Martha Á. Hjálmarsdóttir

Impact of Chain Length on Antibacterial Activity and Hemocompatibility of Quaternary N-Alkyl and N,N-Dialkyl Chitosan Derivatives

A highly efficient method for chemical modification of chitosan biopolymers by reductive amination to yield N,N-dialkyl chitosan derivatives was developed. The use of 3,6-O-di-tert-butyldimethylsilylchitosan as a precursor enabled the first 100% disubstitution of the amino groups with long alkyl chains. The corresponding mono N-alkyl derivatives were also synthesized, and all the alkyl compounds were then quaternized using an optimized procedure. These well-defined derivatives were studied for antibacterial activity against Gram positive S. aureus, E. faecalis, and Gram negative E. coli, P. aeruginosa, which could be correlated to the length of the alkyl chain, but the order was dependent on the bacterial strain. Toxicity against human red blood cells and human epithelial Caco-2 cells was found to be proportional to the length of the alkyl chain. The most active chitosan derivatives were found to be more selective for killing bacteria than the quaternary ammonium disinfectants cetylpyridinium chloride and benzalkonium chloride, as well as the antimicrobial peptides melittin and LL-37.

The Effect of Substituent, Degree of Acetylation and Positioning of the Cationic Charge on the Antibacterial Activity of Quaternary Chitosan Derivatives

A series of water-soluble cationic chitosan derivatives were prepared by chemoselective functionalization at the amino group of five different parent chitosans having varying degrees of acetylation and molecular weight. The quaternary moieties were introduced at different alkyl spacer lengths from the polymer backbone (C-0, C-2 and C-6) with the aid of 3,6-di-O-tert-butyldimethylsilyl protection of the chitosan backbone, thus allowing full (100%) substitution of the free amino groups. All of the derivatives were characterized using 1H-NMR, 1H-1H COSY and FT-IR spectroscopy, while molecular weight was determined by GPC. Antibacterial activity was investigated against Gram positive S. aureus and Gram negative E. coli. The relationship between structure and activity/toxicity was defined, considering the effect of the cationic group’s structure and its distance from the polymer backbone, as well as the degree of acetylation within a molecular weight range of 7–23 kDa for the final compounds. The N,N,N-trimethyl chitosan with 100% quaternization showed the highest antibacterial activity with moderate cytotoxicity, while increasing the spacer length reduced the activity. Trimethylammoniumyl quaternary ammonium moieties contributed more to activity than 1-pyridiniumyl moieties. In general, no trend in the antibacterial activity of the compounds with increasing molecular weight or degree of acetylation up to 34% was observed.

Synthesis of guanidinylated chitosan with the aid of multiple protecting groups and investigation of antibacterial activity.

A new synthetic approach employing two types of protecting groups, tertiarybutyldimethylsilyl (TBDMS) and tertiarybutyloxycarbonyl (Boc) was developed to obtain a series of guanidinylated chitosan derivatives. The synthesis was carried out in organic solvents which allowed quantitative reaction, a good control on the degree of substitution, and 100% substitution of the chitosan amino groups. Similar derivatives carrying the trimethylammonium group were also synthesized as reference compounds. All the derivatives were characterized using (1)H and COSY NMR and IR spectroscopy. The antibacterial effect against clinically relevant strains of S. aureus and E. coli was found to increase with increase in the degree of substitution and decrease in the spacer length of the derivatives in both the series. An optimum activity could be obtained at a degree of substitution above 0.5 for most derivatives. The trimethylammonium derivatives showed slightly higher activity than the corresponding guanidinium derivatives but a similar structure-activity relationship was obtained.

Epidemiology of penicillin-non-susceptible pneumococci in Iceland, 1995–2010

OBJECTIVES The first penicillin-non-susceptible pneumococci (PNSP) were identified in Iceland in 1988. A rapid increase followed, associated with expansion of a single multiresistant clone, Spain(6B)-2, peaking at 19.8% in 1993. After interventions led to reduced antimicrobial use in children, the prevalence of PNSP decreased until 1995. The aim of this study was to follow the evolution of PNSP from 1995 to 2010, the period preceding the introduction of conjugated pneumococcal vaccines into the vaccination programme. METHODS The laboratory at the Landspitali University Hospital serves ∼ 85% of the Icelandic population. All pneumococci isolated from 1995 to 2010 (n = 13,937) were stored (-80 °C). Oxacillin-resistant isolates were serotyped and penicillin MICs were determined. Selected strains were genotyped by PFGE and multilocus sequence typing. RESULTS In 1995, the rate of PNSP was 24.2%, declining to 13.6% in 2001, and then increasing to 38.6% in 2010. Similar changes were observed for resistance to erythromycin and tetracycline. In 1995, 60.7% of PNSP were serotype 6B, mainly the Spain(6B)-2 clone, declining to 5.7% in 2010. PNSP of serotype 19F rapidly increased after 2004 to comprise 85.8% of all serogrouped PNSP in 2010, with most isolates belonging to a single multiresistant PFGE clone identified as sequence type (ST) 271 and ST1968, representing single- and double-locus variants of the international clone Taiwan(19F)-14, respectively. PNSP were most common among young children, from the nasopharynx, middle ear and lower respiratory tract. CONCLUSIONS The epidemiology of PNSP was dominated by two multiresistant clones. The second expanded rapidly when the first one was disappearing, causing higher antibiotic resistance rates among pneumococci than seen before in Iceland.

Topical Formulation Comprising Fatty Acid Extract from Cod Liver Oil: Development, Evaluation and Stability Studies

The purpose of this study was to develop a pharmaceutical formulation containing fatty acid extract rich in free omega-3 fatty acids such as eicosapentaenoic acid and docosahexaenoic acid for topical use. Although the health benefits of cod liver oil and other fish oils taken orally as a dietary supplement have been acknowledged and exploited, it is clear that their use can be extended further to cover their antibacterial properties. In vitro evaluation showed that 20% (v/v) fatty acid extract exhibits good activity against strains of the Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis, Streptoccoccus pyogenes and Streptoccoccus pneumonia. Therefore, free polyunsaturated fatty acids from cod liver oil or other fish oils can be used as safe and natural antibacterial agents. In this study, ointment compositions containing free fatty acids as active antibacterial agents were prepared by using various natural waxes and characterized. The effects of different waxes, such as carnauba wax, ozokerite wax, laurel wax, beeswax, rice bran wax, candelilla wax and microcrystalline wax, in the concentration range of 1% to 5% (w/w) on the ointment texture, consistency and stability were evaluated. The results showed significant variations in texture, sensory and rheological profiles. This was attributed to the wax’s nature and chain composition. Microcrystalline wax gave the best results but laurel wax, beeswax and rice bran wax exhibited excellent texturing, similar sensory profiles and well-balanced rheological properties.

Prevalence of pilus genes in pneumococci isolated from healthy preschool children in Iceland: association with vaccine serotypes and antibiotic resistance

OBJECTIVES The objective of this study was to investigate the prevalence of pilus islets [pilus islet 1 (PI-1) and pilus islet 2 (PI-2)] in pneumococcal isolates from healthy Icelandic preschool children attending day care centres, prior to the introduction of conjugated pneumococcal vaccine, and the association of the pilus islets with vaccine serotypes and antibiotic resistance. METHODS Nasopharyngeal swabs were collected from 516 healthy children attending day care centres in Reykjavik in March and April 2009. Infant vaccination was started in 2011, thus the great majority of the children were unvaccinated. Pneumococci were cultured selectively, tested for antimicrobial susceptibility and serotyped. The presence of PI-1 and PI-2 was detected using PCR. RESULTS A total of 398 viable isolates were obtained of which 134 (33.7%) showed the presence of PI-1. PI-1-positive isolates were most often seen in serotype 19F [30/31 (96.8%)] and were of clade I, and in 6B [48/58 (82.8%)] of clade II. PI-2-positive isolates were most common in serotype 19F [27/31 (87.1%)]; all of them were also PI-1 positive. Of the PI-1-positive and PI-2-positive isolates, 118 (88.1%) and 31 (81.6%), respectively, were of vaccine serotypes. Both PI-1 and PI-2 were more often present in penicillin-non-susceptible pneumococci (PNSP) than in penicillin-susceptible pneumococci [PI-1 in 41/58 (70.7%) and 93/340 (27.4%), respectively, and PI-2 in 28/58 (48.3%) and 10/340 (2.9%), respectively]. CONCLUSIONS Genes for PI-1 and/or PI-2 in pneumococci isolated from healthy Icelandic children are mainly found in isolates of vaccine serotypes and in PNSP isolates belonging to multiresistant international clones that have been endemic in the country.

Cationic quaternized aminocalix[4]arenes: cytotoxicity, haemolytic and antibacterial activities.

This study reports the characterization of three cationic amphiphillic aminocalix[4]arenes as potential antimicrobial agents in vitro. In cytotoxicity tests on mouse macrophage RAW 264.7 cells aminocalix[4]arenes 1 and 3 showed no toxicity up to 200 and 100 μM concentrations, respectively, while 2 was non-toxic only up to 50 μM. With regard to the haemolytic activity on rabbit red blood cells, 1 was not active at concentrations up to 100 μM in contrast to the other two studied macrocycles. Compounds showed negligible ability to protect either mouse macrophage RAW 264.7 cells from anthrax lethal toxin of Bacillus anthracis (B. anthracis) or rabbit red blood cells from α-haemolysin of Staphylococcus aureus (S. aureus) in comparison to amino-β-cyclodextrins. However, all aminocalix[4]arenes showed potential as antimicrobials. Their minimum inhibitory concentrations (MIC) against Escherichia coli (E. coli) and S. aureus were in the 16-32 μg/ml concentration range, while minimum lethal concentrations (MLC) varied from 16 to 256 μg/ml depending on the bacteria and aminocalix[4]arene considered. Macrocycle 1 showed partial synergism against S. aureus in tandem with a model antibacterial drug, fusidic acid, at certain concentration combinations.

Cocolonization of Pneumococcal Serotypes in Healthy Children Attending Day Care Centers: Molecular Versus Conventional Methods.

Objectives: Pneumococci are common colonizer, especially of children, and cocolonization of different serotypes is an important factor for intraspecies genetic exchange. The aim of this study was to analyze pneumococcal carriage and serotype distribution in unvaccinated healthy children in Iceland and compare conventional culture methods and molecular methods using DNA extracted directly from the samples. Methods: Nasopharyngeal swabs were obtained from 514 children aged 2–6 year attending day care centers in Reykjavik in 2009. The swabs were selectively cultured for pneumococci and the isolates serotyped using latex agglutination. DNA was also extracted directly from the swabs and serotyped using a multiplex PCR panel designed to detect vaccine serotypes and the most commonly carried non-vaccine serotypes. Result: Pneumococcal carriage was detected in 391 (76.1%) of the children using polymerase chain reaction (PCR) and in 371 (72.2%) using conventional methods. Cocolonization was detected in 92 (23.5%) of the carriers when PCR method was used and in 30 (8.1%) when conventional methods were used, detecting 500 and 401 strains, respectively (P < 0.0001). The most common serotypes were 23F, 19A, 6B, 6A and 19F, rates 13–8%. The number of isolates of serotypes included in the 10-valent and 13-valent vaccines and detected by PCR were 234 (58.4%) and 363 (90.5%), respectively and by conventional methods 186 (46.4%) and 293 (73.1%), respectively. Conclusion: Cocolonization was detected in a fourth of the children carrying pneumococci using DNA extracted directly from nasopharyngeal swabs. The rate of carriage was very high, but no serotype dominated, and the children were commonly colonized by vaccine serotypes, especially cocolonized children.

N,N,N-trimethyl chitosan as an efficient antibacterial agent for polypropylene and polylactide nonwovens

Abstract The paper presents a method of depositing N,N,N-trimethyl chitosan (TMC) layers onto polypropylene and polylactide nonwovens. A two-step modification procedure is applied: first, grafting the nonwovens with acrylic acid and next layer-by-layer deposition. Turbidimetric measurements confirm the creation of polycomplexes between grafted poly(acrylic acid) and deposited TMC. The created material structure is evaluated using gravimetric analysis, reflectance Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive spectroscopy measurements and pH-metric titration. The modified material exhibits good antibacterial properties against Gram-positive bacteria Staphylococcus aureus.

Comparison of Serotype Prevalence of Pneumococci Isolated from Middle Ear, Lower Respiratory Tract and Invasive Disease Prior to Vaccination in Iceland.

Background Information on pneumococcal serotype distribution before vaccination is a prerequisite for evaluation of vaccine effect. The aim was to investigate the prevalence of pneumococcal serotypes isolated from middle ear (ME), lower respiratory tract (LRT) and from invasive disease (IPD) in Iceland prior to implementation of ten-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV-10) into the infant vaccination program (April 2011). Methods and findings All isolates cultured 2007–2011 from ME, LRT and IPD identified as pneumococci were serotyped and tested for susceptibility at the Clinical Microbiology Department, Landspitali University Hospital that serves approximately 85% of the Icelandic population. Pneumococcal isolates were 1711 and 1616 (94.4%) were available for serotyping and included. Isolates belonging to PHiD-CV10 serotypes (VTs) were 1052 (65.1%). Isolates from ME were 879 (54.4%), with 639 (72.7%) from 0–1 year old patients and 651 of VTs (74%). Isolates from LRT were 564 (34.9%), with 292 (51.8%) from ≥65 years old patients, and 300 (53.2%) of VTs. IPD isolates were 173 (10.7%), although more evenly distributed according to age than isolates from the other sites most were from adults and the youngest age group,101 (58.4%) isolates were of VTs. The most common serotype was 19F, 583 (36.1%). Its prevalence was highest in ME, 400 (45.5%), 172 (30.5%) in LRT and 11 isolates (6.4%), in IPD. Penicillin non-susceptible isolates were 651 (40.3%), mainly belonging to VTs, 611 (93.9%), including 535 (82.2%) of 19F. Conclusions Multiresistant isolates of serotype 19F were highly prevalent, especially from ME of young children but also from LRT of adults. Serotype 14 was the most common serotype in IPD. The rate of VTs was high and almost all PNSP were of VTs. There was great difference in vaccine coverage between sampling sites, also reflecting difference in vaccine coverage by age groups.