Effects of antibiotics on Pseudomonas aeruginosa NK125502 and Pseudomonas fluorescens MF0 biofilm formation on immobilized fibronectin.

The effect of subminimal inhibitory concentration (1/2 MIC) of antibiotics on the biofilm formation on immobilized fibronectin by Pseudomonas was investigated by examining the reference strains NK125502 P. aeruginosa and MF0 P. fluorescens in a microtiter plates assay. When the antibiotics were added during bacterial growth and biofilm development, gentamicin was the only antimicrobial agent tested which decreased significantly the biofilm formation by the two strains. Cefsulodin and chloramphenicol also decreased the P. aeruginosa biofilm development (P<0.01), whereas polymyxin B inhibited biofilm formation by P. fluorescens (p<0.05). When the antibiotics were only present during bacterial growth and not during biofilm development, gentamicin was the only antibiotic tested to decrease significantly the biofilm formation by P. aeruginosa for incubation times of 20 and 72h (P<0.01), whereas P. fluorescens was not affected. This persistent inhibition of P. aeruginosa biofilm formation may be interesting in intermittent antibiotherapy treatments.

alpha5beta1 integrins and fibronectin are involved in adherence of the Pseudomonas aeruginosa ER97314 clinical strain to A549 cells.

The role of fibronectin (Fn) and its natural receptors alpha5beta1 integrins in the interaction of P. aeruginosa with A549 epithelial cells was compared in the clinical isolate ER97314 and the reference PAK strain. Both strains expressed functional type IV pili, as shown by the results of the twitching motility assay. The ER97314 strain was highly adherent to immobilized Fn (640 000+/-20 000 CFU per well) while the PAK strain adhered less efficiently (70 000+/-10 000 CFU per well). Both strains adhered to A549 cells (33 400+/-1200 and 1200+/-100 CFU per well, for PAK and ER97314, respectively), only the PAK strain being significantly internalized (9430+/-2020 CFU per well). Cytochalasin D and genistein significantly decreased bacterial adherence of the 2 strains and caused also a significant decrease in PAK internalization. This inhibitory activity was not related to changes in the expression of alpha5beta1 integrins. Antibodies to Fn and alpha5beta1 integrins inhibited the adherence of the ER97314 strain but had no significant effect on PAK interaction with human cells. These findings suggest that only some P. aeruginosa strains can target Fn and their natural receptors alpha5beta1 integrins for adherence to A549 cells.

Comparison of foetal metabolic differentiation in three cattle muscles.

Metabolic differentiation of Semitendinosus (ST), Cutaneus trunci (CT) and Masseter (MA) in cattle foetuses aged from 110 to 260 days was studied by measuring isocitrate dehydrogenase (ICDH, oxidative) and lactate dehydrogenase (LDH, glycolytic) activities. The five LDH isoenzymes were separated by electrophoresis and assayed by densitometry. ICDH activity increased from 210 days onwards in the three muscles but more intensively in MA (oxidative). LDH activity increased from 170 days onwards in ST, 180 days onwards in CT and only from 210 days onwards in MA and was higher in the glycolytic muscles (ST and CT). The proportion of the LDH-M subunit increased during foetal life in glycolytic muscles. At 110 days, it was higher in CT, intermediate in ST and lower in MA. These results show that 1) metabolic differentiation of bovine muscle begins during the last third of foetal life and 2) the proportion of the LDH-M subunit seems to be related to the contractile type of adult muscle from the first stages of foetal life.

Contractile differentiation of foetal cattle muscles: intermuscular variability.

off actile differentiation was studied in six foetal muscles exhibiting different contractile characteristics in adult cattle: the Masseter, Diaphragma, Biceps femoris, Longissimus thoracis, Semitendinosus and Cutaneus trunci. These muscles were excised from foetuses aged 60-260 days. Fibre types were identified by immunohistochemistry using three monoclonal antibodies raised against types 1, 2a, 2b (or 2x) and foetal myosin heavy chains. The different myosin isoforms were also separated by electrophoresis, identified by immunoblotting and quantified by ELISA. At least two generations of cells were observed in all the muscles studied. The primary, early differentiated one, gave rise to type II fibres in Cutaneus trunci and type I fibres in all remaining muscles. The secondary generation of cells differentiated later than the first generation of cells. Its pattern of differentiation was more complex in particular from 150 to 210 days. It formed slow fibres in slow adult muscles, fast fibres in fast adult muscles and both types in mixed muscles. Precocity of differentiation was muscle-type dependent and related to muscle function at birth.

Electrophoretic separation of bovine muscle myosin heavy chain isoforms.

This study concerns the definition of the optimum conditions for separation of adult and developmental myosin heavy chain (MHC) isoforms in bovine muscle. The various techniques published do not result in good separation of the MHC in this species. The trials carried out concerned the concentration of acrylamide and N, N'-methylene-bis-acrylamide, and more particularly the concentration of Tris in the separating gel. The finding was that analysis of adult isoforms and developmental isoforms require different conditions. A acrylamide gradient of 3.5-10% with 200 mM Tris pH 8.8 gives good resolution for adult isoforms. Under these conditions 3 fast adult isoforms are revealed. However, study of MHC isoforms throughout foetal life in bovines is complex, and requires the combined use of more than one gel (gradient 3.5-10% at 200 mM Tris and gradient 3.5-10% at 250 mM Tris).

Comparative study of metabolic differentiation of foetal muscle in normal and double-muscled cattle.

Studies of the contractile differentiation of foetal muscles in normal and double-muscled cattle have shown that double-muscled foetal muscles display comparative delayed contractile differentiation. We compared the metabolic differentiation of foetal muscles in normal and double-muscled cattle, for which no data were available. The semitendinosus (ST) muscle was excised from normal and double-muscled cattle foetuses aged 110-260 days. Isocitrate dehydrogenase (ICDH) and lactate dehydrogenase (LDH) activities, respectively representative of aerobic oxidative and anaerobic glycolytic metabolism, were assayed. The five LDH isozymes were separated by electrophoresis under non-denaturing conditions and assayed by densitometry. ICDH activity was weaker in the ST of double-muscled foetuses than in that of normal ones throughout gestation, and it increased later in the double-muscled foetuses than in normal ones. In contrast, the LDH activity increased at 180 days in both. It was greater in the double-muscled beyond that time. The proportion of LDH-M subunit was higher at all foetal ages in the double-muscled. Overall, these results show that: (1) the metabolic differentiation of muscle fibre occurs during the last third of foetal growth in both normal and double-muscled cattle; and (2) the differentiation of aerobic oxidative metabolism is delayed in double-muscled foetuses compared with normal ones.

Comparison of the foetal development of muscle in normal and double-muscled cattle.

Muscle differentiation was studied in foetal Semitendinosus muscle from normal cattle and those with the 'culard' gene of muscular hypertrophy sampled at 90, 130, 170 and 210 days of foetal life. The different fibre types were characterized by immunohistochemistry with antibodies specific to different isoforms of myosin heavy chains. The isoforms were separated by electrophoresis, identified by immunoblotting and quantified by ELISA. In double-muscled animals, there was a slower rate of differentiation in the first generation of cells, most markedly apparent at 90 days. At 130 days, differentiation was retarded mainly in the second generation, while at 170 days there was no longer any difference between the two animal types in the differentiation of first generation cells, which were totally slow in both. At the same stage however, type IIC fibres in double-muscled animals were much slower in appearing and continued to be so at 210 days, albeit to a lesser extent. These findings show that differentiation of the muscle fibres occurs at a slower rate in double-muscled foetuses particularly during the first two-thirds of foetal life.

Study of the influence of age and weaning on the contractile and metabolic characteristics of bovine muscle.

Weaning is an interesting period for the study of the nutritional regulation of muscle energy metabolism, since during this stage the nature of the substrates supplied to the muscle and their energy balance are profoundly changed. The aim of this study was to determine the effect of these modifications on the contractile and metabolic characteristics of bovine muscle. Two similar groups of 7 male Montbéliard calves were used with the same age and weight, and with the same energy intake. One group consisted of milk-fed calves, the other of weaned animals. The latter were progressively weaned over a period between 107 and 128 d. The average age at slaughter in the 2 groups was 170 d. Biopsy specimens of semitendinosus (ST) muscle were taken at the ages of 66 d, 94 d (before the beginning of weaning) and 136 d (at the end of weaning) to follow the evolution of muscle characteristics. Samples of longissimus thoracis (LT) muscle were taken 24 h after slaughter and used to study the changes in protein and DNA content. The proportion and area of the different types of fiber, I (slow, oxidative), IIA (fast, oxido-glycolytic), IIB (fast, glycolytic) and IIC (fast/slow, oxidoglycolytic) were measured by immunohistochemistry and image analysis. The metabolism of the muscles was determined by studying isocitrate dehydrogenase (ICDH, oxidative) and lactate dehydrogenase (LDH, glycolytic) activity. The results obtained between 2 and 6 months of life showed an overall increase in the area of the fibers (I, IIA, IIB and IIC) and a conversion of type IIA fibers into type IIB accompanied by a shift in the energy metabolism towards a glycolytic type. Weaning caused temporary stress, whose main consequences were to decrease overall muscle fiber area and the percentage of type IIB fibers, and increase the proportion of type IIC fibers in weaned animals. These effects may have been due to the nutritional and behavioral disturbances that accompany weaning, because 42 d after the end of weaning there was no difference in the size of ST and LT fibers between the 2 groups whereas the proportion of type IIA fibers was still higher in weaned animals.

Presence of an unidentified myosin isoform in certain bovine foetal muscles.

Genetic variation in the establishment of bovine muscle fibre types was studied by comparing muscle differentiation at 210 days of foetal life in normal cattle and in 'culard' animals, which have muscular hypertrophy. The different fibre types were determined by histochemical and immunohistochemical analyses with monoclonal antibodies specific to different myosin heavy chain isoforms. The isoforms were separated by electrophoresis and quantified by the ELISA method. Four muscles with different contractile and metabolic characteristics were studied: Semitendinosus, Longissimus thoracis, Masseter (slow) and Cutaneus trunci (fast). Muscle fibres recognized by none of the antibodies used were observed in 'culard' foetuses in all the muscles studied and also in the Cutaneus trunci of normal animals. Electrophoretic analysis showed no particular myosin isoform in these muscles. It is possible therefore that the fibres contained a mysosin isoform until now unidentified in cattle, with a molecular weight the same as that of known isoforms. This newly observed isoform seems to be specific to muscles rich in IIB fibres such as Cutaneus trunci and to the muscles of adult 'culard' cattle.