A study of coagulase-negative staphylococci isolated from clinically significant infections at an Australian teaching hospital

Some 151 isolates of coagulase-negative staphylococci isolated from patients at an Australian teaching hospital were characterized by biochemical analysis, antibiotic sensitivity patterns and slime production. S. epidermidis was the predominant species (64%) isolated from clinically significant infections, and all S. epidermidis isolates from true bacteremias produced slime. Forty-nine per cent were resistant to methicillin and 61% to gentamicin. S. haemolyticus isolates from clinically significant infections also showed antibiotic resistance and 80% were resistant to more than five antibiotics. The importance of coagulase-negative staphylococci as pathogens in this large teaching hospital was confirmed.     

Slime Production and Expression of the Slime-Associated Antigen by Staphylococcal Clinical Isolates

The ability to produce slime and to express a slime-associated antigen was examined in a collection of staphylococcal clinical isolates. Slime-producing strains were found among coagulase-negative staphylococci in percentages comparable to those reported in other studies; surprisingly, a high percentage of Staphylococcus aureus strains also were able to produce this extracellular material. In the latter case, this ability was strongly dependent on the presence of an additional carbohydrate source in the growth medium. Expression of the slime-associated antigen appeared to be species specific and confined to the Staphylococcus epidermidis sensu stricto isolates; its strong association with the ability of these strains to produce thicker biofilms indicated slime-associated antigen as a possible virulence marker for S. epidermidis.     

Association of slime with pathogenicity of coagulase-negative staphylococci causing nosocomial septicemia.

To assess the role of slime in the pathogenesis of nosocomial bloodstream infections caused by coagulase-negative staphylococci, we compared the characteristics of 27 nosocomial bloodstream isolates with those of 27 skin isolates from non-hospital personnel. Of 27 bloodstream isolates, 14 were judged to be significant by a clinical index, and 13 were contaminants. Slime production was observed in 13 of 14 significant isolates but in only 3 of 13 contaminants (P = 0.0003) and 4 of 27 skin isolates (P = 0.0001). The 14 pathogens were identified as Staphylococcus epidermidis. Only 7 of 13 contaminants and 9 of 27 skin isolates belonged to the same species (P less than 0.006). Slime-producing strains of S. epidermidis represented 13 of 14 pathogens but only 2 of 13 contaminants (P less than 0.0003). Neither adherence to Teflon catheters nor phagocytosis and killing of coagulase-negative staphylococci by polymorphonuclear leukocytes was significantly influenced by slime production. Nevertheless, the identity of the organism and the slime production test predicted the clinical significance of blood isolates of coagulase-negative staphylococci with an overall accuracy of 89%.     

Detection of methicillin-resistant staphylococci by using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect the femA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast, femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related to femA in S. epidermidis and other coagulase-negative staphylococci examined.     

The role of extracellular slime in opsonophagocytosis of Staphylococcus epidermidis

Infections caused by coagulase-negative staphylococci (CNS) are a major problem in immunocompromised patients. It has been claimed that extracellular slime production by CNS predicts pathogenicity and inhibits host defences. Luminol-enhanced neutrophil chemiluminescence (CL) and bacterial killing assays were used to assess the effect of slime production on opsonophagocytosis and killing by polymorphonuclear leucocytes in vitro. There was wide variation in CL induction amongst the 43 strains of Staphylococcus epidermidis examined. The presence of slime had no influence either on the requirement or on the efficiency of opsonisation. Slime-producing and non-slime-producing strains showed a stepwise increase in induced CL up to a serum concentration of 10%, and were dependent on complement for efficient phagocytosis. The bacterial killing assays confirmed the CL results. Our data suggest that extracellular staphylococcal slime has no specific anti-opsonic property in vitro. Opsonophagocytosis may still be hampered in vivo by the physical presence of slime.     

Laboratory, clinical, and epidemiological aspects of coagulase-negative staphylococci.

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, are increasingly important causes of nosocomial infection. Microbiologists and clinicians no longer can afford to disregard clinical isolates of coagulase-negative staphylococci as contaminants. Accurate species identification and antimicrobial susceptibility testing, in a clinically relevant time frame, are important aids in the diagnosis and management of serious coagulase-negative staphylococcal infections. Emphasis in the clinical laboratory should be placed on the routine identification of S. epidermidis and Staphylococcus saprophyticus, with identification of other species of coagulase-negative staphylococci as clinically indicated. The application of newer techniques, such as plasmid analysis and tests for slime production and adherence, contribute to our understanding of the epidemiology and pathogenesis of coagulase-negative staphylococci and may also be helpful in establishing the diagnosis of infection.     

Isolation of methicillin-resistant coagulase-negative staphylococci from chickens.

Methicillin-resistant coagulase-negative staphylococci were isolated from the nares and skin of 1- to 8-week-old healthy chickens in three flocks from a farm. Isolation of methicillin-resistant coagulase-negative staphylococci was positive for 72 (25.7%) of the 280 chickens tested, with the frequency varying from 2.2 to 100% according to flock. A total of 45 appropriate isolates were selected and subjected to identification. Of the 45 methicillin-resistant coagulase-negative staphylococcal isolates selected, 37 were identified as Staphylococcus sciuri, 5 were identified as Staphylococcus epidermidis, and 3 were identified as Staphylococcus saprophyticus. The distribution of the species was different among the flocks. Comparative analysis of the SmaI-digested chromosomal DNA by pulsed-field gel electrophoresis revealed that the isolates could have originated from a single clone of each of S. sciuri and S. saprophyticus and three clones of S. epidermidis. By two methods based on the PCR technique, the mecA gene was detected in all five representative isolates of each methicillin-resistant coagulase-negative staphylococcal clone. The nucleotide sequence of a PCR fragment obtained from an isolate of S. sciuri was completely identical to the corresponding region of mecA genes reported in human methicillin-resistant Staphylococcus aureus isolates and Staphylococcus epidermidis isolates. The representative methicillin-resistant coagulase-negative staphylococcal isolates were resistant to many beta-lactam antibiotics, and some isolates were also resistant to macrolide and aminoglycoside antibiotics. This is the first evidence of the existence of methicillin-resistant coagulase-negative staphylococci from animals possessing the mecA gene.     

In vitro activity of quinupristin/dalfopristin in comparison with five antibiotics against worldwide clinical isolates of staphylococci

Objective   To evaluate the in vitro activity of quinupristin/dalfopristin (Q/D), a streptogramin combination, in comparison with five antibiotics against worldwide clinical isolates of staphylococci.
Methods   A multicenter in vitro study was performed using the E test during a period of 3 months (April to June) in 1997 on fresh, clinically significant, non repetitive strains of staphylococci from patients hospitalized in 23 different hospitals in 18 countries tested.
Results   A total of 2132 staphylococcal isolates including methicillin resistant (MR), methicillin susceptible (MS) S. aureus (1003 MS, 462 MR), S. epidermidis (169 MS, 251 MR), S. haemolyticus (28 MS, 46 MR), S. hominis (28 MS, 16 MR), and coagulase negative staphylococci (86 MS, 43 MR) were analyzed.
Q/D was highly active against all species tested. MIC₉₀ (mg/L) ranged from 0.5 to 2 depending on the species. Strains had MIC≤1 mg/L in 97.6%. For S. aureus , S. epidermidis , S. hominis and other coagulase-negative staphylococci no differences in MIC₉₀ were observed for MS or MR. One dilution difference was observed for S. haemolyticus , which overall was the less susceptible species. Erythromycin resistance was observed among 57– 87% of MR-strains and was lower among MS-strains (18–56%). Erythromycin resistance had no or little influence on MIC of Q/D. In comparison to vancomycin, Q/D was two to four times more active.
Conclusions   The streptogramin combination Q/D showed an excellent in vitro activity against all staphylococcal species tested regardless of the resistance pattern to other drug classes, particularly resistance to methicillin. Q/D was two to four times more active than vancomycin and MIC values varied from 0.5–2 according to the species. The synergy of Q/D was well conserved in macrolide-resistant strains.     

Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR.

In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310- and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA+) staphylococci and to differentiate S. aureus (femA+) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA+, and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.     

Problems with the laboratory identification and evaluation of coagulase-negative staphylococci

During a 105 day period in mid 1984, 796 isolates of coagulase-negative staphylococci were recovered from routine specimens handled in the Microbiology Laboratory, Dunedin Hospital. Of these isolates 504 (63.3%) were from wounds, 170 (21.4%) from urines, 58 (7.3%) from intravascular catheter tips, 44 (5.5%) from blood cultures and 20 (2.5%) from sputa. Presumptive identification of 315 consecutive isolates revealed 175 (55.6% of total) strains of Staphylococcus epidermidis, 44 (14.0%) S. capitis, 36 (11.4%) S. haemolyticus/hominis, 29 (9.2%) S. warneri, 19 (6.0%) S. simulans and 12 (3.8%) members of the S. saprophyticus group. Using laboratory generated criteria, 44.8%, 25.9% and 4.5% of coagulase-negative isolates from catheter tips, urines and blood cultures respectively, were deemed pathologically potentially significant. Although more common, S. epidermidis did not appear to be significantly more virulent than other members of the epidermidis species group or S. simulans; 67% of the S. saprophyticus group isolates from urine were considered pathologically significant. Antibiograms on the 796 coagulase-negative isolates revealed 63.2% resistant to penicillin, 22.6% to methicillin, 34.8% to cephradine, 27.5% to gentamicin, and 14.4% to erythromycin; multiple resistance was common. Methicillin resistance was a feature of S. saprophyticus group strains. With isolates from catheter tips and blood cultures, a significantly higher percentage of those regarded as significant were gentamicin resistant. Apart from penicillin, antibiotic resistance was not so marked in strains of coagulase-positive staphylococci recovered over the same period.     

Species identification of coagulase-negative staphylococci from urinary tract isolates.

A new scheme for identification of coagulase-negative staphylococci was applied to 138 consecutive urinary isolates of coagulase-negative staphylococci. The most common species were Staphylococcus epidermidis (53%), S. hominis (12%), and S. haemolyticus (10%). S. saprophyticus comprised only 5%. The disk method for antibiotic susceptibility for all species grouped together disclosed resistance most commonly to penicillin (35%), tetracycline (33%), methicillin (27%), and sulfonamide (24%). This pattern was also seen specifically with S. epidermidis. Further studies are needed to determine the incidence of species-specific antibiotic resistance and species-specific infection by site. This may be of particular interest in those patients with nosocomial infections due to coagulase-negative staphylococci.     

Identification of Staphylococcus epidermidis by desferrioxamine susceptibility and trehalose fermentation tests

The importance of coagulase-negative staphylococci, especially Staphylococcus epidermidis in clinical and nosocomial infection are recognized increasingly in recent years. A rapid and accurate identification of S. epidermidis is therefore important and necessary. A new test, susceptibility to desferrioxamine, coupled with trehalose fermentation has been recommended for the identification of this organism. However, the medium and method used are different from what has been recommended by the NCCLS. To investigate the feasibility of using the desferrioxamine susceptibility test in conjunction with the routinely used disc agar diffusion test, we employed 111 staphylococcal strains (including 51 S. epidermidis isolates, 15 S. hominis and 45 other coagulase-negative staphylococci) as test organisms, and followed the procedures recommended by the NCCLS in which Mueller-Hinton agar and standard inoculum were used. Results indicated that all strains of S. epidermidis and S. hominis were susceptible to 1 mg desferrioxamine (the diameter of the inhibition zone were 28-37 mm). The minimum inhibitory concentrations of desferrioxamine to S. epidermidis and S. hominis isolates were determined to be 125 micrograms/ml. Further differentiation of S. hominis and S. epidermidis can be made by their ability to ferment trehalose, the former could while the latter could not. We conclude that the desferrioxamine susceptibility test of coagulase-negative staphylococci can be used in conjunction with the routine disc agar diffusion method. S. epidermidis can be identified rapidly and accurately by its susceptibility to 1 mg desferrioxamine and inability to ferment trehalose.     

Species identification of coagulase-negative staphylococcal isolates from blood cultures.

Coagulase-negative staphylococci generally are not fully identified, are called Staphylococcus epidermidis, and are considered contaminants when isolated from blood cultures. In a cancer hospital during 6 months, 46 patients had multiple blood cultures (mean, 3.1) which yielded coagulase-negative staphylococci. Species identification of these showed that 10 of the 46 (22%) were not S. epidermidis. Similarly, 96 coagulase-negative staphylococci isolated from only one of multiple blood cultures from patients and thought to be skin contaminants were identified. Of 96 of the staphylococci, 14 (16%) of the latter group were not S. epidermidis. Species found included S. haemolyticus, S. hominis, S. warneri, S. simulans, and S. xylosus. Eight isolates of these species were methicillin resistant, and all eight were mannitol fermenters. The results suggest that these species invasively infect cancer patients with the same frequency at which the species colonize. No one species was identified as being more pathogenic than the others. Routine species identification of coagulase-negative staphylococci from blood cultures of cancer patients contributed little to management except to occasionally distinguish multiple-episode culture contamination by different species from sustained bacteremia with the same species.     

Outbreak of Mupirocin-Resistant Staphylococci in a Hospital in Warsaw, Poland, Due to Plasmid Transmission and Clonal Spread of Several Strains

An outbreak of mupirocin-resistant (MuR) staphylococci was investigated in two wards of a large hospital in Warsaw, Poland. Fifty-three MuR isolates of Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. xylosus, and S. capitis were identified over a 17-month survey which was carried out after introduction of the drug for the treatment of skin infections. The isolates were collected from patients with infections, environmental samples, and carriers; they constituted 19.5% of all staphylococcal isolates identified in the two wards during that time. Almost all the MuR isolates were also resistant to methicillin (methicillin-resistant S. aureus and methicillin-resistant coagulase-negative staphylococci). Seven of the outbreak isolates expressed a low-level-resistance phenotype (MuL), whereas the remaining majority of isolates were found to be highly resistant to mupirocin (MuH). The mupA gene, responsible for the MuH phenotype, has been assigned to three different polymorphic loci among the strains in the collection analyzed. The predominant polymorph, polymorph I (characterized by a mupA-containing EcoRI DNA fragment of about 16 kb), was located on a specific plasmid which was widely distributed among the entire staphylococcal population. All MuR S. aureus isolates were found to represent a single epidemic strain, which was clonally disseminated in both wards. The S. epidermidis population was much more diverse; however, at least four clusters of closely related isolates were identified, which suggested that some strains of this species were also clonally spread in the hospital environment. Six isolates of S. epidermidis were demonstrated to express the MuL and MuH resistance mechanisms simultaneously, and this is the first identification of such dual MuR phenotype-bearing strains. The outbreak was attributed to a high level and inappropriate use of mupirocin, and as a result the dermatological formulation of the drug has been removed from the hospital formulary.     

Evaluation of restriction endonuclease fingerprinting of chromosomal DNA and plasmid profile analysis for characterization of multiresistant coagulase-negative staphylococci in bacteremic neonates.

A procedure was developed for restriction endonuclease fingerprinting (REF) of the chromosomal DNA of coagulase-negative staphylococci. A total of 48 isolates comprising 29 Staphylococcus epidermidis and 19 Staphylococcus haemolyticus isolates from blood and mucocutaneous sites of 15 premature neonates were characterized by REF, plasmid profile (PP) analysis, antimicrobial susceptibility testing, biotyping, and slime production. On the basis of REF analysis of chromosomal DNA, the 48 coagulase-negative staphylococcal isolates were subdivided into 10 subgroups, whereas PP analysis subdivided the strains into 20 distinct subgroups. REF analysis of total DNA (i.e., chromosome plus plasmid) resulted in the same 20 subgroups as were subdivided by PP analysis. The high discriminatory power of PP analysis was associated with the variability of plasmid content in coagulase-negative staphylococcal strains isolated during the outbreak. REF patterns were found to be stable both in vitro and in vivo. Isolates carried from 2 to 10 plasmids that ranged in molecular size from 0.9 to 39.5 megadaltons. Plasmids were disseminated among the coagulase-negative staphylococci, regardless of the genetic relatedness of their chromosomal DNAs. Hence, a lack of correlation existed between the grouping of isolates by REF analysis of chromosomal DNA and the grouping by PP analysis. There were one and two distinct chromosomal patterns among 4 of 4 blood cultures and 15 of 15 mucocutaneous cultures of S. haemolyticus, respectively. In contrast, a higher proportion of distinct chromosomal patterns was found for S. epidermidis in blood cultures (7 of 11 cultures) compared with those identified for isolates in mucocutaneous cultures (6 of 18 cultures). In summary, REF analysis of chromosomal DNA, rather than total DNA, is a useful marker for epidemiological investigations of coagulase-negative staphylococci. PP analysis can also be used to provide additional epidemiological information regarding the most recent genetic events.     

Development of the quantitative micro-test for slime production by coagulase-negative staphylococci

The macro-test for slime production by coagulase-negative staphylococci was adapted to a spectrophotometric micro-test assay to obtain more objective and quantitative information on slime production. A total of 135 isolates of coagulase-negative staphylococci (70 macro-test-positive and 65 macro-test-negative) were tested by both methods. The quantitative micro-test optical density readings were (mean ±SD) 1.176 ± 0.294 and 0.130 ± 0.095 for the macro-test-positive and -negative groups, respectively. The micro-test was reproducible and demonstrated quantitative differences in slime production among the different species of coagulase-negative staphylococci. Aside fromStaphylococcus epidermidis, the majority of the coagulase-negative staphylococci had very low optical density readings, indicating little or no slime production under the conditions employed in this assay. This test allows one to study the relative production of slime by different strains and species of coagulase-negative staphylococci, and may be useful in studying the effects of different conditions, such as antibiotic exposure, on slime production.     

Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction.

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillin-resistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to beta-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria.     

Emergence of teicoplanin-resistant coagulase-negative staphylococci.

Over a period of 5 years we have recovered 32 clinical isolates of coagulase-negative staphylococci (CoNS) exhibiting either decreased levels of susceptibility or true resistance to teicoplanin (MICs, 16 to 128 micrograms/ml); these isolates make up 0.55% of the total CoNS isolated by us. Twenty-nine of the strains were also methicillin resistant, and all were susceptible to vancomycin. Fourteen of the strains were Staphylococcus epidermidis, fourteen were Staphylococcus haemolyticus, and four were Staphylococcus hominis. In one case, a strain of S. haemolyticus was isolated with a vancomycin-resistant, teicoplanin-resistant Enterococcus faecalis strain. All strains were nosocomially acquired and were isolated from 17 different wards. Teicoplanin resistance occurred as a sporadic phenomenon, and none of the isolates were epidemiologically related. The isolates were from 30 patients, 13 of whom presented with true infections (43%). Five (38%) of the 13 patients with true infections had been previously treated with vancomycin. None of the infected patients were previously treated with teicoplanin. The in vivo development of resistance to teicoplanin among CoNS strains limits the therapy of infections by these microorganisms. There is a need for surveillance of nosocomial isolates of CoNS to determine resistance to glycopeptides.     

Studies on clinical isolates of coagulase-negative staphylococci resistant to methicillin. Evidence of cross-resistance between methicillin and cephalothin

The in vitro susceptibility to cephalothin and cefuroxime of 195 isolates of methicillin-resistant coagulase-negative staphylococci was determined by the agar-diffusion test, using 7.5% NaCl-supplemented agar. The distribution of the inhibition zone diameters for isolates of S. epidermidis (S. biotype 1) as well as for S. haemolyticus (S. biotype 4) was trimodal. While 4% of the isolates were found susceptible to cefuroxime, 39% of the S. epidermidis/S. hominis (S. biotype 1) isolates and 34% of the S. haemolyticus (S. biotype 4) isolates were found susceptible to cephalothin by this method. Eight of these isolates (six S. epidermidis, two S. haemolyticus) were selected for susceptibility testing by the tube-dilution method, together with four isolates (three S. haemolyticus, one S. epidermidis) found resistant to cephalothin by the agar-diffusion test. The first-mentioned isolates were all found susceptible to cephalothin with MICs less than or equal to 2 micrograms/l, while the last-named all were resistant with MICs greater than or equal to 16 micrograms/ml. Population analyses revealed sub-populations of highly resistant bacteria in all methicillin-resistant isolates of S. epidermidis (S. biotype 1), as well as in all isolates of S. haemolyticus (S. biotype 4). We thus concluded that methicillin-resistance in isolates of coagulase-negative staphylococci implies resistance to cephalosporins and that the difference between S. epidermidis and S. haemolyticus as regards cephalosporin-susceptibility is quantitative and not qualitative. Eighty-nine per cent of the 195 methicillin-resistant isolates in this study were resistant to penicillin and at least one more antibiotic. We therefore think that resistance to penicillin and one or more non-beta-lactam antibiotics strongly suggests methicillin-resistance and that such isolates should be further tested on hypertonic media.     

Identification, clinical distribution, and susceptibility to methicillin and 18 additional antibiotics of clinical Staphylococcus isolates: nationwide investigation in Italy.

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.