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The bacteriolytic activity of Staphylococcus aureus and Staphylococcus albus strains was tested with various media. Whereas S. aureus strains were found to be active under all conditions, the percentage of active S. albus strains was significantly influenced by the composition of the medium. Ionic strength and concentration of the organic nitrogen source were found to be the main factors affecting the expression of bacteriolytic activity of straphylococci. Virtually all of 318 S. aureus and 603 S. albus strains were active on a medium containing 3% peptone, 0.3% glucose, 0.2% yeast extract, 0.1% disodium phosphate, 2.2% sodium chloride and 0.9% agar. The optimal conditions for the bacteriolytic activity of S. aureus strains were different from those of most S. albus strains. Within S. albus, optimal conditions differed also from strain to strain. It is suggested that further studies on this subject may prove useful for the identification and taxonomy of staphylococci. A possible relationship between the production of extracellular bacteriolytic enzymes and pathogenic properties of staphylococci is also considered.

Objective:To assess the incidence of resistance to erythromycin and to the three other macrolide antibiotics most extensively used in Italy (azithromycin, clarithromycin and roxithromycin) among clinical strains of Streptococcus pyogenesfreshly isolated from throat cultures of pediatric patients in an area of Central Italy.

Both human lysozyme (HL) and hen egg white lysozyme (HEWL) inhibited the proliferative response of peripheral blood lymphocytes to T cell mitogens such as the lectins phytohemagglutinin and concanavalin A. This inhibition was observed both when HL or HEWL was added to the lymphocyte cultures in combination with phytohemagglutinin or concanavalin A and when lymphocytes were pretreated with either lysozyme and extensively washed prior to culture with mitogens. Under both conditions, the effects were strictly dose dependent; the lysozyme concentrations yielding maximal inhibitory effect were 5 μg/ml for HL and 1 μg/ml for HEWL, while both lower and higher concentrations were less effective. Specific antilysozyme rabbit sera completely prevented the inhibitory effects of both HL and HEWL on the proliferative response of lymphocytes to phytohemagglutin or concanavalin A. Chitotriose (a lysozyme inhibitor) caused a strong reduction in the inhibitory effects of the two lysozymes on the lymphocyte response to either lectin. HL and HEWL also were found to markedly inhibit the polyclonal B cell proliferation and differentiation induced by pokeweed mitogen and T cells. A less marked inhibition was also obtained when T cells, but not B cells, were pretreated with HL or HEWL. Again, as in the experiments with T cell mitogens, the effects were dose dependent and 5 μg/ml HL and 1 μg/ml HEWL proved to be the most effective concentrations. The possible mechanisms by which lysozyme inhibits the lymphocyte response to mitogenic lectins are considered and discussed. The enzymatic activity seemed to perform an essential function, as shown by the loss of effect when the heat- or trypsin-inactivated lysozymes were used and by the fact that only the enzymatically active compound, among certain semisynthetic derivatives of HEWL, inhibited the lymphocyte response to the mitogens. However, the cationic properties of the lysozyme molecule appeared to be essential too, since enzymes with a similar specificity of action showed effects similar to those observed with HL or HEWL only when they carried a strong positive charge. It is suggested that lysozyme, which is naturally secreted by monocytes and macrophages, might interact with lymphocyte surface receptor sites and participate in the complex mononuclear phagocyte-lymphocyte interactions and in the modulation of lymphocyte activation.

A simplified system for routine identification of the six lyogroups of human staphylococci is described. The method is based on the determination of the lytic activity on five different test media and of the phosphatase activity of each isolate. A total of 689 staphylococci isolated from clinical material over a 6-month period were identified according to this simplified system. The same strains were identified in parallel using three other methods designed for routine identification of staphylococci: the Kloos and Schleifer abbreviated scheme (J Clin. Microbiol. 1:82-88, 1975) the scheme proposed by the Subcommittee on the Taxonomy of Staphylococci and Micrococci (Int. J. Syst. Bacteriol. 26:332-334, 1976), and the API STAPH micromethod. Isolated whose identification was in disagreement were identified on a taxonomic level according to the extensive classification scheme of Schleifer and Kloos. In addition, 42 reference staphylococci of known identity were reidentified using the simplified lyogroup system as well as the three other routine methods. The results of this comparative study was detailed and discussed. A primary advantage of the simplified lyogroup system is that it also provides separation of staphylococci from micrococci, which must be performed separately when the other methods are used. Other significant advantages concerned with reliability, speed, and practicality are shown.

The purpose of this study was to determine whether lysostaphin would enhance its anti-staphylococcal efficacy in combination with lysozyme. Minimum inhibitory concentrations (MICs) of lysostaphin and lysozyme were separately determined for 41 strains belonging to 10 different species of human staphylococci. Lysozyme was virtually inactive and showed MICs of 15 mg/ml. On the contrary, all strains were susceptible to lysostaphin and showed MICs ranging from 2.5 to 60 micrograms/ml for the different Staphylococcus species. When the MIC of lysostaphin was determined in media containing submultiples of the MIC of lysozyme, the values obtained were much lower. The reduction of the lysostaphin MIC ranged from 16- to 200-fold in the different species tested. In Staphylococcus aureus, in particular, the combination of lysostaphin with 1.5 mg of lysozyme per ml reduced the MIC of lysostaphin by 25-fold. The activities of two combinations of the two enzymes were evaluated: one combination was expected to be active on S. aureus only, and the other combination was expected to inhibit all Staphylococcus strains. The first combination (0.5 micrograms of lysostaphin plus 0.5 mg of lysozyme per ml) was inhibitory to all of the 84 S. aureus strains tested, whereas 137 of 151 strains of other Staphylococcus species were unaffected. On the contrary, all of the 235 Staphylococcus strains tested were inhibited by the second combination (4 micrograms of lysostaphin plus 5 mg of lysozyme per ml). The possible mechanisms of lysostaphin potentiation by lysozyme are considered, and the potential use of a lysostaphin-lysozyme combination for topical therapy of staphylococcal infections resistant to other antibiotics is discussed.

A simplified application of the polymerase chain reaction (PCR) to the routine detection of human immunodeficiency virus type 1 (HIV-1) transcripts from peripheral lymphocytes of infected subjects is described. This technique is simpler than previously described assays and was shown to be highly sensitive after ethidium bromide staining of polyacrylamide gel electrophoresis of amplified material. The method can be used for the virologie evaluation of HIV-1-infected subjects, thus allowing early identification of seropositive patients with signs of active infection.

A multicentric study of clinical Staphylococcus isolates was performed by seven operative units working in different areas of Italy. Over a 6-month period, a total of 3,226 staphylococci, isolated from in- and outpatients, were identified and tested for antimicrobial susceptibility by a protocol agreed upon by all units. On the basis of their bacteriolytic-activity patterns and other conventional tests, the isolates were identified by lyogroups , which closely correlate with human Staphylococcus species. Lyogroup I (Staphylococcus aureus) and lyogroup III (Staphylococcus capitis) were the most and the least frequently isolated staphylococci, respectively. Significant differences depending on strain origin from in- or outpatients were only observed with lyogroup IV (i.e., novobiocin- resistant staphylococci), whose isolation from outpatients was three times greater than from inpatients. Lyogroup I was predominant among isolates from most clinical sources. Lyogroup IV predominated in strains isolated from the urinary tract; lyogroup V (Staphylococcus epidermidis) predominated in strains from blood, cerebrospinal fluid, and indwelling artificial devices; and lyogroup VI ( Staphylococcus hominis, Staphylococcus haemolyticus, and Staphylococcus warneri ) predominated in strains from bile and the male genital tract. The incidence of methicillin resistance within the different lyogroups varied from unit to unit, suggesting epidemiological differences among different hospitals and different geographical areas. On the whole, methicillin resistance was more frequent in coagulase-negative staphylococci than in S. aureus and ranged from 19% for lyogroups I and III to 30% for lyogroup II (Staphylococcus simulans). Laboratory testing with 18 additional antibiotics suggested the occurrence of some specific differences in susceptibility among the different lyogroups . The rate of organisms resistant to the various antibiotics was greater among methicillin-resistant than among methicillin -susceptible staphylococci; particularly marked differences occurred with cephalosporins, rifampin, gentamicin, and tobramycin. The results suggested an increasing spread in Italy, during the last few years, of staphylococcal resistance to methicillin and to many other antibiotics. Some questions about the actual reliability of laboratory tests for the determination of staphylococcal susceptibility to methicillin and other beta-lactam antibiotics were raised by parallel test performances in which both unsupplemented and 5% NaCl-supplemented Mueller-Hinton agars were used. The presence of NaCl heightened, on the whole, the number of resistant strains detected; however, a few isolates resistant in the unsupplemented medium and susceptible in the salt-supplemented medium were also encountered. This was true not only for methicillin but also for all other beta-lactam antibiotics tested except cefamandole. With cefamandole, the presence of 5% NaCl reduced the number of resistant strains detected.

The antistaphylococcal activity of rifapentine, a new rifamycin SV derivative, was evaluated in vitro and compared with that of rifampin. A total of 313 staphylococcal strains freshly isolated from clinical material and including representatives of all currently recognized Staphylococcus species of human origin were used. The susceptibility to methicillin of all the test strains was determined preliminarily. Despite minor differences with some species, the MICs of rifapentine were found to be substantially similar to those of rifampin. Methicillin-resistant strains of all species were most resistant to rifapentine and rifampin than were their methicillin-susceptible counterparts. For most strains tested, the MBCs of both rifamycins exceeded by twofold the respective MICs. Both the checkerboard dilution and time-kill methods were used to determine the interactions of rifapentine or rifampin with six different antibiotics: cefamandole, vancomycin, teicoplanin, gentamicin, erythromycin, and fusidic acid. No significant differences between the two rifamycins in the combinations were observed against either methicillin-susceptible or methicillin-resistant strains. Minor differences were noted depending on the second antibiotic tested or the staphylococcal species examined. Antagonism was never observed, and indifference was the prevalent response. Cases of synergism were observed occasionally with the checkerboard method and slightly more often with the time-kill method.

The antibacterial activity of teichomycin, a glycopeptide antibiotic similar to vancomycin, has been evaluated in vitro and compared with that of vancomycin. Test strains included 130 staphylococci and 132 streptococci, with representatives of the major currently recognized species or groups, and lesser numbers of clostridia, propionibacteria, and group JK bacteria. Teichomycin was found to be more active than vancomycin. Its minimum inhibitory concentration (MIC) was two- to fourfold lower than that of vancomycin with staphylococci and anaerobic bacteria, and two- to eightfold lower with streptococci. No significant differences were observed with group JK bacteria. For most strains tested, minimum bactericidal concentrations (MBCs) of both teichomycin and vancomycin either equalled or exceeded by twofold the respective MICs. Higher MBC-to-MIC ratios were obtained for enterococci and pneumococci with both antibiotics. Both teichomycin and vancomycin showed similar in vitro interactions with rifampin in combination tests. Neither antagonism nor (with very few exceptions) synergism occurred.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.