In vivo and in vitro effects of lactoferrin on Yersinia pseudotuberculosis.

Iron is a growth requirement for virtually all microbes. In the human body, extracellular iron is sequestered from microbes by binding to proteins such as lactoferrin. In this study the effect of lactoferrin and human milk on Yersinia pseudotuberculosis was investigated. Its growth in vitro was inhibited by iron-free, but not iron saturated, pure lactoferrin or human milk. Iron-free human milk and to a less extent normal human milk were bactericidal for Y. pseudotuberculosis cells that were suspended in deionized water. The in vivo studies also show that iron-saturated lactoferrin enhanced growth, whereas, the viable count was reduced by iron-free pure lactoferrin and EDDA. Nine envelope proteins were decreased or disappeared upon growth in iron-deficient medium, whereas one new high molecular weight protein appeared under the same conditions.

Effect of some physical and chemical factors on the bactericidal activity of human lactoferrin and transferrin against Yersinia pseudotuberculosis.

The iron-chelating proteins lactoferrin and transferrin have been shown to be bactericidal for a variety of organisms. In this study, the effect of pH, temperature, their concentration, and magnesium and calcium on the bactericidal activity against Yersinia pseudotuberculosis was investigated. The bactericidal activity of lactoferrin was higher at acid pH, whereas, the bactericidal activity of transferrin was higher at alkaline pH. Both were not efficient at 4 degrees, 15 degrees, and 25 degrees C, but they were efficient at 37 degrees C. Lactoferrin, but not transferrin, was very efficient at 42 degrees C. The activity of both were time and concentration dependent. Calcium did not effect their activity up to 60 mM, whereas, magnesium reduced the activity of lactoferrin only.

Effect of temperature on the lipid and fatty acid composition of Yersinia pseudotuberculosis.

In Yersinia pseudotuberculosis, the total lipid content is increased by decreasing the temperature of the growth medium. Thin layer chromatography of phospholipids revealed the presence of phosphatidy glycerols (PG) and phosphatidylethanolamine (PE) in the bacterium when grown at 26 degrees and 37 degrees C. Cardiolipin (CL) is present only in the bacterium grown at 37 degrees C, while lysophosphatidylglycerol (LPG) is present only in the bacterium grown at 26 degrees C. Unidentified phospholipids designated X1 was present in the bacterium at the two temperatures and X2 was only detected at 26 degrees C. Fatty acid unsaturation increased with decreasing temperature. Hexadecenoic acid (C16:1) content increased as the temperature was reduced to 26 degrees C. At lower temperature, stearic, oleic, linoleic, eicosanoic and linolenic acids were decreased. These observations support the view that regulation of membrane adaptation and the mechanism behind the responses might be more complex than is generally believed.

Antibiotic resistance, plasmid content, phage type, and capsule type of Staphylococcus aureus isolates at a children's hospital.

One hundred clinical isolates of Staphylococcus aureus were obtained from Al-Solimaniah Children's Hospital, Riyadh, Saudi Arabia. The isolates were screened for their antibiotic susceptibility, plasmid profiles, phage types, and capsular polysaccharide types. A total of 29 antibiotic resistance patterns were obtained. Nine plasmids were detected and 18 plasmid profiles were obtained. Thirteen isolates were non-phage-typeable and 14 different phage-typing-patterns were obtained from the other 87 isolates. Thirty eight isolates were capsular polysaccharide type 8 and 56 were capsular polysaccharide type 5; the other 6 isolates were non typeable. According to the above typing criteria, the isolates were divided into 13 groups. Plasmid profiles were found to be superior to phage typing, whereas, phage typing was superior to the antibiogram as a technique for determining similarities and differences among S. aureus hospital isolates.

Effect of ethylenediamine di-o-hydroxyphenylacetic acid and transferrin on the growth of some bacterial strains in vitro.

The ability of some bacterial strains to obtain iron from ethylenediamine di-o-hydroxyphenylacetic acid (EDDA) or iron-free transferrin, and accordingly grow in their presence, was studied. Growth of Staphylococcus aureus and Yersinia pseudotuberculosis was inhibited by EDDA or by iron-free transferrin. Growth of Streptococcus faecalis, however, was inhibited by iron-free transferrin, but not by EDDA. The other bacterial strains, i.e.; Escherichia coli, Salmonella typhimurium and Shigella dysenteriae were able to grow both in the presence of EDDA or iron-free transferrin. All of the above bacterial strains grow in the presence of iron-saturated transferrin which was not able to bind the iron of the medium and accordingly left the iron of the medium available to them.

Effect of medium salinity on some chemical constituents of two halophilic Bacillus spp. from Saudi Arabia.

The cell envelope amino acids of two moderately halophilic Bacillus isolates (BST and BSF) varied according to medium salinity. Cystine and proline were mostly effected. In both isolates growing in the presence of 6 and 18% NaCl there were more dicarboxylic amino acids than basic amino acids which makes the cell envelope proteins quite acidic. The concentrations of the cell-associated cations (Na+, K+, and Mg2+) were high in both isolates, and varied according to the NaCl concentration. The two isolates contained glucosamine and muramic acid in their cell walls. The amounts of these two sugar derivatives, however, varied with the NaCl concentration. Thin-layer chromatography of phospholipids revealed the presence of cardiolipins, phosphatidylglycerols and phosphatidylethanolamines in the two isolates irrespective of medium salinity. Phosphatidylglycerols and the phosphatidylethanolamines increased on increasing the NaCl concentration of the growth medium. Lysophosphatidylglycerols were detected only in the 6% grown BST cells. Unidentified phospholipids designated X1 (in isolates BSF and BST), X2 (in isolate BST) and X3 (in isolate BSF) were also detected; the concentrations of X1 and X3 were salinity dependent.

Antibiotic resistance and plasmids of some human nasal isolates of Staphylococcus aureus.

The Staphylococcus aureus isolates were all susceptible to vancomycin. More than 90% of the isolates were susceptible to rifampicin, ampiclox, methicillin, erythromycin and clindamycin. The isolates were highly resistant to the beta-lactamase-sensitive penicillins, that is 91, 93.2 and 70.4% of the isolates were resistant to penicillin, ampicillin and carbinicillin. Twelve plasmids were found in the isolates, the 35 and 11 Mdal plasmids coded for aminoglycosides and tetracycline resistances, respectively.

Association of coagulase and/or pigmentation with the virulence of a capsule-lacking Staphylococcus aureus in iron-compromised mice.

The effect of pigmentation and coagulase on the virulence of a capsule-lacking Staphylococcus aureus were studied using iron-compromised mice. The presence of either coagulase or pigmentation maintained the viability of this bacterium in the peritoneal cavity. Both coagulase and pigmentation, however, lead not only to maintained viability, but also to its proliferation in the peritoneal macrophages. The iron was found to enhance the virulence of this bacterium, but iron enhancement was contingent on the presence of either pigmentation or coagulase or preferably both.

Effects of heat and pH stresses on the recovery of Staphylococcus aureus on medium-110.

The effects of heat and pH stresses on Staphylococcus aureus strain ATCC 10390 (coagulase positive) and strain ATCC 6020 (coagulase negative) were studied. The coagulase negative strain was more sensitive to heat and low or high pH than the coagulase positive strain. Both strains, however, were injured by heat and by pH 4.5 and 9 as estimated by the decrease in their tolerance to the selective agents (sodium chloride and sodium azide) present in the selective medium. The heat or pH injured cells, however, regained their tolerance to the selective agents if they were pregrown in a non-selective medium before they were plated on the selective medium.

Role of pre-incubation in non-selective medium in recovery of Staphylococcus aureus from swimming pools and beaches.

Staphylococcus aureus was isolated from eleven open swimming pools and eleven swimming beaches in certain areas of Saudi Arabia. Pre-incubation of the water samples in a non-selective medium prior to their growth in the selective medium increased the rate of recovery over that of direct plating on the selective medium. The average increase was from 20.68% to 46.1% for pools and from 32.7% to 37.3% for beaches. Pre-incubation, therefore, is very important for the pool water which appears to contain more stressed bacteria.

Isolation of Erysipelothrix rhusiopathiae from a 55 year old man and a positive tracing of the infection to chicken-raising premise.

Erysipelothrix rhusiopathiae was isolated from the swollen finger of a 55 year old man. The swelling was due to a peck by an infected chicken. Tracing the infection to the chicken-raising premises has proven that the bacterium was present in some of the chicken and manure samples.

Effect of the virulence associated 47 megadalton plasmid of Yersinia pestis on permeability to gentian violet and sensitivity to novobiocin.

The loss of the 47 megadalton (Mdal) plasmid of Yersinia pestis resulted in increased gentian violet uptake and increased sensitivity to novobiocin. The addition of calcium caused a decrease in gentian violet uptake and a decrease in novobiocin sensitivity independent of the plasmid. Differentiation of cells bearing and lacking the plasmid was accomplished using novobiocin in the presence of calcium at 26 degrees C. The novobiocin resistance levels of cells lacking the 47 Mdal plasmid and cells with the plasmid, but with an insertion in or a deletion in, or encomposing, the calcium dependence region of this plasmid, indicated that the resistance to novobiocin was not associated with the calcium dependence region, but was associated with a separate 47 Mdal plasmid region. Examination of Yersinia pseudotuberculosis and Yersinia enterocolitica isolates demonstrated that functionally similar sites are present on the plasmids that encode for calcium dependence in these species.

A permeability mutant of Yersinia pestis with increased susceptibility to phagocytosis which retains potential for intraphagocytic growth and virulence.

Gentian violet resistance was used to select mutants of Yersinia pestis with altered cell envelope permeability. Mutants in one class lacked the 6 and 61 megadalton (Mdal) plasmids, but retained the 47 Mdal plasmid associated with calcium dependence. Mutants in a second class retained all three plasmids. One mutant in the latter class, EV76S7, lacked major outer membrane protein J and yielded reduced levels of minor outer membrane proteins A and C. Protein J is known to interact differently with lipopolysaccharide (LPS) from cells bearing and cells lacking the 47 Mdal plasmid following growth at 37 degrees C. The 47 Mdal plasmid is known to influence gentian violet uptake, novobiocin sensitivity, susceptibility to phagocytosis, the growth in macrophages and the virulence of Y. pestis. Gentian violet uptake was lower and novobiocin sensitivity higher in the mutant than in the parental strain, but the underlying effects of the 47 Mdal plasmid on these parameters had not changed. EV76S7 cells were phagocytized to a greater extent than the parental cells following growth at 37 degrees C, but survival and growth within peritoneal phagocytes and the LD50's of cells with and without the 47 Mdal plasmid were not altered by the loss of protein J, we conclude that protein J--LPS interactions are not required for virulence, and that the reduced virulence of cells lacking the 47 Mdal plasmid does not result from the altered protein J--LPS interactions which are known to result from loss of the plasmid.

The calcium dependence region of the 47 megadalton plasmid of Yersinia pestis is required for growth within macrophages.

Yersinia pestis cells bearing the 6,47 and 61 megadalton (Mdal) plasmids have been shown previously to be more resistant to novobiocin and to take up less gentian violet than cells lacking the 47 Mdal plasmid. Mutants with lesions in the calcium dependence region of the 47 Mdal plasmid, which are known to have resistance to high levels of novobiocin, were found to have rates of gentian violet and deoxycholate sensitivities identical to cells with intact plasmids. Cells bearing the 6, 47 and 61 Mdal plasmids, but not cells without the 47 Mdal plasmid, were known to grow within peritoneal macrophages. Cells with mutations in the calcium dependence region of the 47 Mdal plasmid had greatly reduced virulence in iron compromised mice and were unable to proliferate in the peritoneal macrophages of these mice. Cells lacking both the 6 and 61 Mdal plasmids, selected in the presence of gentian violet, were found to have reduced gentian violet uptake but unexpectedly had increased sensitivity to novobiocin and deoxycholate. These cells had an LD50 in iron compromised mice similar to that of cells with all three plasmids and proliferated in the peritoneal macrophages of iron compromised mice.