Investigation of Biofilm-Associated Antibiotic Susceptibilities of Methicillin-Resistant Staphylococci Isolated from Catheter-Related Nosocomial Infections

Introduction: The primary aetiology of chronic periapical abscesses, including E. faecalis, S. mutans, S. sanguinis, and P. gingivalis, can be eliminated using intracanal medicaments such as calcium hydroxide. The purpose of this study was to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of calcium hydroxide paste in the vulnerable initial 7-day of treatment against E. faecalis ATCC 29212, S. mutans ATCC 25175, S. sanguinis ATCC 10556, and P. gingivalis ATCC 33277. Methods: An in-vitro laboratory experiment using a spectrophotometer as a microdilution method was conducted to determine bacteria's MIC and MBC values on calcium hydroxide paste. The sample used in this study was four bacteria using intracanal medicament of calcium hydroxide (Ca(OH)2), incubated for seven days at 37°C, and then bacterial growth was observed. The cell inhibition percentage was calculated using optical density measurements to determine the MIC value. The low MIC and MBC were defined as sensitive bacteria to calcium hydroxide. Results: Calcium hydroxide paste against E. faecalis (ATCC 29212) with MIC values at a concentration of 750 μg/ml and MBC values at a concentration of 96,000 μg ml; S. mutans (ATCC 25175) with MIC value at a concentration of 3,000 ug/ml and MBC value at a concentration of 48,000 ug/ml; S. sanguinis (ATCC 10556) with MIC value at a concentration of 3,000 ug/ml and MBC value at a concentration of 6,000 ug/ml; P. gingivalis (ATCC 33277) with MIC value at a concentration of 6,000 ug/ml and MBC value at a concentration of 48,000 ug ml. Conclusions: Calcium hydroxide can inhibit bacterial growth activity. E. faecalis (ATCC 29212) and S. sanguinis (ATCC 10556) are more sensitive to calcium hydroxide paste than other bacteria, with the lowest MIC and MBC on seven days of incubation since the maximum calcium and hydroxyl ions are released.

Most Burkholderia cepacia strains are resistant to many, or all, of the antibacterial agents commonly used in cystic fibrosis (CF), and selection of appropriate antibiotics for treatment of pulmonary exacerbations is therefore difficult. We developed a technique for rapid in vitro testing of multiple antibiotic combinations for B. cepacia isolates. For each of 119 multi-drug-resistant isolates of B. cepacia, our multiple combination bactericidal test (MCBT) studied the bactericidal activity of 10 to 15 antimicrobial agents using 225 +/- 97 single, double, and triple antibiotic combinations. Of the 119 isolates, 50% were resistant to all single antibiotics tested, 8% were resistant to all two-drug antibiotic combinations, but all were inhibited by at least one bactericidal triple-drug combination. When used alone, meropenem, ceftazidime and high-dose tobramycin (200 microg/ml) were bactericidal against only 47, 15, and 14% of in vitro isolates, respectively. Using a double antibiotic combination improved bactericidal activity; meropenem-minocycline, meropenem-amikacin, and meropenem-ceftazidime combinations were bactericidal against 76, 73, and 73% of isolates, respectively. However, 47% of isolates demonstrated antagonism (growth of an organism when a second antibiotic was added to a bactericidal single antibiotic). Triple antibiotic combinations that contained tobramycin, meropenem, and an additional antibiotic were most effective, and were bactericidal against 81 to 93% of isolates. We conclude that triple-antibiotic combinations are more likely than double and single antibiotic combinations to be bactericidal against B. cepacia in vitro. MCBT testing is a useful technique to help clinicians decide on appropriate nonantagonistic combination antibiotic therapy for patients with CF infected with B. cepacia.

Background: Nosocomial infections caused by methicillin-resistant Staphylococci could lead to increased morbidity and mortality, but little is known about the prevalence of infections with these organisms in healthcare facilities and in the community in Tripoli. This study investigated the in vitro susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase negative staphylococci (MRCNS) to antimicrobial agents, and determined the molecular characteristics of MRSA. Methods: This is a retrospective observational study aiming at determining the prevalence and antibiotic resistance pattern of (MRSA) and (MRCNS) isolated from non-duplicated clinical specimens in Tripoli Central Hospital (TCH) between June 2013 and June 2014. Isolates were identified using standard laboratory procedures. Antimicrobial susceptibility tests were carried out by disk diffusion method and automated systems. DNA of the MRSA isolates was used for PCR to determine the molecular analysis. Results: 218 isolates of Staphylococci were obtained, 71.6% were coagulase positive staphylococci (CPS) and 28.4% were coagulase negative staphylococci (CNS). 39.7% of CPS were MRSA, while 75.8% of CNS were MRCNS. The rates of hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) among MRSA isolates were 61.3% and 38.7% respectively. A similar trend was detected among MRCNS isolates, where 74.5% were HA-MRCNS and 25.5% were CA-MRCNS. All the MRSA and MRCNS isolates were susceptible (100%) to vancomycin, tigecycline, linezolid, quinupristin/dalfopristin, daptomycin and moxifloxacin. Generally, hospital-acquired strains showed higher resistance rates than community-acquired ones to the most commonly tested non-beta-lactam antibiotics. 35.5% of all staphylococcal isolates exhibited mecA+ gene and 12.9% expressed mecC+. Meanwhile, 38.7% of MRSA isolates harbored both mecA and mecC. However, 12.9% of MSSA isolates were negative for both mecA and mecC. The mecA gene was detectable in 59.1% and 40.9 % of HA-MRSA and CA-MRSA isolates respectively. Conclusion: Hospital-acquired MRSA and MRCNS isolates had higher resistance rates to non-beta lactam antimicrobial drugs than the respective community-acquired isolates. This was shown by early detection of mecC gene among MRSA isolates.

The effect of sulphur on the antibacterial properties of succinic acid-Cu(II) and mercaptosuccinic acid-Cu(II) MOFs

Background: Pseudomonas aeruginosa is an important environmental, opportunistic, and nosocomial pathogen with a significant threat to public health. Combination therapy has many advantages due to the simultaneous action of two drugs on two separate cellular targets. In the present study, the effect of the combination of doxycycline and natural products against planktonic cells, biofilm, and virulence factors of P. aeruginosa was evaluated. Methods: To perform this work, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of doxycycline and natural products were determined by broth microdilution method. The microtiter plate assay method was used to determine the minimal biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC). The effect of doxycycline and natural products against pyocyanin, swarming motility, and swimming motility was evaluated. The checkerboard method was used to evaluate the effect of the combination of doxycycline with natural products against planktonic and biofilm cells. Results: The MIC of doxycycline ranges between 2 µg/mL and 128 µg/mL with an average of 35.89 µg/mL. Sinapic acid shows the best inhibitory activity against planktonic cells with an average MIC of 27.79 µg/mL. At the sub-inhibitory concentrations, the pyocyanin production, swarming motility, and swimming motility decrease. Out of the six combinations tested, the combination formed by doxycycline and sinapic acid exhibited synergistic activity for the prevention of biofilm formation with a 7-fold reduction in MBIC of doxycycline. Conclusion: This study revealed that the doxycycline and sinapic acid combination could be considered as a promising candidate for the development of therapy against P. aeruginosa infections.

BackgroundAlthough the most widely accepted mechanism of action for polymyxins is related to bacterial lysis via disruption, we hypothesized that this antimicrobial drug class could have other effects on Pseudomonas aeruginosa planktonic and sessile cells. Little is known regarding oxidative burst and zeta potential (ZP) data associated with the interaction between polymyxin B and P. aeruginosa cells. The present study evaluated endogenous reactive oxygen species (ROS) production and changes in the net charges of biofilm and planktonic cells in response to polymyxin B.ResultsPolymyxin B induced concentration-dependent killing at all concentrations tested in planktonic and sessile cells from P. aeruginosa strains. Sublethal concentrations of polymyxin B induced oxidative burst. ROS production was higher in resistant planktonic cells than in biofilm cells but this was not observed for susceptible cells. Moreover, no net surface charge alterations were observed in planktonic cells from a susceptible strain treated with polymyxin B, but a significant increase of ZP was noted in planktonic cells from a resistant strain.ConclusionOxidative burst generated by planktonic and sessile cells from P. aeruginosa strains against polymyxin B indicates that ROS may have an important role in the mechanism of action of this drug. ZP data revealed that electrostatic interactions of the cationic peptide with the anionic surface of the cells are strain-dependent. Therefore, we suggested that the intracellular effects of polymyxin B should be further investigated to understand polymyxin B-induced stress in P. aeruginosa.

Effects of oregano essential oil and carvacrol on biofilms of Staphylococcus aureus from food-contact surfaces

IntroductionCryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity.MethodsIn the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans.ResultsBTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 μg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 μg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties.ConclusionThese results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.

Acinetobacter baumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living) and sessile (biofilm) forms of A . baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures) and sessile (biofilm) cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A . baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

The aim of this study was to investigate the susceptibility of Mutans streptococci in both planktonic and biofilm states to erythrosine.S. mutans was cultured in brain-heart infusion (BHI) broth.Erythrosine was diluted in BHI broth and prepared at a concentration range of 0.02 -10000 g/L.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured using the microdilution method.After forming biofilms on 96-well plates, the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were measured.S. mutans was susceptible to erythrosine in both planktonic and biofilm states.MIC and MBC values were both 19.5 g/L for the planktonic state, while MBIC and MBEC values were 313 g/L and 2500 g/L, respectively, for the biofilm state.Erythrosine (19.5 g/L) exhibited a bactericidal effect on S. mutans (killing 99.9%) in the planktonic state.For biofilms, erythrosine inhibited biofilm growth and eradicated 99.9% of biofilm bacteria at higher concentrations than MIC and MBC.These MBIC and MBEC concentrations are much lower than known noxious doses, and the MIC, MBC, and MBIC values were even lower than clinical concentrations.

ABSTRACTBackgroundMalassezia genus includes lipodependent commensal yeasts of humans and animals' skin and mucous membranes. It can cause dermatological pathologies, and azoles are mainly used for treatment. However, in vitro susceptibility testing has shown decreased sensitivity to these antifungals. Some publications have suggested that resistance mechanisms to azoles include biofilm formation and efflux pump expression, which are proteins encoded by the ATM1 gene, among others.ObjectiveThis work aimed to characterise Colombian isolates of Malassezia spp. resistant to azoles.MethodsTwenty‐six Malassezia spp. isolates were identified via PCR, ribosomal gene sequencing and phylogenetic analyses. Susceptibility tests were performed on planktonic and sessile cells by microdilution against azoles and by adding efflux pump inhibitors. The relative expression levels of the ATM1 gene in fluconazole‐resistant isolates were evaluated via RT‐qPCR.ResultsIt was observed that 42% of the isolates in their planktonic form were resistant to voriconazole, 31% to fluconazole, 23% to itraconazole and 15% to ketoconazole. The minimum inhibitory concentration (MIC) was higher in sessile cells than planktonic cells, especially for fluconazole. The MICs of itraconazole, ketoconazole and voriconazole decreased in the presence of haloperidol, promethazine and tacrolimus, while this effect did not occur with fluconazole. The expression of the ATM1 gene was markedly greater in Malassezia spp. isolates resistant to fluconazole than in those susceptible (p < 0.05), both in those exposed and not exposed to the antifungal agent.ConclusionsWe observed resistance of Colombian Malassezia spp. isolates to azoles, mainly fluconazole, through the expression of efflux pumps and biofilm formation.

Environmental resistance is an important factor for understanding the epidemiology of leptospirosis. Recently, new Leptospira hosts were identified, including also marine mammals. Moreover, halotolerant Leptospira strain, isolated from the environment and animals, highlighted the capability of this microorganism to persist in the seawater. The aim of this research was to investigate the bacteriostatic and bactericidal effect of salt on Leptospira strains belonging to 16 different serovars. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were verified through the microdilutions method starting from a 20% sodium chloride concentration. MIC values obtained were between 0.3125% and 10% of salt, while MBC values between 0.625% and >20%. Icterohaemorrhagiae (MIC: 0.3125%; MBC: 0.625%) resulted the most inhibited serovar, while the most resistant was Tarassovi (MIC: 10%; MBC: >20%). Interestingly, trends were reported for Pomona (MIC: 1.25%; MBC: >20%) and Bratislava (MIC: 0.625%; MBC: 20%), highlighting low MIC values but high MBC values. This is the first investigation aimed at the in vitro effect of salt on the growth of Leptospira spp. reference strains.

Aggregatibacter actinomycetemcomitans (Aa) plays a vital role in some destructive forms of periodontitis. While mechanical and chemical plaque control is the first step in periodontitis treatment, side effects of adjunctive chemical agents such as chlorhexidine (CHX) mouthwash have led to the application of natural alternatives with minimal side effects. Therefore, this study evaluated the antibacterial effect of the hydroalcoholic extract of Quercus infectoria (Qi) galls on Aa in vitro. The hydroalcoholic extract of Qi was obtained by the maceration method, and Aa bacterial strain was cultured. The inhibition zone diameter was measured through the agar well diffusion method. Also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined by the broth microdilution method. All the experiments were repeated three times. 0.2% CHX was used as a control. The inhibition zone diameter of Aa increased with increasing concentration of Qi extract. While MIC and MBC values for CHX were 0.0039 and 0.0078 mg/mL, respectively, both MIC and MBC values of the Qi extract for this bacterium were similar, i.e., 2.5 mg/mL, which was significantly higherd. Since other in vivo studies have confirmed the other properties of this extract and its safety in terms of cytotoxicity and mutagenicity, hydroalcoholic extract of Qi may be used in mouthwashes or local delivery systems to affect periodontal biofilm.

Estudio multicéntrico sobre la actividad in vitro de ceftarolina frente a Staphylococcus aureus aislados en España

The development of biofilms is responsible for 80% of microbial infections, which are highly resistant to conventional antibiotics. The study aimed to assess the antimicrobial activity of essential oils of Mimosa verrucosa Benth. and Illicium verum Hook.f. against planktonic and sessile cells and evaluate toxicity. Thirty compounds were identified, the main ones being a-pinene (12.6%), b-pinene (16.7%) and (E)-caryophyllene (14.2%) for M. verrucosa, and estragole (4.2%) and anethole (86.8%) for I. verum. The minimum inhibitory concentrations of the EO of I. verum (29.40 µg/ml) and M. verrucosa (24.89 µg/ml) against planktonic cells showed 99% efficacy against all cells tested (sensitive and resistant Staphylococcus aureus and Escherichia coli). In sessile cells, essential oils of I. verum and M. verrucosa showed efficacy against sensitive S. aureus. The minimum bactericidal concentration (MBC) test revealed that I. verum caused cell death in sensitive S. aureus and E. coli. However, M. verrucosa only showed bactericidal activity against planktonic cells. Considering the expanding resistance to antimicrobials, the EOs tested represent an important therapeutic option, especially against S. aureus and E. coli, which can produce biofilms on various surfaces, becoming a serious public health problem.