Localization and activity of lipoxygenase in the ovule of Larix kaempferi (Lamb.) Carr. during female gametophyte maturation.

KEY MESSAGE: Lipoxygenase activity and localization vary throughout the development of Larix kaempferi ovules, with the highest enzyme activity observed in ovules at the cellular stage and the most intense immunogold reaction noted at the mature archegonium stage of gametophyte development. Lipoxygenases are a family of oxidoreductases with a significant role in biological systems, widespread in living organisms e.g. mammals, fish, corals, plants, mosses, algae, fungi, yeasts, and bacteria. Lipoxygenase activity in plants leads to the formation of phytooxylipins, i.e. signaling molecules, which play a crucial role in many significant physiological processes such as male and female gametophyte maturation, germination and seedling growth, pathogen resistance, abiotic stress response, fruit ripening, and senescence. The activity and localization of lipoxygenase change during plant growth and development. The localization of lipoxygenase in a developing ovule of Larix kaempferi was analyzed using the immunogold labeling method, and the activity was determined spectrophotometrically with linolenic acid as a substrate. Among the investigated stages, the immunogold reaction was the most intense at the mature archegonium stage in the ovule. Lipoxygenase was found in all parts of the L. kaempferi ovule. The largest number of immunogold particles was detected in the integument cells of all the analyzed stages of ovule development. Only one isoform of lipoxygenase with an optimum at pH 8 was active in the ovules during female gametophyte maturation. The highest enzyme activity was determined at the cellular stage, whereas the mature archegonium stage was characterized by its lowest level, which means that LOX activity in developing ovules of the Japanese larch is not correlated with the number of antibody-labeled molecules of the enzyme.

Occurrence and Antimicrobial Resistance among Staphylococci Isolated from the Skin Microbiota of Healthy Goats and Sheep.

Staphylococci colonize the skin and mucous membranes of different animals. The purpose of this study was to determine the staphylococcal composition of the skin microbiota of healthy, non-vet visiting, and antimicrobially non-treated sheep and goats. In total, 83 strains (44 from goats and 39 from sheep) were isolated and identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The diversity of the isolated Staphylococcus species was relatively high, and only coagulase-negative staphylococci (CoNS) were isolated. In sheep, S. vitulinus (9/39, 23.1%) was the most common species, followed by S. equorum (8/39, 20.5%), S. lentus (7/39, 17.9%), S. sciuri (6/39, 15.4%), S. xylosus (6/39, 15.4%), S. warneri (1/39, 2.6%), S. simulans (1/39, 2.6%), and S. nepalensis (1/39, 2.6%). In the goats, the most common species was S. sciuri, which was detected in 13 (29.5%) animals. The goat skin was also inhabited by S. equorum (7/44, 15.9%), S. vitulinus (6/44, 13.6%), S. cohnii (5/44, 11.4%), S. lentus (4/44, 9.1%), S. suscinus (3/44, 6.8%), S. caprae, (2/44, 4.5%), S. auricularis (2/44, 4.5%), S. warneri (1/44, 2.3%), and S. xylosus (1/44, 2.3%). Only one S. xylosus strain of goat origin carried the enterotoxin gene (sea). Antimicrobial resistance was not common among the isolated staphylococci. Only 31 (37.3%) strains were resistant to at least one antimicrobial agent, with the highest frequency of resistance to penicillin (16.8%), followed by clindamycin (9.6%), erythromycin (8.4%), moxifloxacin (8.4%), and tetracycline (7.2%). All isolates were susceptible to eight antibiotics (amikacin, gentamycin, ciprofloxacin, levofloxacin, rifampicin, chloramphenicol, trimethoprim-sulfamethoxazole, and tigecycline), representing six different classes. Three isolates displayed a multi-resistance phenotype (MDR): the goat isolates S. cohnii and S. sciuri, as well as the ewe isolate S. xylosus. The MDR S. cohnii isolate was found to be methicillin-resistant and carried the mecA gene. Moreover, the staphylococci isolated from the healthy animals carried genes conferring resistance to ß-lactams (mecA, blaZ), tetracyclines (tetL, tetK), macrolides (ermB, ermC), lincosamides (lnu), and fluoroquinolones (grlA). However, the prevalence of these genes was low.

Staphylococcal species composition in the skin microbiota of domestic pigeons (Columba livia domestica).

Staphylococci are a natural component of the skin microbiota of many organisms, including humans and birds. As opportunistic pathogens, they can cause a variety of infections in humans. The close contact between domestic pigeons and their owners provide an opportunity for exchange of skin-associated bacteria. In this study, 41 healthy racing pigeons were tested. Staphylococci were detected on the skin of each bird (41/41, 100%). Isolates were identified at the species level using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The diversity of the Staphylococcus species was relatively high and coagulase-negative staphylococci (CoNS) were predominantly isolated. In total, ten different staphylococcal species were identified. S. lentus (19/41, 46.3%) was noted most frequently. The pigeon skin was also inhabited by S. xylosus (6/41, 14.6%), S. equorum (4/41, 9.8%), S. hyicus (3/41, 7.3%), S. intermedius (2/41, 4.9%), S. sciuri (2/41, 4.9%), S. vitulinus (2/41, 4.9%), S. lugdunensis (1/41, 2.4%), S. hominis (1/41, 2.4%), and S. auricularis (1/41, 2.4%). Our results indicate that domestic pigeons may carry pathogens with zoonotic potential. All strains were susceptible to 12 antibiotics (ciprofloxacin, clindamycin chloramphenicol, erythromycin, fosfomycin, gentamicin, levofloxacin, norfloxacin, rifampicin, tobramycin, trimethoprim/sulfamethoxazole, vancomycin) representing 8 different classes. None isolate displayed a multidrug-resistant phenotype. Resistance to tetracycline (6/41, 14.6%) and to penicillin (4/41, 9.7%) was shown. The mecA gene was not detected in the examined strains and no methicillin-resistant staphylococci were found on the skin of the healthy pigeons.

Occurrence and Characteristics of Staphylococcus aureus Isolated from Dairy Products.

Food, particularly milk and cheese, may be a reservoir of multi-drug resistant Staphylococcus aureus strains, which can be considered an important issue in terms of food safety. Furthermore, foods of animal origin can be a cause of staphylococcal food poisoning via the production of heat-stable enterotoxins (SE). For this reason, we investigated the prevalence of and characterized Staphylococcus aureus strains isolated from milk and fresh soft cheese obtained from farms located in Wielkopolskie and Zachodniopomorskie Provinces in Poland. Overall, 92% of S. aureus isolates were positive for at least one of the 18 enterotoxin genes identified, and 26% of the strains harbored 5 to 8 enterotoxin genes. Moreover, the S. aureus strains contained genes conferring resistance to antibiotics that are critically important in both human and veterinary medicine, i.e., ß-lactams (mecA), aminoglycosides (aac(6')/aph(2″), aph(3')-IIIa, ant(4')-Ia) and MLSB (erm(A), msr(A), lun(A)). The antimicrobial susceptibility of S. aureus to 16 antibiotics representing 11 different categories showed that 74% of the strains were resistant to at least 1 antibiotic. Moreover, 28% of the strains showed multidrug resistance; in particular, two methicillin-resistant S. aureus strains (MRSA) exhibited significant antibiotic resistance. In summary, our results show that dairy products are contaminated by S. aureus strains carrying genes encoding a variety of enterotoxins as well genes conferring resistance to antibiotics. Both MRSA strains and MSSA isolates showing multidrug resistance were present in foods of animal origin.

Effect of Low Temperature on Changes in AGP Distribution during Development of Bellis perennis Ovules and Anthers.

Arabinogalactan proteins (AGPs) are a class of heavily glycosylated proteins occurring as a structural element of the cell wall-plasma membrane continuum. The features of AGPs described earlier suggest that the proteins may be implicated in plant adaptation to stress conditions in important developmental phases during the plant reproduction process. In this paper, the microscopic and immunocytochemical studies conducted using specific antibodies (JIM13, JIM15, MAC207) recognizing the carbohydrate chains of AGPs showed significant changes in the AGP distribution in female and male reproductive structures during the first stages of Bellis perennis development. In typical conditions, AGPs are characterized by a specific persistent spatio-temporal pattern of distribution. AGP epitopes are visible in the cell walls of somatic cells and in the megasporocyte walls, megaspores, and embryo sac at every stage of formation. During development in stress conditions, the AGP localization is altered, and AGPs entirely disappear in the embryo sac wall. In the case of male development, AGPs are present in the tapetum, microsporocytes, and microspores in normal conditions. In response to development at lower temperature, AGPs are localized in the common wall of microspores and in mature pollen grains. Additionally, they are accumulated in remnants of tapetum cells.

Sida hermaphrodita seeds as the source of anti - Candida albicans activity.

Sida hermaphrodita is a perennial herbaceous plant with potential economic importance; however, there is no information about its antimicrobial properties. The aim of our study was to analyze the morphology and metabolic activity of Candida albicans cells after exposure to the extract from S. hermaphrodita seeds, determine its cytotoxicity against human skin fibroblasts and carry out chemical analysis of the extract. Microscopic analysis showed that the crude seed extract (CSE) caused a significant decrease in the metabolic activity of fungal cells, clear cell deformation, and budding disturbances. The analysis of cytotoxicity showed no influence of the extract on the fibroblasts. The CSE and seed extract after dialysis (DSE) were analyzed using electrophoretic, chromatographic, and spectroscopic methods. SDS-PAGE electrophoresis showed the presence of proteins and carbohydrate compounds in the extract. The Raman spectroscopy analysis of the DSE confirmed the presence of proteins, while FTIR analyses revealed the occurrence of albumin-type proteins. The NMR and GC-MS analyses showed the presence of carbohydrates in the seed extract. The MALDI and ESI LC-MS/MS analysis of the CSE and the DSE fractions revealed the occurrence of vicilin-type and plant lipid transfer proteins. The seed extract is a promising formulation to use in C. albicans infections.

Analysis of AGP contribution to the dynamic assembly and mechanical properties of cell wall during pollen tube growth.

Arabinogalactan proteins as cell wall structural proteins are involved in fundamental processes during plant development and growth. The aim of this study was to evaluate AGP function in the distribution of pectin, cellulose and callose along Fragaria x ananassa pollen tube and to associate the cell wall structure with local mechanical properties. We used Yariv reagent which interacts with AGPs and allows the observation of the assembly of cell walls without AGPs performing their function. Cytochemical, immunofluorescence labelling and atomic force microscope have been used to characterize the changes in cell wall structure and stiffness. It was shown that disordering of the structure of AGP present in cell walls affects the localization of cellulose, pectins and the secretion of callose. Changes in cell wall assembly are relevant to pollen tube mechanical properties. The stiffness gradient lengthwise through the axis of the pollen tube has demonstrated a significantly higher Young's modulus of the shank region than the growth zone. It has been revealed that the apex of the pollen tube cultured in the presence of Yariv reagent is stiffer (1.68 MPa) than the corresponding region of the pollen tube grown under control conditions (0.13-0.27 MPa). AGP affects the structure of the cell wall by changing the distribution of other components and the modification of their localization, and hence it plays a significant role in the mechanical properties of the cell wall.

Structural network of arabinogalactan proteins (AGPs) and pectins in apple fruit during ripening and senescence processes.

The cell wall is an essential framework determining the overall form of the plant cell. Our study was focused on the distribution of arabinogalactan proteins (AGPs), arabinan, and homogalacturonan in fruit cells during ripening and storage with emphasis on quantitative analysis of their presence in particular regions of the cell wall - plasma membrane. The localization of the examined compounds was determined with immunohistochemistry techniques and immunogold labelling. Spatio-temporal colocalization between AGPs epitopes - [ßGlcA(1→3)-αGalA(1→2)Rha] recognized by JIM13 and MAC207 antibodies, and arabinan labelled by the LM16 antibody was detected in the inner cell wall layer, in association with the plasma membrane. The specific arrangement of AGP and arabinan epitopes differentiated them from homogalacturonan epitopes, consisting of GalA residues recognized by LM19 and LM20 antibodies in all the examined fruit maturity stages. The disruption of cell wall - plasma membrane continuum, observed during ripening-associated softening process, was associated with both the substantial decrease of AGPs, pectins content and with remodeling of their arrangement. The results indicate that the textural properties of fruit during growth and postharvest storage, an attribute of fruit quality becoming selection criteria for consumers, depend on the existence of dynamic network organizing polysaccharides and glycoproteins in the extracellular matrix.

Multifactorial mechanisms of the pathogenesis of methicillin-resistant Staphylococcus hominis isolated from bloodstream infections.

Staphylococcus hominis is a species of the coagulase-negative staphylococci. It has been designated as a potential pathogen but so far the pathogenic mechanisms of this bacterium have not been determined. We studied 30 clinical isolates of methicillin-resistant S. hominis, which were previously examined for biofilm forming properties. The results of this study revealed that all these S. hominis strains had the ability to adhere to HeLa cells. Over 40% of the S. hominis strains invaded epithelial cells. The invasion index ranged from 0 to 41.5%. All isolates exhibited the cytotoxic activity of extracellular factors, which caused the destruction of epithelial cells. More than 90% of these methicillin-resistant strains contained at least one aminoglycosides resistance gene. The ant(4')-I gene was found in 63% of the isolates, aac(6')/aph(2″) in 20% and aph(3')-IIIa in 47%. Two strains were assigned to SCCmec type VIII and three to SCCmec type III. The remaining isolates (83%) harboured a non-typeable SCCmec type. The mec complex A was predominant in this species. The results indicate that the pathogenicity of S. hominis may be multifactorial, involving adhesion, invasion and the activity of extracellular toxins, which cause damage to the host epithelium.

Arabinogalactan proteins: Immunolocalization in the developing ovary of a facultative apomict Fragaria x ananassa (Duch.).

BACKGROUND AND AIMS: Arabinogalactan proteins are present in the extracellular matrix and their occurrence is developmentally regulated. The studies were carried out to localise arabinogalactan proteins in ovary cells of Fragaria x ananassa Duchesne (strawberry) during megasporogenesis, megagametogenesis, and formation of the embryo. METHODS: The research included studies of ovary histochemistry and immunofluorescence labelling of AGP epitopes was performed with antibodies JIM13, JIM15 and MAC207. The use of the immunogold labelling method allowed specific detection of AGP epitopes at the subcellular level. KEY RESULTS: The localization of AGPs was studied in the cells of the ovary wall and elements building the developing ovule i.e. the integument, nucellus, archespore, megaspores, embryo sac, and embryo of a facultative apomict Fragaria x ananassa cv. 'Mount Everest'. For the first time the presence of AGP epitopes at the stage of a multicellular archespore was described. The occurrence of AGPs in the functional megaspore walls is related to selection of a megaspore continuing development; during later stages of development, AGPs are also evident markers of the female gametophyte. The intense fluorescence indicates the presence of AGPs in the embryo sac wall as well as in the cytoplasm compartment of the egg apparatus and around the secondary nucleus of the central cell. The localization of AGPs in the ovule of F. x ananassa resembles the distribution of these proteins in amphimictic plants. CONCLUSIONS: Arabinogalactan proteins occur in similar parts of the ovule of amphimictic and apomictic plants. The results confirm the participation of AGPs in reproductive structures as a useful marker during development of female gametophyte.

Effect of subinhibitory concentrations of tigecycline and ciprofloxacin on the expression of biofilm-associated genes and biofilm structure of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of foreign body-associated infections. This is related to the bacterium's ability to form biofilms on synthetic materials. Bacteria within a biofilm may be exposed to subinhibitory concentrations (sub-MICs) of antibiotics because of an agent's limited penetration into the biofilm core. Here, we investigated the effect of sub-MICs of tigecycline and ciprofloxacin on the expression of biofilm-associated genes, i.e. icaA, altE and sigB, and the biofilm structure of five clinical isolates of S. epidermidis. For most tested isolates, the expression of these genes increased after exposure to 0.25 MIC and 0.5 MIC tigecycline. A slight decrease in icaAmRNA levels was observed only in two isolates in the presence of 0.25 MIC tigecycline. The effect of ciprofloxacin exposure was isolate-dependent. At 0.5 MIC, ciprofloxacin induced an increase of sigB and icaAmRNA levels in three of the five tested isolates. At the same time, expression of the altE gene increased in all isolates (from 1.3-fold to 42-fold, depending on the strain). Confocal laser scanning microscopy analysis indicated that sub-MIC ciprofloxacin decreased biofilm formation, whereas tigecycline stimulated this process. Our data suggest that sub-MIC tigecycline may have bearing on the outcome of infections.

Coagulase-negative staphylococci: pathogenesis, occurrence of antibiotic resistance genes and in vitro effects of antimicrobial agents on biofilm-growing bacteria.

Coagulase-negative staphylococci (CoNS) are opportunistic pathogens that particularly cause infections in patients with implanted medical devices. The present research was performed to study the virulence potential of 53 clinical isolates of Staphylococcus capitis, Staphylococcus auricularis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus cohnii and Staphylococcus caprae. All clinical strains were clonally unrelated. Isolates carried genes encoding resistance to ß-lactam (mecA) (15 %), aminoglycoside [aac(6')/aph(2″)(11 %), aph (3')-IIIa (15 %), ant(4')-Ia (19 %)] and macrolide, lincosamide and streptogramin B (MLSB) [erm(A) (4 %), erm(B) (13 %), erm(C) (41 %), msr(A) (11 %)] antibiotics. CoNS isolates (64 %) were able to form biofilms. Confocal laser scanning microscopy revealed that these biofilms formed a three-dimensional structure composed mainly of living cells. All biofilm-positive strains carried the ica operon. In vitro studies demonstrated that a combination treatment with tigecycline and rifampicin was more effective against biofilms than one with ciprofloxacin and rifampicin. The minimum biofilm eradication concentration values were 0.062-0.5 µg ml-1 for tigecycline/rifampicin and 0.250-2 µg ml-1 for ciprofloxacin/rifampicin. All CoNS strains adhered to the human epithelial cell line HeLa, and more than half of the isolates were able to invade the HeLa cells, although most invaded relatively poorly. The virulence of CoNS is also attributed to their cytotoxic effects on HeLa cells. Incubation of HeLa cells with culture supernatant of the CoNS isolates resulted in cell death. The results indicate that the pathogenicity of S. capitis, S. auricularis, S. lugdunensis, S. cohnii and S. caprae is multi-factorial, involving the ability of these bacteria to adhere to human epithelial cells, form biofilms and invade and destroy human cells.

Clonality, virulence and the occurrence of genes encoding antibiotic resistance among Staphylococcus warneri isolates from bloodstream infections.

Coagulase-negative Staphylococcuswarneri is an opportunistic pathogen capable of causing several infections, especially in patients with indwelling medical devices. We evaluated the virulence-associated properties of 23 clinical isolates recovered from blood specimens. In addition, the carriage of biofilm-associated genes, as well as antibiotic-resistant genes, was identified. S. warneri isolates appeared to be clonally unrelated and revealed a high degree of genetic diversity. All isolates revealed adhesion to epithelial cells, and 43.5 % of strains invaded the cells. Moreover, 52 % of isolates formed biofilm in vitro. PCR analysis demonstrated the presence of the ica operon, in two of the 12 biofilm-positive isolates. This indicated that biofilm formation, in this species, is not restricted to strains harbouring icaADBC genes, encoding polysaccharide intercellular adhesion. Analysis by confocal laser scanning microscopy revealed that biofilm-forming strains formed a three-dimensional structure, composed of mainly living cells. All strains revealed cell-contact cytotoxicity that was strongly associated with biofilm formation. Moreover, cell-free supernatants, of 95 % of the isolates, expressed a cytotoxic activity which caused the destruction of HeLa cells. S. warneri capable of forming biofilm carried significantly more genes encoding resistance to beta-lactams, aminoglicosides and macrolide-lincosamide streptogramin B antibiotics than biofilm-negative isolates. We have shown that tigecycline/rifampicin is effective against bacteria growing as a biofilm. The biofilm inhibitory concentration range of tigecycline/rifampicin was ≤1 µg ml-1. Results indicated that S. warneri have the ability to adhere, form biofilm, invade and destroy epithelial cells, which could be important mechanisms contributing to the development of diseases.

Diversity of staphylococcal cassette chromosome mec elements in nosocomial multiresistant Staphylococcus haemolyticus isolates.

Staphylococcus haemolyticus is the second, most frequently isolated coagulase-negative staphyloccus (CoNS) from patients with hospital-acquired infections, and it is usually resistant to methicillin and other semisynthetic penicillins. The purpose of this study was to characterize staphylococcal cassette chromosome mec (SCCmec) elements and assess the in-vitro activity of antibiotics against 60 S. haemolyticus strains recovered from hospitalized patients. All these strains expressed methicillin resistance and carried a mecA gene. Moreover, all strains possessed a multiresistant phenotype, i.e., exhibited resistance to more than three classes of antibiotics. Eleven strains (18 %) harbored the SCCmec type V, containing ccrC and mec complex C. Three isolates harboring the ccrC gene did not contain a known mec complex. One strain positive for mec complex C was not typeable for ccr. This suggests that ccrC and mec complex C may exist autonomously. Only four strains carried mec complex B, whereas none of the S. haemolyticus harboured mec complex A. A new combination, which is mec complex B-ccrAB ship, was found in S. haemolitycus. The ccrAB ship was also identified in two strains of S. haemolitycus in which the mec gene complex was not identified. The results of the present study indicate that in S. haemolyticus the mec gene complex and the ccr genes are highly divergent. However, ccr sequence analysis does not allow the identification of a new allotype, based on a cut-off value of 85 % identity. The ccr genes in the S. haemolitycus strain showed ≥96 % sequence identity to the ccrAB2 genes.

Characterization of Staphylococcal Cassette Chromosome mec (SCCmec) in Methicillin-Resistant Staphylococcus epidermidis Strains Isolated from Biomaterial-Associated Infections and their Antibiotic Resistance Patterns.

This work aims to provide an insight into staphylococcal cassette chromosome mec elements and antibiotic resistance in clinical isolates of Staphylococcus epidermidis. The dominating type was SCCmec ­ IV. Fifteen isolates were assigned to SCCmec type III, two isolates to SCCmec type II. Most isolates were resistant to at least three of the non-ß-lactam antibiotics tested. None of the strains exhibited resistance to new generation antibiotics, such as daptomycin and linezolid. Also, none of these strains showed resistance to tigecycline and only four strains were resistant to rifampin i.e. antibiotics which are very efficient in treating biofilm-associated infections.

Molecular basis of resistance to macrolides, lincosamides and streptogramins in Staphylococcus hominis strains isolated from clinical specimens.

Coagulase-negative staphylococci (CoNS) are the most frequently isolated bacteria from the blood and the predominant cause of nosocomial infections. Macrolides, lincosamides and streptogramin B (MLSB) antibiotics, especially erythromycin and clindamycin, are important therapeutic agents in the treatment of methicillin-resistant staphylococci infections. Among CoNS, Staphylococcus hominis represents the third most common organism. In spite of its clinical significance, very little is known about its mechanisms of resistance to antibiotics, especially MLSB. Fifty-five S. hominis isolates from the blood and the surgical wounds of hospitalized patients were studied. The erm(C) gene was predominant in erythromycin-resistant S. hominis isolates. The methylase genes, erm(A) and erm(B), were present in 15 and 25% of clinical isolates, respectively. A combination of various erythromycin resistance methylase (erm) genes was detected in 15% S. hominis isolates. The efflux gene msr(A) was detected in 18% of isolates, alone in four isolates, and in different combinations in a further six. The lnu(A) gene, responsible for enzymatic inactivation of lincosamides was carried by 31% of the isolates. No erythromycin resistance that could not be attributed to the genes erm(A), erm(B), erm(C) and msr(A) was detected. In S. hominis, 75 and 84%, respectively, were erythromycin resistant and clindamycin susceptible. Among erythromycin-resistant S. hominis isolates, 68% of these strains showed the inducible MLSB phenotype. Four isolates harbouring the msr(A) genes alone displayed the MSB phenotype. These studies indicated that resistance to MLSB in S. hominis is mostly based on the ribosomal target modification mechanism mediated by erm genes, mainly the erm(C), and enzymatic drug inactivation mediated by lnu(A).

Characterization of Staphylococcal Cassette Chromosome mec (SCCmec) in Methicillin-Resistant Staphylococcus epidermidis Strains Isolated from Biomaterial-Associated Infections and their Antibiotic Resistance Patterns.

This work aims to provide an insight into staphylococcal cassette chromosome mec elements and antibiotic resistance in clinical isolates of Staphylococcus epidermidis. The dominating type was SCCmec - IV. Fifteen isolates were assigned to SCCmec type III, two isolates to SCCmec type II. Most isolates were resistant to at least three of the non-ß-lactam antibiotics tested. None of the strains exhibited resistance to new generation antibiotics, such as daptomycin and linezolid. Also, none of these strains showed resistance to tigecycline and only four strains were resistant to rifampin i.e. antibiotics which are very efficient in treating biofilm-associated infections.

In Vitro Activity of Rifampicin Combined with Daptomycin or Tigecycline on Staphylococcus haemolyticus Biofilms.

Staphylococcus haemolyticus is of increasing concern as a cause of several biofilm-associated infections, and today, it represents the second most common organism among clinical isolates of coagulase-negative staphylococci. However, little is known regarding the treatment of infections caused by these bacteria. In this study, we characterize the biofilm formed by S. haemolyticus strains isolated from bloodstream infections and assess in vitro the activity of rifampicin combined with daptomycin or tigecycline against bacteria growing in a biofilm. The results of our studies indicated that the majority (78 %) of methicillin-resistant Staphylococcus haemolyticus strains have the ability to form a biofilm in vitro. None of these strains carried icaADBC genes indicating that they form biofilm via ica-independent mechanisms. The molecular characterization of the biofilm showed that proteins are the predominant matrix component and play a major role in biofilm structure. Extracellular DNA and polysaccharides, other than polysaccharide intercellular adhesin, are also present in the biofilm matrix, but they play a minor role. The images obtained by confocal laser scanning microscopy showed that most S. haemolyticus strains formed a dense biofilm with a low number of dead cells. In vitro study demonstrated excellent activity of tigecycline in combination with rifampicin against cell growth in the proteinous biofilm. The BIC (biofilm inhibitory concentration) value for tigecycline/rifampicin ranged from 0.062 to 1 µg/ml, whereas for daptomycin/rifampicin from 0.125 to 2 µg/ml. These results indicated that the tigecycline/rifampicin combination was more effective against ica-independent biofilm, formed by S. haemolyticus strains, than the daptomycin/rifampicin combination.

Virulence and the presence of aminoglycoside resistance genes of Staphylococcus haemolyticus strains isolated from clinical specimens.

We examined thirty methicillin-resistant Staphylococcus haemolyticus isolates cultured from clinical specimens for antibiotic resistance, various important interactions of the bacteria with epithelial cells and putative virulence determinants. All strains were resistant to oxacillin and carried the mecA gene. Aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene encoding nucleotidyltransferases was detected in 43 %, aminocyclitol-6'-acetyltransferase-aminocyclitol-2″-phosphotransferase (aac(6')/aph(2″)) gene encoding bifunctional acetyltransferases/phosphotransferases in 33 %, aminocyclitol-4'-adenylyltransferase (ant(4')-Ia) gene encoding phosphotransferases in 20 %. The coexistence of resistance to methicillin and aminoglycosides was investigated in multi-resistant strains. Coexisting (aac(6')/aph(2″)) and (aph(3')-IIIa) genes were detected in 33 % of isolates, whereas 63 % of isolates had at least one of these genes. All strains revealed adherence ability and most of them (63 %) were invasive to epithelial cells. Electron microscopy revealed that the bacteria were found in vacuoles inside the cells. We observed that the contact of the bacteria with host epithelial cells is a prerequisite to their cytotoxicity at 5 h-incubation. Culture supernatant of the strains induced a low effect of cytotoxicity at the same time of incubation. Cell-free supernatant of all isolates expressed cytotoxic activity which caused destruction of HEp-2 cells at 24 h. None of the strains was cytotonic towards CHO cells. Among thirty strains, 27 % revealed lipolytic activity, 43 % produced lecithinase and 20 % were positive for proteinase activity. Analyses of cellular morphology and DNA fragmentation exhibited typical characteristic features of those undergoing apoptosis. The Pearson linear test revealed positive correlations between the apoptotic index at 24 h and percentage of cytotoxicity. Our results provided new insights into the mechanisms contributing to the development of S. haemolyticus-associated infections. The bacteria adhered and invaded to non-professional phagocytes. The invasion of epithelial cells by S. haemolyticus could be similar to phagocytosis that requires polymerization of the actin cytoskeleton. The process is inhibited by cytochalasin D. Moreover, they survived within the cells by residing in membrane bound compartments and induced apoptotic cell death.

Biofilm formation by Staphylococcus hominis strains isolated from human clinical specimens.

Staphylococcus hominis is the third species of coagulase-negative staphylococci (CoNS) most frequently isolated from specimens of patients with hospital-acquired infections. Many infections caused by CoNS appeared to be associated with biofilms. Nevertheless, the knowledge of the ability of S. hominis to form a biofilm is limited. The aim of this study was to analyze the formation of the biofilm by 56 S. hominis strains isolated from clinical cases. The biofilm three-dimensional structure was reconstructed by confocal laser scanning microscopy. We found that most of S. hominis strains carried icaADBC genes encoding polysaccharide intercellular adhesin (PIA), which plays a crucial role in the formation of biofilms in staphylococci strains. However, only a half of the ica-positive strains had an ability to form a biofilm in vitro. In this study, we also accessed the sensitivity of biofilms of S. hominis strains to sodium metaperiodate, proteinase K and DNase. We found that polysaccharides and proteins are the major components of the extracellular matrix of the biofilm formed by S. hominis. DNase did not have a significant effect on biofilms, which suggested that nucleic acid plays a minor role in the mature biofilm.