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Some observations on the effects of age of calves on the phagocytosis and killing of Staphylococcus aureus by polymorphonuclear leucocytes.

The effects of age on bovine polymorphonuclear (PMN) cell function were investigated by comparing the efficiencies of phagocytosis and killing of Staphylococcus aureus by peripheral blood leucocytes sequentially obtained from 15 calves between the ages of less than 1 and 84 days. One group of seven calves was kept in a controlled environmental chamber with air temperature of 5 degrees C and 58% relative humidity (RH) and another group of eight calves was kept at 16 degrees C and 58% RH. The calves were given a diet a liquid milk substitute and dry food, and were weaned abruptly from the liquid diet at 35 days of age. The in-vitro efficiencies of phagocytosis, and of killing, Staphylococcus aureus by peripheral blood leucocytes were similar for calves in air temperatures of 5 degrees C and 16 degrees C (P greater than 0.05). Peripheral blood leucocytes obtained from calves of less than 1 day of age were more efficient in phagocytosing S. aureus than those obtained when the same calves were 14-84 days of age (P less than 0.001). Peripheral blood leucocytes obtained when the calves were 42 and 56 days of age were significantly less efficient in phagocytosing and killing S. aureus than those obtained when the same calves were less than 1, 14, 28, 70 and 84 days of age (P less than 0.001).

Aging↗

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen.

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.

Antibodies, Monoclonal↗

Concurrent estimation of the kinetics of adhesion and ingestion of Staphylococcus aureus by human polymorphonuclear leukocytes (PMNs).

Direct recording of the kinetics of early phagocytic events, recognition or adhesion and ingestion, may better characterize some forms of polymorphonuclear leukocyte (PMN) dysfunction. This report describes a method and criteria to discriminate concurrently between adhesion and ingestion of Staphylococcus aureus by human PMNs at the level of the light microscope. The criteria were confirmed by several lines of direct evidence: low temperature and cytochalasin b treatment of PMNs; lysostaphin digestion of target Staphylococcus aureus after PMN incubation; and differential ingestibility of colony types I and III N. gonorrheae. Concurrent determinations of the initial kinetics of adhesion and ingestion of bacteria by PMNs demonstrated generalized first order kinetics, sensitive to bacterial challenge ratio and total particle density. Most importantly, the method may be applied to determine if phagocytically defective PMNs actually fail to recognize opsonized bacteria.

Blood Bactericidal Activity↗

Development of an enzyme-linked immunosorbent assay for staphylococcal protein A produced in Escherichia coli by pUC8 based plasmids containing the Staphylococcus aureus Cowan I protein A gene.

Staphylococcal protein A, a cell wall component of several strains of Staphylococcus aureus has found many uses as a research tool, as a diagnostic reagent and even as a possible therapeutic agent in cancer. These uses have arisen exclusively out of its almost unique property of binding specifically to the Fc region of many immunoglobulin molecules. As Staphylococcus aureus is pathogenic, it has been desirable for industrial purposes to clone the gene coding for protein A into Escherichia coli and to develop a sensitive assay free from interference by bacterial components. We describe an ELISA assay which is capable of detecting staphylococcal protein A in bacterial lysates at levels as low as 1.0 ng/ml and which is free of interference from lysozyme, lysostaphin, endogenous peroxidases or other bacterial antigens.

Animals↗

Bacterial prostatitis caused by Staphylococcus saprophyticus.

Staphylococcus saprophyticus has been shown to be an important uropathogen in urinary tract infections in young women. Thirty-five isolates from 27 patients with staphylococci in the prostatic fluid of men with bacterial prostatitis were evaluated for the presence of S. saprophyticus. Three patients (11 per cent) with this organism were identified by novobiocin resistance (disk diffusion test), absence of hemolysis, and coagulase. These patients tended to be younger, more symptomatic, and more responsive to appropriate antibiotic therapy than those with staphylococcus epidermidis. S. saprophyticus appears to be an important pathogen in prostatic infections.

Adult↗

Seven surgical silicones retain Staphylococcus aureus differently in vitro.

In an in vitro study, we have quantitatively evaluated the capability of seven different types of silicone to retain a Staphylococcus aureus strain, isolated from a surgical wound. All the silicone specimens were taken from prostheses already used in plastic or ophthalmological surgery. Two polymers were used as controls: polystyrene, because of its known capability to favour in vitro bacterial recovery, and nylon, for its bacterial repellence. The results show that all silicones are suitable substrata for Staphylococcus aureus. However, there are some differences among silicone types. The amounts of bacteria retained from silicone oils are greater than or equal to those obtained from the positive control material.

Biocompatible Materials↗

Systemic profiles of antigen-specific lymphocytes in animals chronically exposed to staphylococcus antigen in the mammary region.

Systemic profiles of lymphocytes were assessed in goats exposed chronically with Staphylococcus antigens in the supramammary region. Animals were inoculated three times subcutaneously in the right supramammary region with heat-killed Staphylococcus aureus antigen (HKS) at 1 month intervals. Prior to immunization and 1 week following each injection, 3 and 6 day cultures of peripheral blood mononuclear cells were made to determine proliferative responses of lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). Peripheral blood lymphocytes responded significantly to both HKS and PHA in 3 day cultures after the second injection and showed peak responses after the final immunization, suggesting that repeated local injection of S. aureus antigen at the supramammary region, can induce an anamnestic response to the antigen in the peripheral blood of these animals with a concomitant increase in the responsiveness to the polyclonal mitogen, PHA. In contrast, initial antigen challenge induced little, if any, increase in responses to the specific antigen or mitogen when compared to pre-injection states. These data may also suggest that non-reactivity of peripheral blood lymphocytes to the HKS antigen immediately after the primary injection of antigen may be the result of local retention of antigen-reactive cells at the sites of infection.

Animals↗

In vivo effect of teicoplanin and vancomycin upon haemolytic and bactericidal activity of serum against Staphylococcus aureus.

The present study was undertaken to determine whether teicoplanin and vancomycin influence the hemolytic and bactericidal activity of serum obtained from mice (Swiss, aged 15 +/- 2 weeks). Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units (CFU/ml) of Staphylococcus aureus that survived after 24 h of incubation in the presence of serum. The results indicate that (1) teicoplanin and vancomycin increase the haemolytic activity of serum; (2) in the serum from animals treated with both teicoplanin or vancomycin, the number of CFU/ml of Staphylococcus aureus declines; and (3) these antibiotics appear to have a similar effect upon the complement system.

Animals↗

Staphylococcus saprophyticus found to be a common contaminant of food.

The mode of transmission of Staphylococcus saprophyticus, a urinary tract pathogen, was investigated in three related studies. The presence of this organism was sought, during a period of 1 year, in 1331 specimens of various foods, in 920 beef and pork carcasses and on 107 cultures which had been inoculated directly from abattoir workers' protective gloves. Staphylococcus saprophyticus was found to contaminate 16.4% of the various food samples with a high prevalence of 34% in raw beef and pork. It was common in both domestic and imported raw meat products. There was no seasonal variation in the presence of S. saprophyticus in the samples obtained from carcasses. The bacterium was found in 69% of all cultures from the workers' protective gloves. We conclude that S. saprophyticus, originating from slaughtered animals, contaminates food and eventually colonizes the human intestinal tract.

Abattoirs↗

Use of enzyme-linked immunosorbent assay to measure bovine milk and serum antibodies to alpha toxin, beta toxin, and capsular antigens of Staphylococcus aureus.

An enzyme-linked immunosorbent assay (ELISA) procedure was used to quantitate milk and serum antibodies (IgG) to Staphylococcus aureus alpha and beta toxins, and S. aureus 2-8 and Smith diffuse strain capsular antigens. Milk samples were collected on two occasions. A comparison was made between levels of milk antibodies specific for the two toxins and capsular antigens for 41 cows that were infected with S. aureus on both sampling dates, and 18 cows not S. aureus-infected on either date. Staphylococcus aureus-infected cows were grouped according to somatic cell counts. All groups of infected cows, regardless of somatic cell counts, had significantly higher milk antibody levels to alpha and beta toxins than did the non-infected cows (P less than .002). Serum samples taken for 13 infected and 4 non-infected cows also indicated that significant elevations in anti-alpha toxin and anti-beta toxin IgG were present in S. aureus-infected cows, compared to non-infected cows. A similar immune response was not seen to capsular antigens, however. No significant differences were present between the two groups of cows for either milk or serum antibodies to Smith diffuse strain capsular antigens. Milk antibodies to 2-8 capsule were significantly elevated only in infected cows with somatic cell counts greater than 10(6)/ml, compared to non-infected cows; no differences were present for serum antibodies to 2-8 capsule between infected and non-infected cows. These results indicate that significant increases in milk (and possibly serum) antibodies to alpha and beta toxins are present in cows with chronic staphylococcal mastitis, apparently resulting from a systemic immune response to these toxins. There does not appear to be a similar immune response to capsular antigens.

Animals↗

Histone H1 structure probed by Staphylococcus aureus V8-proteinase.

Proteolytic digestion of calf thymus histone H1 with Staphylococcus aureus V8-proteinase under structuring conditions generates one major limit peptide P1 which consists of approx. 170 residues. Edman degradation establishes the N-terminal sequence as: Leu-Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys. Chymotryptic fingerprinting shows that the C-terminal part of the H1 molecule is fully preserved. The peptide therefore comprises the residues H1 (42-210). The Glu-41 cleavage is extremely unusual as it occurs in the structured G-domain which is known to be resistant to proteinases (Hartman, P. G., Chapman, G. E., Moss, T. and Bradbury, E. M. (1977) Eur. J. Biochem. 77, 45-71; Böhm, L., Sautière, P., Cary, P. D. and Crane-Robinson, C. (1982) Biochem. J. 203, 577-582). The V8-proteinase cleavage product H1 (42-210) shows only 20% folding as compared to 95-99% folding shown by the peptides H1 (34-121), H1 (31-210) and H1 (33-210). Folding of the G-domain thus critically depends upon the presence of the eight residues 33-41 amongst which the Gly-Pro-Pro sequence at position 36-38 and a beta-turn predicted at position 35 are considered to be particularly important. The location of the cleavage site in the G-domain renders Staphylococcus aureus V8-proteinase suitable as a structural probe.

Amino Acid Sequence↗

Characterization of Staphylococcus spp. and Micrococcus spp. isolated from Iberian ham throughout the ripening process.

The Iberian dry cured ham is an uncooked meat product highly appreciated because of its characteristic flavour. This product is obtained from highly marbled Iberian pig hindlegs after 18-24 months of maturation under natural environmental conditions. The role of Micrococcaceae in the development of the aroma characteristics of this products remains unclear. Identification of Gram-positive, catalase-positive cocci isolated from Mannitol Salt Agar plates showed that Staphylococcus xylosus followed by Staphylococcus equorum are the predominant organisms, even after 16 months of maturing. A remarkable variety of types of both staphylococci and micrococci are detected at any sampling time. The metabolic activities of these organisms could contribute to the characteristics of the final product.

Animals↗

A survey of methicillin-resistant Staphylococcus aureus affecting patients in England and Wales.

For a six-month period between October 1987 and March 1988, 660 isolates of methicillin-resistant Staphylococcus aureus (MRSA) from 570 patients were sent to the Staphylococcus Reference Laboratory at Colindale to supplement the National reporting survey of MRSA in England and Wales. The isolates were characterized by phage typing, antibiotic susceptibility and by selected biochemical tests. Patient details were also surveyed. Fourteen strains affected more than one hospital and were called multi-hospital epidemic strains. One strain, EMRSA-1, accounted for more than 40% of isolates and of patients. Other epidemic strains were defined. Ten additional strains were restricted to single hospitals. Only 25 primary isolates were non-typable but 67 sporadic typable strains occurred. The patients affected were approximately equally either infected or colonized. The sexes were represented equally. Orthopaedic and geriatric wards were over-represented. Epidemic strains were clumping factor positive while some sporadic strains were weak producers. Urea alkalinization and protein A production could supplement phage typing and antibiotic resistance in strain recognition.

Adult↗

Investigation of nosocomial prosthetic valve endocarditis due to antibiotic-resistant Staphylococcus epidermidis.

A reservoir of antibiotic-resistant Staphylococcus epidermidis strains in our cardiac surgery unit appeared to be the source of organisms responsible for three cases of early prosthetic valve endocarditis. Staphylococcus epidermidis isolates recovered from the skin of 13 patients before and after surgery were compared. All were typed by plasmid profile, antimicrobial susceptibility and slime production. The three strains from early prosthetic valve endocarditis resembled the antibiotic-resistant nosocomial strains recovered from the skin of eight patients following surgery and the environment of the operating theatres. These strains expressed resistance to oxacillin, gentamicin, kanamycin and tobramycin and most produced slime, whereas those isolated from the skin of patients at the time of admission were predominantly susceptible to antibiotics and few produced slime.

Adult↗

Efficacy of mupirocin nasal ointment in eradicating Staphylococcus aureus nasal carriage in chronic haemodialysis patients.

Topical 2% mupirocin ointment eradicated chronic Staphylococcus aureus nasal carriage immediately post-therapy in 17 (77%) of 22 haemodialysis patients. Mean time to recurrence was 3.8 weeks. Similar pre-therapy and post-therapy phage types occurred in 12 (71%) of 17 patients. Staphylococcus aureus infections developed in none of 17 successfully treated patients, two of five treatment failures (P = 0.05), and 10 of 46 untreated patients studied concurrently (P = 0.03).

Anti-Bacterial Agents↗

Long-term carriage, and transmission of methicillin-resistant Staphylococcus aureus after discharge from hospital.

The purpose of this study was to determine whether patients who become carriers of methicillin-resistant Staphylococcus aureus (MRSA) during their stay in hospital, remain colonized after discharge. Thirty-six patients colonized with MRSA during one of three outbreaks at Utrecht University Hospital (1986-89) were screened 2 or 3 years after they had become carriers. Patients were also interviewed to determine factors contributing to persistent carriage, such as antibiotics, re-admissions to the hospital, presence of skin lesions and chronic diseases. At the same time transmission of MRSA to family members was determined. The epidemic MRSA strain was still found in three patients (8%). One was a cystic fibrosis patient who had had frequent re-admissions to the hospital and had received several course of antibiotic treatment. Both of the other patients had skin lesions: a fistula and a colostomy respectively. None of the 44 family members of the patients was colonized or infected with MRSA. We conclude that long-term MRSA carriage occurs with low frequency and is comparable to persistent carriage of methicillin-sensitive Staphylococcus aureus (MSSA). Transmission of MRSA to healthy individuals in an antibiotic-free environment is a rare event.

Adolescent↗

Molecular epidemiology of a multiple strain outbreak of methicillin-resistant Staphylococcus aureus amongst patients and staff.

Since methicillin-resistant Staphylococcus aureus (MRSA) isolates are not endemic in our hospital, which is a tertiary referral centre, the finding of 13 MRSA isolates from 12 patients associated with an acute vascular surgery ward between October 1993 and December 1993 prompted further epidemiological and laboratory investigations. Two strains were distinguished by antibiogram and phage-typing. One strain, resembling EMRSA-16, colonized six patients and was probably introduced from another hospital in the Oxford Region. Five other patients were colonized by a second strain, gentamicin-resistant and non-typable by phage-typing, probably introduced into the hospital 12 months previously by a patient from Nairobi, Kenya. A 12th patient was colonized by both strains simultaneously. Of 46 staff members screened three were colonized--one by an EMRSA-16 strain, a second by the gentamicin-resistant 'Nairobi'-strain and a third member carried yet a further distinct MRSA strain. The healthcare worker colonized by the 'Nairobi'-strain had been carrying the isolate 12 months previously and was the likely source of this strain. These isolates were also characterized by the repetitive extragenic palindromic-polymerase chain reaction (REP-PCR), a novel PCR-based methodology which has not been previously used in characterizing Staphylococcus aureus in an outbreak. This method corroborated the strain classifications provided by the traditional methods, confirming that there had been spread of two strains simultaneously. Our study demonstrates that multiple strains of MRSA may circulate amongst patients and staff during an outbreak, patients may be colonized by more than one strain simultaneously and long-term staff carriage (> 12 months) may be an important source of colonization in patients. REP-PCR is a rapid and effective molecular typing method for MRSA.

Bacterial Typing Techniques↗

Colonization by Staphylococcus species resistant to methicillin or quinolone on hands of medical personnel in a skilled-nursing facility.

We report here a pilot survey of colonization with methicillin- and/or ciprofloxacin-resistant Staphylococcus species on hands of nursing personnel in a private skilled-nursing facility. We found only one nurses aide who carried methicillin-resistant Staphylococcus aureus and one who carried ciprofloxacin-resistant S. aureus, each on only one of the surveys. None of the control nonmedical personnel were found to carry methicillin-resistant S. aureus or ciprofloxacin-resistant S. aureus. The colonization rate of methicillin-resistant coagulase-negative staphylococci on the hands of medical personnel was 59%, compared with 13% for the nonmedical personnel, and the counts of methicillin-resistant coagulase-negative staphylococci were also significantly higher for nursing personnel. For ciprofloxacin-resistant coagulase-negative staphylococci, 30% of nursing personnel had positive cultures whereas no ciprofloxacin-resistant coagulase-negative staphylococci strains were recovered from the nonmedical control cohort. Three of the patients had presumptive infections with methicillin- or ciprofloxacin-resistant coagulase-negative staphylococci, all urinary tract infections. Personnel hands represent a likely mode of transmission of such strains between patients, and skilled-nursing facility patients may represent a reservoir for carrying the coagulase-negative staphylococci back to acute care facilities.

Adult↗