A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17–30 and LFampin268–284, a shorter fragment of LFampin265–284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10⁵ CFU/ml, could be killed by 5–10 μM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.     

Sensitivity of bacterial coaggregation to chelating agents

Coaggregation between pairs of microorganisms was found to be inhibited by chelating agents, such as acetylacetone, citrate, EDTA and carboxymethylcellulose. Assays were conducted on eight pairs of periodontopathogens and one pair consisting of Escherichia coli and Saccharomyces cerevisiae. The inhibitory effects of the chelating agents were reversible except for Actinomyces naeslundii 12104, the adhesin of which was irreversibly inactivated. Even though the bacteria possessed different kinds of adhesins, their sensitivity to chelating agents appears to be a common property. Non-toxic chelating agents, such as carboxymethylcellulose and citrate, may prove to be useful anti-adhesins.     

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei

Deposits on surfaces in water-bearing systems, also known as ‘fouling’, can lead to substantial losses in the performance of industrial processes as well as a decreased product quality. Early detection and localization of such deposits can, to a considerable extent, save such losses. However, most of the surfaces that become fouled, for example, in process water pipes, membrane systems, power plants, and food and beverage industries, are difficult to access and analyses conducted on the water phase do not reveal the site or extent of deposits. Furthermore, it is of interest to distinguish biological from non-biological deposits. Although they usually occur together, different countermeasures are necessary. Therefore, sensors are required that indicate the development of surface fouling in real-time, non-destructively, and in situ, preferably allowing for discrimination between chemical and/or biological deposits. In this paper, an optical deposit sensor is presented which fulfills these requirements. Based on multiple fluorescence excitation emission matrix analysis, it detects autofluorescence of amino acids as indicators of biomass. Autofluorescence of nicotinamide adenine dinucleotide?+?hydrogen is interpreted as an indicator of biological activity, thus it acts as a viability marker, making the method suited for assessing the efficacy of disinfection treatments. Scattering signals from abiotic deposits such as calcium carbonate or corrosion products can clearly be distinguished from biotic substances and monitored separately. The sensor provides an early warning of fouling, allowing for timely countermeasures to be deployed. It also provides an assessment of the success of cleaning treatments and is a promising tool for integrated antifouling strategies.     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.     

Growing Burkholderia pseudomallei in Biofilm Stimulating Conditions Significantly Induces Antimicrobial Resistance

Background

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear.

Methodology/Principal Findings

The change in resistance of B. pseudomallei to doxycycline, ceftazidime, imipenem, and trimethoprim/sulfamethoxazole during biofilm formation were measured as minimum biofilm elimination concentration (MBEC) in 50 soil and clinical isolates and also in capsule, flagellin, LPS and biofilm mutants. Almost all planktonic isolates were susceptible to all agents studied. In contrast, when they were grown in the condition that induced biofilm formation, they were markedly resistant to all antimicrobial agents even though the amount of biofilm production was not the same. The capsule and O-side chains of LPS mutants had no effect on biofilm formation whereas the flagellin-defective mutant markedly reduced in biofilm production. No alteration of LPS profiles was observed when susceptible form was changed to resistance. The higher amount of N-acyl homoserine lactones (AHLs) was detected in the high biofilm-producing isolates. Interestingly, the biofilm mutant which produced a very low amount of biofilm and was sensitive to antimicrobial agents significantly resisted those agents when grown in biofilm inducing condition.

Conclusions/Significance

The possible drug resistance mechanism of biofilm mutants and other isolates is not by having biofilm but rather from some factors that up-regulated when biofilm formation genes were stimulated. The understanding of genes related to this situation may lead us to prevent B. pseudomallei biofilms leading to the relapse of melioidosis.     

Endogenous prolactin modulated the calcium absorption in the jejunum of suckling rats

Prolactin has been reported to stimulate intestinal calcium absorption in young and mature, but not aging rats. The present study was performed on suckling rats to elucidate the actions of endogenous prolactin on calcium absorption in various intestinal segments. Before measuring the calcium fluxes, 9-day-old rats were administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg bromocriptine, i.p., twice daily to abolish secretion of endogenous prolactin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, s.c. Thereafter, the 16-day-old rats were experimented upon by instilling the 45Ca-containing solution into the intestinal segments. The results showed that, under a physiological condition, the jejunum had the highest rate of calcium absorption compared with other segments (1.4 +/- 0.35 micromol.h-1.cm-1, p < 0.05). The duodenum and ileum also manifested calcium absorption, whereas the colon showed calcium secretion. Lack of endogenous prolactin decreased lumen-to-plasma and net calcium fluxes in jejunum from 2.07 +/- 0.31 to 1.19 +/- 0.12 and 1.40 +/- 0.35 to 0.88 +/- 0.18 micromol.h-1.cm-1 (p < 0.05), respectively, and exogenous prolactin restored the jejunal calcium absorption to the control value. Endogenous prolactin also had an effect on the duodenum but, in this case, exogenous prolactin did not reverse the effect of bromocriptine. However, neither ileal nor colonic calcium fluxes were influenced by prolactin. Because luminal sodium concentration has been demonstrated to affect calcium absorption in mature rats, the effect of varying luminal sodium concentrations on calcium fluxes in suckling rats was evaluated. The jejunum was used due to its highest rate of calcium absorption. After filling the jejunal segments with 124 (control), 80, 40 mmol/L Na+-containing or Na+-free solution, increases in calcium absorption were found to be inversely related to luminal sodium concentrations in both control and bromocriptine-treated rats. The plasma concentration of 45Ca under luminal sodium free condition was also higher than that of the control condition (2.26% +/- 0.07% vs. 2.01% +/- 0.09% administered dose, p < 0.05). However, 3H-mannitol, a marker of the widening of tight junction that was introduced into the lumen, had a stable level in the plasma during an increase in plasma 45Ca, suggesting that the widening of tight junction was not required for enhanced calcium absorption. In conclusion, calcium absorption in suckling rats was of the highest rate in the jejunum where endogenous prolactin modulated calcium absorption without increasing the paracellular transport of mannitol.     

Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from Kluyveromyces lactis: Studies on batch and continuous UF membrane-fitted bioreactors

A study of galacto-oligosaccharides (GOS) synthesis from lactose with beta-galactosidase from Kluyveromyces lactis (Maxilact L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40 degrees C and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40 degrees C, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAE-PAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal GOS).