pathogenic and Opportunistic Yeasts in Pigeon Excreta by MALDI-TOF Mass Spectrometry and Their Prevalence in Chon Buri Province, Thailand.

Pigeon excreta can cause environmental and public health issues, particularly in urban and public areas. They are reservoirs of several human pathogens including fungi, bacteria, and viruses. Epidemiological data of pathogenic and opportunistic yeasts in pigeon droppings in Chon Buri, one of the most reputable tourist cities of Thailand, are scarce. The present study aimed to identify yeasts in pigeon droppings by MALDI-TOF mass spectrometry, and to study their prevalence in Chon Buri, Thailand. A total of 200 pigeon fecal samples were collected randomly from all 11 districts of Chon Buri. A sum of 393 yeast-like colonies were isolated on Sabourand's dextrose agar and CHROMagar media. These isolates were further confirmed for their species by MALDI-TOF MS. Twenty-four yeast species belonging to 11 different genera were identified in pigeon fecal samples. Candida spp., predominantly C. krusei (14.32%), were the most prevalent yeast species. Other yeast species, including C. glabrata (12.73%), C. metapsilosis (11.93%), Lodderomyces elongisporus (10.87%), C. tropicalis (7.16%), C. albicans (5.83%), and Cryptococcus neoformans (4.77%) were identified. This study provides valuable epidemiological data and diversity of yeasts in pigeon droppings in Chon Buri, Thailand, and also supports the use of MALDI-TOF MS for yeast identification and epidemiological surveillance.

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium.

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.

Optimizing Concentration of Polyethelene Glycol for Exosome Isolation from Plasma for Downstream Application.

BACKGROUND: Exosomes are ubiquitous extracellular nanovesicles secreted from almost all living cells that are thought to be involved in several important cellular processes, including cell-cell communication and signaling. Exosomes serve as a liquid biopsy tool for clinical and translational research. Although many techniques have been used to isolate exosomes, including ultracentrigation, size-exclusion chromatography, and immunocapturing-based techniques, these techniques are not convenient, they require expensive instrumentation, and they are unhandy for clinical samples. Precipitation techniques from available commercial kits that contain polyethelene glycol (PEG) are now widely used, but these kits are expensive, especially if a large number of biological samples are to be processed. OBJECTIVE: the purpose of this study is to compare and optimize the efficacy of different concentrations of PEG with two commercial kits ExoQuick (SBI) and Total Exosome Isolation (TEI) from Invitrogen in human plasma. METHODS AND MATERIALS: we determined exosome quantity, size distribution, marker expression, and downstream application. RESULTS: among the precipitation methods, we found the size of particles and concentrations with 10-20% PEG are similar to ExoQuick and better than TEI. Interestingly, we detected cfDNA with ExoQuick and 10-20% PEG but not TEI and 5% PEG. Moreover, 10% PEG detection of miR-122 and miR-16 expression was superior to ExoQuick and TEI. Furthermore, in proteomics results it also found the identified proteins better than commercial kits but there was a high level of contamination of other proteins in serum. CONCLUSIONS: together, these findings show that an optimal concentration of 10% PEG serves as a guide for use with clinical samples in exosome isolation for downstream applications.

Genome analysis of secondary metabolite­biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Specific and sensitive, ready-to-use universal fungi detection by visual color using ITS1 loop-mediated isothermal amplification combined hydroxynaphthol blue.

Being ubiquitous, fungi are common opportunistic pathogens to humans that can lead to invasive and life-threatening infections in immunocompromised individuals. Eukaryote-resembling cell membrane and filamentous branches make the fungal diagnosis difficult. This study therefore developed a ready-to-use ITS1 loop-mediated isothermal amplification combined with hydroxynaphthol blue (LAMP-HNB) for rapid, sensitive and specific colorimetric detection of universal fungi in all phyla. The ITS1 LAMP-HNB could identify every evolutionary phylum of fungi according to sequence analyses. We tested a total of 30 clinically relevant fungal isolates (representing three major human pathogenic phyla of fungi, namely Zygomycota, Ascomycota and Basidiomycota) and 21 non-fungal isolates, and the ITS1 LAMP-HNB properly identified all isolates, with a detection limit of as low as 4.6 ag (9.6 copies), which was identical to ITS1 and 18S rDNA PCR. The assays were also validated on the feasibility of point-of-care diagnostic with real food (dry peanuts, chili and garlics) and blood samples. Furthermore, the shelf life of our ready-to-use ITS1 LAMP activity (≥50%) was more than 40 days at 30 °C with 3-5% polyvinyl alcohol or glycerol additive. The results supported the ready-to-use ITS1 LAMP-HNB for simple detection of fungi contamination with high sensitivity in local and resource-constrained areas to prevent opportunistic fungal species infections.

Fluorometric Paper-Based, Loop-Mediated Isothermal Amplification Devices for Quantitative Point-of-Care Detection of Methicillin-Resistant Staphylococcus aureus (MRSA).

Loop-mediated isothermal amplification (LAMP) has been widely used to detect many infectious diseases. However, minor inconveniences during the steps of adding reaction ingredients and lack of simple color results hinder point-of-care detection. We therefore invented a fluorometric paper-based LAMP by incorporating LAMP reagents, including a biotinylated primer, onto a cellulose membrane paper, with a simple DNA fluorescent dye incubation that demonstrated rapid and accurate results parallel to quantitative polymerase chain reaction (qPCR) methods. This technology allows for instant paper strip detection of methicillin-resistant Staphylococcus aureus (MRSA) in the laboratory and clinical samples. MRSA represents a major public health problem as it can cause infections in different parts of the human body and yet is resistant to commonly used antibiotics. In this study, we optimized LAMP reaction ingredients and incubation conditions following a central composite design (CCD) that yielded the shortest reaction time with high sensitivity. These CCD components and conditions were used to construct the paper-based LAMP reaction by immobilizing the biotinylated primer and the rest of the LAMP reagents to produce the ready-to-use MRSA diagnostic device. Our paper-based LAMP device could detect as low as 10 ag (equivalent to 1 copy) of the MRSA gene mecA within 36-43 min, was evaluated using both laboratory (individual cultures of MRSA and non-MRSA bacteria) and clinical blood samples to be 100% specific and sensitive compared to qPCR results, and had 35 day stability under 25 °C storage. Furthermore, the color readout allows for quantitation of MRSA copies. Hence, this device is applicable for point-of-care MRSA detection.

Nitro-Carba test, a novel and simple chromogenic phenotypic method for rapid screening of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: In this study, a rapid and simple chromogenic method for screening of carbapenemase-producing Enterobacteriaceae (CPE), namely the Nitro-Carba test (NCT), was developed. METHODS: The NCT was validated using a total of 31 carbapenemase-producing isolates [9 Klebsiella pneumoniae carbapenemase (KPC), 11 metallo-ß-lactamase (MBL) and 11 OXA-48] and 56 non-carbapenemase-producing isolates. The assay relies on the hydrolysis of nitrocefin by carbapenemases in the presence of carbapenem antibiotics. Carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial isolate were also investigated. RESULTS: The NCT detected all 31 CPE within a timeframe of only 10s to 12min. All carbapenemase-producers hydrolysed nitrocefin in all wells. No colour change in wells with carbapenems was observed in non-carbapenemase-producers. The sensitivity for all three carbapenems was 100%, whilst the specificity of IPM, MEM and ETP was 64.3%, 91.1% and 100%, respectively. IPM, MEM and ETP had minimum inhibitory concentrations (MICs) against all carbapenemase-producing strains ranging from 0.5µg/mL to ≥256µg/mL, 0.25µg/mL to ≥256µg/mL and 1µg/mL to ≥256µg/mL, respectively. OXA-48-producing isolates showed lower MICs compared with MBL- and KPC-producing isolates. CONCLUSION: This assay is a promising method for detecting CPE rapidly. The NCT is a simple and reliable method capable of detecting CPE even in carbapenem-susceptible strains.

A nitrocefin disc supplemented with ertapenem for rapid screening of carbapenemase-producing Enterobacteriaceae.

Reliable, simple and rapid methods for laboratory detection of carbapenemases are important for an appropriate antibiotic administration. A nitrocefin disc containing ertapenem for rapid screening of carbapenemase production among Enterobacteriaceae is developed in the present study. A total of 87 molecularly-confirmed Enterobacteriaceae including 31 carbapenemase producers and 56 non-carbapenemase producers, were tested with nitrocefin discs supplemented with and without ertapenem (20 µg/disc). Nitrocefin discs with ertapenem successfully discriminated all 31 carbapenemase and all non-carbapenemase producers within 30 minutes. The sensitivity and specificity of the method were 100%. The minimum inhibitory concentrations (MICs) of ertapenem against all carbapenemase-producing isolates ranged from 1 to ≥256 µg/mL. This simple test could help to minimize the treatment failure and control the dissemination of infections caused by carbapenemase-associated resistant bacteria. It is a promising approach that could be performed routinely in any laboratory.

Boesenbergia rotunda (L.) Mansf. extract potentiates the antibacterial activity of some ß-lactams against ß-lactam-resistant staphylococci.

OBJECTIVES: The purpose of this study was to investigate the effect of Boesenbergia rotunda (L.) Mansf. extract (BRE) and peptidoglycan inhibitor antibiotics, alone and in combination, against ß-lactam-resistant staphylococci. METHODS: Antibacterial and synergistic activities of BRE alone and in combination with ampicillin (AMP), cloxacillin (CLX), cefazolin (CZO) or vancomycin (VAN) were evaluated against two ß-lactam-resistant Staphylococcus aureus (BRSA) isolates and one ß-lactam-resistant Staphylococcus epidermidis (BRSE) isolate. The activities were confirmed by killing curve assays. The preliminary antimicrobial action was elucidated by transmission electron microscopy (TEM) and cytoplasmic membrane (CM) permeability assay. RESULTS: All tested staphylococci were inhibited by BRE at a minimum inhibitory concentration (MIC) of 16µg/mL. Two BRSA strains showed high resistance to CLX, AMP and CZO, whilst BRSE was resistant to CLX and AMP. All tested isolates remained susceptible to VAN. Chequerboard assay demonstrated a fractional inhibitory concentration index (FICI) of 0.50 for the BRE+CLX combination against the BRSA strains. Killing curve determinations confirmed the antibacterial and synergistic activities. TEM revealed collapse of the CM in BRE-treated cells and damage both of the CM and peptidoglycan (PG) in BRE+CLX-treated cells. The CM permeability assay showed that either BRE or nisin alone as well as BRE+CLX significantly induced leakage of OD260nm-absorbing materials. CONCLUSIONS: BRE potentiated the activity of ß-lactams, particularly CLX, against ß-lactam-resistant staphylococci by damaging the CM and PG layer, leading to leakage of intracellular material. Combination of BRE and ß-lactams provides a potential way forward in developing novel antistaphylococcal agents.

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended-spectrum-ß-lactamases, AmpC ß-lactamases and co-ß-lactamases in Enterobacteriaceae.

A promising means of rapid screening of extended-spectrum-ß-lactamase (ESBL), AmpC ß-lactamase, and co-production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed ß-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, nine co-producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co-ß-lactamase-producing Enterobacteriaceae are endemic.

A demonstration of athermal effects of continuous microwave irradiation on the growth and antibiotic sensitivity of Pseudomonas aeruginosa PAO1.

Stress, caused by exposure to microwaves (2.45 GHz) at constant temperature (37 ± 0.5°C), alters the growth profile of Pseudomonas aeruginosa PAO1. In the absence of microwave treatment a simple, highly reproducible growth curve was observed over 24 h or more. Microwave treatment caused no reduction in growth during the first 6 h, but at a later stage (>12 h) the growth was markedly different to the controls. Secondary growth, typical of the presence of persisters clearly became apparent, as judged by both the dissolved oxygen and the cell density profiles. These treated cells showed distinct morphological changes, but on regrowth these cells reverted to normal. The microwave induced persisters were subject to antibiotic challenge (tobramycin) and showed increased sensitivity when compared to the unstressed planktonic cells. This is in marked contrast to antibiotic induced persisters which show increased resistance. This provides evidence for both a nonthermal effect of microwaves and a previously undescribed route to a novel form of antibiotic susceptible persister cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:37-44, 2017.

Phenotypic detection of AmpC ß-lactamases, extended-spectrum ß-lactamases and metallo-ß-lactamases in Enterobacteriaceae using a resazurin microtitre assay with inhibitor-based methods.

Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC ß-lactamase, extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL) is clinically significant. A simple and relatively quick method for the detection of these resistance phenotypes would greatly improve chemotherapeutic recommendation. This technology would provide valuable input in our surveillance of resistance on a global stage, particularly if the methodology could be applicable to resource-poor settings. A resazurin microtitre plate (RMP) assay incorporating cloxacillin, clavulanic acid and EDTA for the rapid phenotypic identification of AmpC, ESBL and MBL and the co-existence of ß-lactamases has been developed. A total of 47 molecularly characterized Enterobacteriaceae clinical isolates producing AmpCs, ESBLs, co-producers of ESBL and AmpC, MBLs and co-producers of ESBL and MBL were phenotypically examined using the RMP assay. The ceftazidime- and cefotaxime-based RMP assays successfully detected all 16 AmpC, 14 ESBL and 9 MBL producers, 6 ESBL-AmpC co-producers and 2 ESBL-MBL co-producers without false-positive results. The ceftazidime-based assay was more reliable in detecting AmpC alone, while the cefotaxime-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in the detection of ESBL and MBL producers. The findings of the present study suggest that use of the RMP assay with particular ß-lactamase inhibitors explicitly detects three different ß-lactamases, as well as co-existence of ß-lactamases, within 6 h of initial isolation of the pathogen. This assay is applicable to carry out in any laboratory, is cost-effective and is easy to interpret. It could be implemented in screening patients and controlling infection and for surveillance purposes.

The synergy and mode of action of quercetin plus amoxicillin against amoxicillin-resistant Staphylococcus epidermidis.

BACKGROUND: Staphylococcus epidermidis is one of the most multiple resistances to antibiotics in the recent years. Therefore, practically-prescribed antibiotics in the treatment of these strains are not effective. Plant-derived antibacterial is one of the most interesting sources of new therapeutics. The present study was to investigate antibacterial, synergy and modes of action of quercetin and amoxicillin against amoxicillin-resistant Staphylococcus epidermidis (ARSE). METHODS: The MICs, checkerboard assay, viability curves, cytoplasmic membrane (CM) permeability, enzyme assay, transmission electron microscopy, confocal microscopy and FT-IR microspectroscopy measurement was performed. RESULTS: The MICs of amoxicillin, penicillin, quercetin and kaempferol against all ARSE strains were 16, 200, 256-384 and >1024 µg/mL respectively. Synergistic effects were exhibited on amoxicillin plus quercetin and penicillin plus kaempferol against these strains at FIC index 0.50 and <0.38 respectively. The synergistic activity of quercetin plus amoxicillin was confirmed by the viable count. This combination increased CM permeability, caused marked morphological, peptidoglycan and cytoplasmic membrane damage, increased protein amide I and II, but decreased fatty acid in bacterial cells. The quercetin had an inhibitory activity against ß-lactamase. CONCLUSIONS: So, these findings are the first report that quercetin has the synergistic effect with amoxicillin against ARSE via four modes of actions, inhibit peptidoglycan synthesis and ß-lactamases activity, increase CM permeability and protein amide I and II but decrease fatty acid in bacterial cells. Of course, this flavonol has the dominant potential to develop a brand-new collateral phytochemical agent plus amoxicillin to treat ARSE. Future work should focus on the bioavailability, efficacy and toxicity in animal and human studies, as well as, the synergistic effect on blood and tissue should be evaluated and achieved.

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus.

BACKGROUND: Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin. RESULTS: The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 µg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 µg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5 x 10(5) cfu/ml to 10(3) cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 µg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against ß-lactamase in concentrations manner. CONCLUSIONS: So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits ß-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.