Log Reduction In Colony Forming Units Research Articles

Synergistic effects of polymyxin and vancomycin combinations on carbapenem- and polymyxin-resistant Klebsiella pneumoniae and their molecular characteristics.

The emergence and spread of polymyxin resistance, especially among Klebsiella pneumoniae isolates, threaten the effective management of infections. This study profiled for polymyxin resistance mechanisms and investigated the activity of polymyxins plus vancomycin against carbapenem- and polymyxin-resistant K. pneumoniae. The entire genome sequences of seven isolates were profiled for resistance and virulence determinants. The effects of combination therapy were evaluated using the checkerboard technique, time-kill assay, and population profile analysis. Protein profiles of the isolates treated with monotherapy were compared to that of combination therapy. The whole-genome sequencing data revealed that the isolates harbored β-lactams, carbapenems, aminoglycoside, fluoroquinolones, macrolides, and tetracycline resistance genes, with several virulence-associated genes. The plasmids including the bla OXA-232-bearing ColKP3 plasmid were also identified in our isolates. Profiling for polymyxin resistance mechanism revealed a missense mutation in the crrB gene that resulted in a Q180L variant that conferred a deleterious effect on protein function. The combination assay indicated fractional inhibitory concentration index ranging from 0.31 to 1.13, whereas the time-kill assay demonstrated synergistic log reduction in colony-forming units per milliliter. Furthermore, population analysis profiling using dual antibiotics indicated enhancement in bacterial log reduction at lower antibiotics concentrations, compared to higher concentrations of single polymyxins. For protein profiling, 796 proteins were identified, and 56 and 94 of them were increased and decreased in the combined drug treatment groups, respectively, while other differentially produced proteins were detected in all treatment groups, except for the control group. The results demonstrated that the vancomycin combination might benefit the antimicrobial activities of polymyxins. IMPORTANCE This study provides insights into the mechanisms of polymyxin resistance in K. pneumoniae clinical isolates and demonstrates potential strategies of polymyxin and vancomycin combinations for combating this resistance. We also identified possible mechanisms that might be associated with the treatment of these combinations against carbapenem- and polymyxin-resistant K. pneumoniae clinical isolates. The findings have significant implications for the development of alternative therapies and the effective management of infections caused by these pathogens.

Nanotherapeutic delivery of antibiotic cocktail enhances intra-macrophage killing of Mycobacterium marinum

Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in cold-blooded animals and is an opportunistic pathogen in humans. M. marinum is the closest genetic relative of the Mycobacterium tuberculosis complex and is a reliable surrogate for drug susceptibility testing. We synthesized and evaluated two nanoparticle (NP) formulations for compatibility with rifampicin, isoniazid, pyrazinamide, and ethambutol (PIRE), the front-line antimycobacterial drugs used in combination against active tuberculosis infections. Improved in vitro antimicrobial activity was observed with encapsulated rifampicin alone or in a cocktail of drugs formulated through co-encapsulation in amphiphilic polyanhydride NPs. Broth antimicrobial testing revealed that the encapsulation of PIRE in NP resulted in a significant increase in antimicrobial activity, with the benefit over soluble formulations at biologically relevant concentrations ranging from >10 to >3,000 fold. M. marinum-infected human macrophages treated with NP-PIRE were cleared of viable bacteria in 48 h following a single treatment, representing a >4 log reduction in colony-forming units and a >2,000-fold increase in antimicrobial activity. The amphiphilic polyanhydride nanoparticles demonstrated the ability to co-encapsulate PIRE antibiotics and enhance their antimicrobial activity against M. marinum in infected macrophages in culture and in vitro. These data suggest that polyanhydride nanoparticles are a promising nanotherapeutic for combatting Mycobacterium infections through improved intracellular targeting of encapsulated antibiotics.

A Novel Irrigant to Eliminate Planktonic Bacteria and Eradicate Biofilm Superstructure With Persistent Effect During Total Hip Arthroplasty.

Numerous studies have examined the use of topical and irrigation-related adjuvants to decrease the risk of periprosthetic joint infection (PJI) after total hip arthroplasty. Many issues related to their use remain to be investigated. These include cost, antibiotic stewardship, bactericidal effect on planktonic bacteria, host cytotoxicity, necessity to irrigate/dilute potentially cytotoxic agents after their application, and impact on biofilm. Bacterial strains of microorganisms were grown in optimal medium. After the growth phase, the organisms were exposed to the novel irrigation solution (XPerience) or phosphate buffer solution (PBS) for 5minutes before a neutralizing broth was added. The colony-forming units per milliliter and the log reduction in colony-forming units in the treated sample vs the control were then determined. Subsequently, biofilms of microorganisms were grown on hydroxyapatite-coated glass slides. Each slide was then exposed to irrigation solutions for various contact times. Biofilm quantification was performed and the log10 density of each organism was obtained. Invitro testing of the irrigant demonstrated 6-log reductions in planktonic bacteria in 5minutes, and 4-log to 8-log reductions in biofilms. Laboratory tissue testing has demonstrated minimal cytotoxic effects to host tissue allowing for solution to remain in contact with the host without need for subsequent irrigation, creating a barrier to biofilm for up to 5hours after its application. This novel irrigant demonstrates high efficacy against both planktonic bacteria and bacterial biofilms in laboratory testing. Large series invivo data are necessary to further establish its efficacy in reducing primary and recurrent surgical site infections.

Comparative Evaluation of the Antimicrobial Effect of Mangosteen, Triphala, Chitosan, and 2% Chlorhexidine on Mono- and Dual-Species Biofilms of - and Candida albicans: An in Vitro Study.

Objective:The purpose of this in vitro study was to compare the antimicrobial effectiveness of 2% chlorhexidine gel, 0.5% chitosan, ethanol extract of mangosteen pericarp, and Triphala used as intracanal medicaments against Enterococcus faecalis and Candida albicans in mono- and dual-species biofilms.Methods:Bioactive components in the ethanol extract of mangosteen pericarp and Triphala were evaluated by gas chromatography-mass spectrometry (GCMS). Quantitative assessment of the biofilm formations of E. faecalis and C. albicans and as a dual-species in the presence of test medicaments was carried out using a crystal violet (CV) assay in a microtiter plate. Following this, 246 single-rooted premolar teeth were collected, and root specimens were prepared. C. albicans and E. faecalis mono- and dual-species biofilms were grown in the root specimens. At the end of 21 days, the samples were divided into five groups and subjected to different types of medicaments: Control group- distilled water; Chlorhexidine group- 2% chlorhexidine gel; Chitosan group- 0.5% chitosan; Mangosteen group- ethanol extract of mangosteen pericarp; and Triphala group- ethanol extract of Triphala. Colony-forming units (CFUs) were assessed on the first and fifth day after medicament placement.Results:Microbial population reduction was measured by one-way analysis of variance, followed by post-hoc Tukey’s multiple comparison test (P<0.05). Chlorhexidine showed maximum log reduction in CFUs of microorganisms, followed by chitosan, which showed a similar log reduction (P>0.05) for both mono- and dual-species biofilms. However, in the mangosteen and Triphala extract groups, the CFU/mL for dual-species on both days did not have a significant reduction in count (P<0.05) when compared to chlorhexidine and chitosan.Conclusion:Chlorhexidine showed maximum antimicrobial activity, followed by chitosan, on both mono- and dual-species biofilms. Mangosteen and Triphala had good antimicrobial action on the mono-species biofilm.

Antibacterial photodynamic activity of photosensitizer-embedded alginate-pectin-carboxymethyl cellulose composite biopolymer films.

Antimicrobial photodynamic therapy (APDT) is a promising approach for treatment of wounds infected with antibiotic-resistant bacteria. In this approach, delivery of appropriate concentration of photosensitizer (PS) at the infected site is a critical step; it is therefore essential that PS need to be administered at the infected site in a suitable formulation. Here, we report preparation of PS-embedded composite biopolymer films and their photobactericidal properties against methicillin-resistant Staphylococcus aureus (MRSA) and biocompatibility. Sodium alginate (SA), pectin (PC), and carboxymethyl cellulose (CMC) were used for preparing films containing chlorin p6 (Cp6, anionic PS) or methylene blue (MB, cationic PS). Films containing 1% CMC (15 mm diameter; 110 ± 09 μm thickness) showed ~ 55% light transmission in 500 to 750 nm region and high swelling rate as indicated by ~ 38% increase in diameter within 1 h. Absorption spectroscopic studies of PS-embedded films revealed that while Cp6 existed mainly in monomeric state, MB existed in both dimeric and monomeric forms. MRSA incubated with the film for 1 h displayed substantial uptake of Cp6 and MB as indicated by the presence of Cp6 fluorescence and MB staining in cells under the microscope. Furthermore, photodynamic treatment (660 nm, 10 J/cm2) of MRSA with Cp6 embedded in film or free Cp6 resulted in ~ 3 log reduction in colony-forming units (cfu), whereas decrease in cfu was less (~ 1 log) for MB-embedded film than for free MB (~ 6 logs). Studies on human keratinocyte (HaCaT) cells showed that there was no significant change in the viability of cells when they were incubated with solubilized films (plain) for 24 h or subjected to treatment with PS-containing films followed by PDT. These results suggest that films are biocompatible and have potential application in photodynamic treatment of MRSA-infected wounds.

Spectrum of bactericidal action of amylmetacresol/2,4-dichlorobenzyl alcohol lozenges against oropharyngeal organisms implicated in pharyngitis.

PurposePharyngitis is commonly caused by a self-limiting upper respiratory tract infection (URTI) and symptoms typically include sore throat. Antibiotics are often inappropriately used for the treatment of pharyngitis, which can contribute to antimicrobial resistance, therefore non-antibiotic treatments which have broad antiseptic effects may be more appropriate. Amylmetacresol (AMC) and 2,4-dichlorobenzyl alcohol (DCBA) are present in some antiseptic lozenges and have established benefits in providing symptomatic relief and some in vitro antiviral action.MethodsSeven bacterial species associated with pharyngitis, namely Streptococcus pyogenes, Fusobacterium necrophorum, Streptococcus dysgalactiae subspecies equisimilis, Moraxella catarrhalis, Haemophilus influenza, Arcanobacterium haemolyticum and Staphylococcus aureus, were exposed to an AMC/DCBA lozenge dissolved in artificial saliva. In vitro bactericidal activity was measured as a log reduction in colony-forming units (CFUs).ResultsBactericidal activity was recorded against all organisms after 1 minute. Greater than 3 log10 reductions in CFUs were observed at 1 minute for S. pyogenes (log10 reduction CFU/mL ± SD, 5.7±0.1), H. influenza (6.1±0.1), A. haemolyticum (6.5±0.0) and F. necrophorum (6.5±0.0), at 5 minutes for S. dysgalactiae (6.3±0.0) and M. catarrhalis (5.0±0.9) and at 10 minutes for S. aureus (3.5±0.1).ConclusionAn AMC/DCBA lozenge demonstrated a greater than 99.9% reduction in CFUs against all tested species within 10 minutes, which is consistent with the time a lozenge remains in the mouth. Patients with uncomplicated bacterial pharyngitis may benefit from the antibacterial action of antiseptic AMC/DCBA lozenges. Furthermore, AMC/DCBA lozenges may be more relevant and appropriate than antibiotics for pharyngitis associated with a self-limiting viral URTI.

Efficacy of multipurpose contact lens solutions against ESBL-positive Escherichia coli, MRSA, and Candida albicans clinical isolates.

The antimicrobial effects of multipurpose contact lens solutions (MPSs) have been evaluated according to ISO 14729 standards. The aim of this study was to assess the efficacy of commercially available MPSs against extended-spectrum beta lactamases (ESBL)-producing Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans clinical isolates. Three commercially available contact lens solutions (Opti-Free Expresss, ReNu MultiPlus, and Solo Care Aqua) were tested against 18 ESBL-positive E. coli clinical strains, 20 MRSA clinical strains, and 20 C. albicans clinical strains. The stand-alone assays for bacteria and fungi were performed according to ISO 14729 criteria, and all samples were evaluated after 2, 4, and 24 hours of incubation. The numbers of viable microorganisms were evaluated by the plate-counting method. All MPSs demonstrated at least 3 log reduction in colony-forming units (CFU) for all bacterial isolates and 1 log reduction in CFU for all yeast isolates, which meets ISO 14729 standards. Although no statistically significant differences were obtained among MPSs for bacterial isolates, variable responses were observed against clinical isolates: 5% povidone-iodine was more effective compared with Solo Care Aqua for C. albicans clinical strains (P<0.05); and all solutions were effective after an incubation time of only 2 hrs. The MPSs showed good activity against S. aureus, E. coli, and C. albicans. Although effective log reductions were provided with all MPSs, the reduction was variable depending on the strains tested. Multipurpose contact lens solutions should be tested under ISO 14729 standards for both standard and clinical strains.

Glycerol monolaurate antibacterial activity in broth and biofilm cultures.

BackgroundGlycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria.Methodology/Principal FindingsGML is ≥200 times more effective than lauric acid in bactericidal activity, defined as a ≥3 log reduction in colony-forming units (CFU)/ml, against Staphylococcus aureus and Streptococcus pyogenes in broth cultures. Both molecules inhibit superantigen production by these organisms at concentrations that are not bactericidal. GML prevents biofilm formation by Staphylococcus aureus and Haemophilus influenzae, as representative gram-positive and gram-negative organisms, tested in 96 well microtiter plates, and simultaneously is bactericidal for both organisms in mature biofilms. GML is bactericidal for a wide range of potential bacterial pathogens, except for Pseudomonas aeruginosa and Enterobacteriaceae. In the presence of acidic pH and the cation chelator ethylene diamine tetraacetic acid, GML has greatly enhanced bactericidal activity for Pseudomonas aeruginosa and Enterobacteriaceae. Solubilization of GML in a nonaqueous delivery vehicle (related to K-Y Warming®) enhances its bactericidal activity against S. aureus. Both R and S, and 1 and 2 position lauric acid derivatives of GML exhibit bactericidal activity. Despite year-long passage of Staphylococcus aureus on sub-growth inhibitory concentrations of GML (0.5 x minimum bactericidal concentration), resistance to GML did not develop.Conclusions/SignificanceGML may be useful as a broad-spectrum human or animal topical microbicide and may be useful as an environmental surface microbicide for management of bacterial infections and contamination.

Antimicrobial Activity of SinuSurf

Objective: 1) Determine the antimicrobial potential of a novel topical sinonasal surfactant, SinuSurf. 2) Evaluate whether the addition of SinuSurf™ with mupirocin or gentamicin creates an additive/synergistic effect on recalcitrant chronic rhinosinusitis (CRS) pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aurginosa (PA). Method: MRSA and PA01 cultures were grown in 96-well plates containing 10-fold serial dilutions of mupirocin (1x = 150ug/ml) or gentamicin(1x = 80ug/ml), diluted in normal saline with or without the addition of SinuSurf. Colony forming units (CFUs) were determined 20 hours later. Results: In the absence of antibiotics, SinuSurf evinced a 6 log reduction in CFUs for PA and 3 log reduction for MRSA. At the clinically used concentrations of respective antibiotics (80 ug/ml gentamicin, 150 ug/ml mupiricin) no growth was evident. In combination with SinuSurf, mupirocin at 1:10 dilution demonstrates complete eradication not seen with mupiricin alone. Similarly, SinuSurf in conjunction with gentamicin at 1:100 dilution demonstrates no growth while gentamicin alone at that dilution yielded growth. Furthermore, addition of SinuSurf resulted in a 5 log reduction in CFUs at a 1:100 dilution of mupirocin and 1:1000 dilution of gentamicin. Conclusion: Bacterial biofilms have recently been demonstrated in the sinonasal cavities of CRS patients and implicated in recalcitrant disease. Bacteria in the biofilm state require 100X to 1000X concentrations of antibiotics for effective therapy. Our data demonstrate the addition of SinuSurf increases the effective in vitro concentration of antibiotics significantly.

Efficiency of pyrimidine dimer formation in Escherichia coli across UV wavelengths

Inactivation of Escherichia coli as a function of ultraviolet (UV) wavelength was investigated by using the endonuclease-sensitive site (ESS) assay to quantify pyrimidine dimer formation. Ultraviolet dose-response curves were determined based on both log reduction in colony-forming units (CFU) and endonuclease-sensitive sites per kb DNA (ESS/kb) for monochromatic 254-nm low-pressure (LP) UV, polychromatic medium-pressure (MP) UV, 228 and 289-nm UV irradiation. UV irradiation from LP and MP UV sources were approx. equal in both CFU reduction and pyrimidine dimer formation at all UV doses studied; 228-nm irradiation was less effective than LP or MP, and 289-nm irradiation was the least effective in both CFU reduction and pyrimidine dimer formation. These results are in qualitative agreement with the absorption spectrum of pyrimidine bases in DNA. Results indicated an approx. linear relationship between ESS/kb and log CFU reduction. Formation of pyrimidine dimers in genomic DNA is primarily responsible for UV inactivation of E. coli. This work contributed to fundamental understanding of UV disinfection and aids in UV reactor design.

Effect of increasing silver content in phosphate-based glasses on biofilms of Streptococcus sanguis.

Silver is a powerful antibacterial ion that may be useful for dealing with localized infections, such as periodontitis. However, the use of silver in this role could be significantly improved by the development of an effective means of delivery. Phosphate-based glasses may provide a means of delivering the ions in a controlled manner. In our study, we have examined the effect of increasing silver content in phosphate-based glasses on biofilms of Streptococcus sanguis. Glasses of nominally the same dissolution rate were doped with silver at 1, 5,10, and 15 mol % and the number of colony-forming units (CFUs) determined after 6 and 24 h in a constant depth film fermenter (CDFF). The 1 mol % silver showed little change between 6 and 24 h. However, only 0.5 log CFUs were present on the glass containing 5 mol % at 6 h, and this reduced to virtually zero at 24 h. Few viable bacteria were found on the 10 mol % glass at both 6 and 24 h. The 15 mol % glass was investigated in this experiment, but no viable counts were detected. In a second set of experiments, glasses with 10 and 15 mol % silver were tested in the CDFF for up to 192 h. For the 10 mol % silver glass, there was approximately 0.8 log CFUs on this glass, which dropped to almost zero at 50 h. This was approximately 1.5-2.0 log reduction in CFUs compared to controls, and this difference was maintained for the first 50 h. After 50 h, there was a slow increase in the CFUs on all samples. However, CFUs on the 10 mol % silver glass were still suppressed up to 192 h compared to the controls. However, both controls also exhibited a decrease in viable counts at 50 h; this may have been due to carryover of silver into the control sample holders. However, this was minimized by the specimen layout in the CDFF and by having gaps between specimen sets. For the 15 mol % silver glass, counts for both this glass and the controls decreased to virtually zero between 24 and 48 h, but the numbers slowly increased up to 170 h, but the number of CFUs was suppressed compared to the 10 mol % glass at the same time point. The decrease seen is clearly the effect of the silver ions; however, the slow increase in CFUs may be accounted for by the biofilms forming thick layers on top of the glass discs inhibiting the release of ions from the glass by forming a "sacrificial layer" through which further ions have to diffuse.