Antimicrobial and antibiofilm activity of highly soluble polypyrrole against methicillin-resistant Staphylococcus aureus.

AIMS: The purpose was to evaluate the antimicrobial activity of highly soluble polypyrrole (Hs-PPy), alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. METHODS AND RESULTS: Ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 µg ml-1 and bactericidal action at 62.5 µg ml-1, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. CONCLUSIONS: Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

Multidrug Efflux System-mediated resistance in Staphylococcus aureus under a One Health approach.

The aim of the study was to track the spread of antimicrobial resistance among the different sectors of One Health through the detection of Multidrug-Efflux-System in multidrug-resistant Staphylococcus aureus isolates. Multidrug-resistant (MDR) and methicillin-resistant (MRSA) S. aureus isolates were selected: 25 of human, one of animal and eight of food origin. The efflux system genes norA, norB, norC, LmrS, tet38 and msrA were screened by PCR. The activity of the efflux systems was determined by the minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin in the presence and absence of CCCP and in the quantification of ethidium bromide efflux. Furthermore, biofilm formation was determined in the presence and absence of the CCCP. The molecular epidemiology of the isolates was traced with the aid of PFGE. The gene norC was the most prevalent, detected in all isolates and msrA was the least prevalent, detected in only two isolates from humans. There was no difference in the MICs of tetracycline and ciprofloxacin in the presence of CCCP, but 55.9% of isolates showed ethidium bromide efflux. The presence of CCCP decreased the biofilm formation. Regarding the molecular epidemiology, in three clusters was a mixture of the isolates from different origins. Therefore, S. aureus MDR with active multidrug efflux systems are circulating between One Health domains and it is necessary to consider strategies to decrease this circulation in order to prevent the dissemination of resistance mediated by MES.

Profiles of Staphyloccocus aureus isolated from goat persistent mastitis before and after treatment with enrofloxacin.

BACKGROUND: Staphylococcus aureus is one of the main causative agents of mastitis in small ruminants. Antimicrobial use is the major treatment, but there are many flaws linked to resistance, tolerance or persistence. This study aimed to verify changes in resistance, virulence and clonal profiles of S. aureus isolated from persistent mastitis goat milk before and after enrofloxacin treatment. RESULTS: MIC increased to at least one antimicrobial in S. aureus isolates after enrofloxacin treatment compared to before. The most detected resistance genes before and after treatment were tetK, tetM, and blaZ, with more resistance genes detected after enrofloxacin treatment (p < 0.05). Occasional variations in efflux system gene detection were observed before and after treatment. Nine virulence genes (hla, fnbA, fnbB, eta, etb, sea, sec, seh, and sej) were detected at both times, and between these, the hla and eta genes were detected more in isolates after treatment. All isolates of S. aureus belonged to the same sequence type (ST) 133, except for two S. aureus isolates prior to enrofloxacin treatment which were classified as ST5 and the other as a new one, ST4966. Isolates of S. aureus 4, 8, and 100 from before and after treatment had identical pulse types, while others obtained from other animals before and after treatment were classified into distinct pulse types. CONCLUSION: There were occasional changes in the studied profiles of S. aureus isolated before and after treatment of animals with enrofloxacin, which may have contributed to the permanence of bacteria in the mammary gland, even when using traditional treatment, resulting in persistent mastitis.

Biofilm-Forming Ability and Clonality in Acinetobacter baumannii Strains Isolated from Urine Samples and Urinary Catheters in Different European Hospitals.

OBJECTIVE: Biofilm formation has been associated with the persistence of Acinetobacter baumannii in hospital settings and its propensity to cause infection. We investigated the adhesion ability and clonality of 128 A. baumannii isolates recovered from urine and urinary catheters of patients admitted to 5 European hospitals during 1991-2013. METHODS: Isolates identification was confirmed by rpoB sequencing and by the presence of blaOXA-51. The presence of carbapenemases was detected by PCR. Clonality was determined by Sequence Group (SG) identification, Pulsed field gel electrophoresis (PFGE) and Multilocus sequence typing. Adhesion ability was defined by quantitative biofilm production assay and biofilms were characterized by Confocal Laser Microscopy and Scanning Electron Microscopy. RESULTS: The 128 isolates, either resistant (85.9%) or susceptible (14.1%) to carbapenems, and belonging to 50 different PFGE types and 24 different STs, were distributed among SG1 (67.2%), SG2 (10.2%) and other allelic profiles (22.7%). ST218 was the most frequent ST, corresponding to 54,5% of the isolates collected between 2011 and 2013. Among the 109 isolates showing resistance to at least 1 carbapenem, 55% revealed the presence of an acquired carbapenem-hydrolyzing class D - lactamases (CHDL): blaOXA-23 were the most frequent gene detected from 2008 onwards (75%). Among all the clinical isolates, 42.2% were strong biofilm producers, with the older isolates having the highest adhesion ability. Most isolates recovered later, belonging to ST218 and harbouring blaOXA-23, were homogeneously less adhesive. CONCLUSIONS: An evolution towards a decrease in adhesion ability and a CHDL content change was observed along the years in several European countries.

Antibacterial Activity of 7-Epiclusianone and Its Novel Copper Metal Complex on Streptococcus spp. Isolated from Bovine Mastitis and Their Cytotoxicity in MAC-T Cells.

Mastitis is an inflammation of mammary gland parenchyma that adversely affects bovine health and dairy production worldwide despite significant efforts to eradicate it. The aim of this work was to characterize the antimicrobial activity of 7-epiclusianone (7-epi), a compound extracted from the Rheedia brasiliensis fruit, its complex with copper against Streptococcus spp. isolated from bovine mastitis, and to assess their cytotoxicity to bovine mammary alveolar cells (MAC-T). The complex 7-epiclusianone-Cu (7-epi-Cu) was an amorphous green solid with optical activity. Its vibrational spectrum in the infrared region showed absorption bands in the high-frequency region, as well as bands that can be attributed to the unconjugated and conjugated stretching of the free ligand. The complex was anhydrous. One of the tested bacterial strains was not sensitive to the compounds, while the other three had MIC values of 7.8 µg mL-1 and minimum bactericidal concentration (MBC) values between 15.6 and 31.3 µg mL-1. These two compounds are bacteriostatic, did not cause damage to the cell wall and, at sub-inhibitory concentrations, did not induce bacterial adhesion. The compounds were not cytotoxic. Based on these results, 7-epi and 7-epi-Cu exhibited desirable antimicrobial properties and could potentially be used in bovine mastitis treatment.

Toothpaste containing TiF4 and chitosan against erosive tooth wear in situ.

OBJECTIVE: This study compared the protective effect of an experimental TiF4/Chitosan toothpaste with a commercial toothpaste on the prevention of erosive tooth wear (ETW) in situ. METHODS: Fifteen subjects took part in this crossover and double-blind study, in which they wore a palatal appliance containing 4 bovine enamel and 4 dentin in 3 phases (5 days each). Half of the samples were subjected to erosive challenges (90 s in 0.1 % citric acid, pH 2.5, 4 times/day), and the other half to erosive plus abrasive challenges (15 s plus 45 s of contact, 2 times/day). The phases corresponded to the application of the different toothpastes: 1) TiF4 (1400 ppm F-) plus Chitosan, 2) Elmex®, Erosion Protection (1400 ppm F-, Chitosan), and 3) Placebo (negative control). Tooth wear was measured using contact profilometry (µm) and submitted to two-way RM ANOVA/Tukey test (p < 0.05). RESULTS: No significant differences were detected between the experimental and commercial toothpastes, regardless of the challenge on both tissues. Both significantly reduce ETW compared to negative control (p < 0.0006). Tooth wear was increased by brushing only on eroded enamel (p < 0.01), but not on dentin (p = 0.6085). TiF4/Chitosan [erosion 2.98 ± 1.12 µm vs. erosion and abrasion 3.12 ± 1.33 µm] and Elmex® toothpastes [erosion 2.35 ± 0.93 µm vs. erosion and abrasion 2.98 ± 1.0 µm] minimized the impact of brushing compared to placebo on enamel [erosion 4.62 ± 1.48 µm vs. erosion and abrasion 5.15 ± 1.50 µm]. CONCLUSIONS: TiF4 plus chitosan toothpastes showed to be effective in minimizing the ETW as the commercial toothpaste is in situ. CLINICAL RELEVANCE: The experimental toothpaste has similar effect against ETW compared to the commercial toothpaste. Considering the increased ETW prevalence worldwide, this result supports clinical trials and a possible application of this experimental anti-erosive toothpaste in the future.

Synergism between molecules derived from Garcinia brasiliensis and antimicrobial drugs on field isolates of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the smallest free-living bacteria found in nature; it has an extremely small genome and lacks a cell wall. It is the main etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease with worldwide distribution that causes significant losses in swine production. Due to the great economic impact caused by EP, new strategies for treating and controlling this agent are researched. The objective of this study was to verify the anti-M. hyopneumoniae activity of compounds derived from Garcinia brasiliensis and the synergism with the main antimicrobials used in the treatment of EP; this is the first study assessing the synergism between bioactive molecules and antimicrobial compounds in vitro against isolates of M. hyopneumoniae. The minimum inhibitory concentrations (MICs) of the antimicrobials tiamulin, valnemulin, and enrofloxacin, as well as the bioactive compounds guttiferone-A (Gut-A), 7-epiculsone (7-Epic), copper 7-epiculsone (7-Epic-Cu), and benzophenone, were determined. Subsequently, the interactions of antibiotics with the compounds were evaluated using the checkerboard method. Three field M. hyopneumoniae isolates were used, and the J strain was used as a control. The MIC values of the antimicrobials compared to the field isolates were equal to and lower than those of the reference strain J. Among the compounds used, 7-Epic-Cu showed the lowest MIC value. Synergistic association was observed for Gut-A with tiamulin and valnemulin, whereas 7-Epic and 7-Epic-Cu showed synergistic action with enrofloxacin. No synergistic effect was observed for benzophenone. Despite being an initial study, the results suggest that these combinations hold promise for the treatment of infections caused by M. hyopneumoniae.

The draft genome of Staphylococcus warneri TRPF4, a bacteriocin producer with potent activity against the causative agent of Legionnaires' Disease.

In this work, we present the draft genome sequence of Staphylococcus warneri strain TRPF4 consisting of 2,634,550 bp with a G + C content of 32.4%. The genome sequence includes 2466 protein-coding genes, 11 rRNAs and 62 tRNAs, in 33 contigs. Applying the Rapid Annotation using Subsystem Technology (RAST) a total of 1322 protein-coding genes were assigned to 393 subsystems. Also, a set of 1286 protein-coding genes with designated functions were assigned to 21 categories in the Cluster of Orthologous Groups (COG) database. Further analysis of BAGEL3 software demonstrated that the TRPF4 genome contains two gene clusters responsible for the synthesis of three bacteriocins, one warnericin RK and two delta-lysins. Besides, a novel delta-lysin of 3.48 kDa was identified for the first time. The three predicted bacteriocins were chemically synthesized and screened for the antimicrobial activity against a range of pathogens, exhibiting a potent and specific antimicrobial activity counter to L. pneumophila, with minimum inhibitory concentrations (MIC) ranging from 1.9 to 7.8 µg mL-1. These results indicate that the strain TRPF4 can produce bacteriocins with anti-Legionella activity. This was verified by the extracting the bacteriocins from the fermentation broth and testing against L. pneumophila. Additionally, the strain TRPF4 exhibited no cytotoxicity in mammalian cell lines. In summary, the genomic sequences and in vitro assays demonstrated the potential application of bacteriocins from S. warneri TRPF4 as a scaffold for further development of drugs against L. pneumophila, the causative agent of Legionnaires' Disease.

Bacillus subtilis TR47II as a source of bioactive lipopeptides against Gram-negative pathogens causing nosocomial infections.

This study aimed to investigate the antimicrobial, antibiofilm, and cytotoxic effects of biosurfactant lipopeptides synthesized by Bacillus subtilis TR47II. For this purpose, the lipopeptides were partially purified using a three-step process and characterized. In the first step, the crude extract obtained from acid precipitation exhibited strong antibacterial activity against the Gram-negative opportunistic pathogens Alcaligenes faecalis ATCC 8750, Achromobacter xylosoxidans ATCC 13138, Pseudomonas alcaligenes ATCC 14909, and Pseudomonas putida ATCC 15175. Moreover, partial inhibition was observed against Klebsiella aerogenes ATCC 13048 (42%), Escherichia coli ATCC 25922 (16%), and Pseudomonas aeruginosa ATCC 27853 (47%). The lipopeptides in the crude extract were extracted with methanol and fractioned on a silica gel chromatography column, rendering four TLC-pooled chromatographic fractions, named F1, F2, F3, and F4. The chromatographic fraction F4 was the most bioactive, with MIC values between 300 and 600 µg mL-1. Besides, F4 at sub-MIC doses dislodged the biofilms of A. faecalis, A. xylosoxidans, and P. alcaligenes by about 100, 85, and 81%, respectively. No cytotoxic effect was observed in mammalian cells at MIC. MALDI-TOF-MS analysis revealed that F4 contained cyclic lipopeptides belonging to two families: iturins (m/z 1004 to 1087) and fengycins (m/z 1424 to 1545). The dual effect of F4 on planktonic and sessile growth could suggest that the synergistic application of these biosurfactants could be efficient in the control of these opportunistic pathogens.

Antimicrobial and Synergistic Activity of 2,2',4-Trihydroxybenzophenone Against Bacterial Pathogens of Poultry.

In poultry farming, the spread of bacterial pathogens results in disease outbreaks causing significant economic losses to this industry. Many of these pathogenic bacteria are zoonotic and have a substantial impact on public health. Antimicrobials are essential for the prevention and treatment of these bacterial infections. However, the indiscriminate use of these agents provides favorable conditions for selection, propagation and persistence of bacteria and development of antimicrobial resistance. We developed a new antimicrobial candidate that could be used alone or in synergy with research protocols for therapeutic, prophylactic and growth promoter uses in the poultry industry. The present study aimed at evaluating the antimicrobial activity of the synthetic compound 2,2',4-trihydroxybenzophenone against pathogenic bacteria that cause important diseases in poultry and public health. We tested the hemolytic effect of this compound, studied its synergistic effect with conventional antimicrobials and analyzed the site of action on the bacteria. The results of our study showed antimicrobial activity of benzophenone against Gram-positive and Gram-negative bacteria with a similar effect in ATCC (American type culture collection) and field isolates. This compound was non-hemolytic. 2,2',4-trihydroxybenzophenone acted on the bacterial cell wall. We identified the synergistic effect between 2,2',4-trihydroxybenzophenone and bacitracin, this effect indicate that antimicrobial synergism may be useful for the treatment of necrotic enteritis in poultry. This compound may also be used as a growth promoter by reducing the dose of bacitracin and thus decreasing the pressure of bacterial resistance in poultry which would circumvent the development of cross-resistance in humans.

Synergism of Plant Compound With Traditional Antimicrobials Against Streptococcus spp. Isolated From Bovine Mastitis.

Mastitis is an inflammation of the mammary gland that causes major losses in the dairy industry. Streptococcus spp. are among the main agents of this disease. Increased resistance to antibiotics is one of the causes of therapeutic failure. Plants, due to their broad chemodiversity, are an interesting source of new molecules with antibacterial activity. Using these compounds along with traditional antibiotics is a possible method for reversing resistance. The objective of this work was to determine the interactions between the activities of guttiferone-A and 7-epiclusianone, two active substances isolated from the fruits of Garcinia brasiliensis, and traditional antibiotics against Streptococcus spp. isolated from bovine mastitis and known to be resistant to them. First, the MIC for the antibiotics and bioactive compounds was determined, followed by their activities, alone and in combination. Then, their cytotoxicity was measured in bovine mammary epithelial cells. Finally, molecular docking simulations were performed to elucidate molecular details of the interactions between ß-lactamase and the compounds binding to it (clavulanic acid, ampicillin, 7-epiclusianone, and guttiferone-A). The bacterial isolates were resistant to ampicillin and gentamicin. Both antibiotics showed predominantly synergistic antibacterial activities in combination with guttiferone-A or 7-epiclusianone. These two active substances were not cytotoxic at synergistic concentrations and both showed strong binding to ß-lactamase, which may explain the reversal of ampicillin resistance. These substances are promising for the treatment of bovine mastitis.