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J Clin Microbiol. 2017 February; 55(2): 585–595.
Published online 2017 January 25. Prepublished online 2016 December 7. doi:  10.1128/JCM.02092-16
PMCID: PMC5277529

Clinical and Microbiological Aspects of β-Lactam Resistance in Staphylococcus lugdunensis

Nathan A. Ledeboer, Editor
Nathan A. Ledeboer, Medical College of Wisconsin;


Antimicrobial susceptibility results from broth microdilution MIC testing of 993 Staphylococcus lugdunensis isolates recovered from patients at a tertiary care medical center from 2008 to 2015 were reviewed. Ninety-two oxacillin-susceptible isolates were selected to assess the accuracy of penicillin MIC testing, the penicillin disk diffusion test, and three β-lactamase tests, including the cefoxitin-induced nitrocefin test, penicillin cloverleaf assay, and penicillin disk zone edge test. The results of all phenotypic tests were compared to the results of blaZ PCR. The medical records of 62 patients from whom S. lugdunensis was isolated, including 31 penicillin-susceptible and 31 penicillin-resistant strains, were retrospectively reviewed to evaluate the clinical significance of S. lugdunensis isolation, the antimicrobial agents prescribed, if any, and the clinical outcome. MIC testing revealed that 517/993 (52.1%) isolates were susceptible to penicillin and 946/993 (95.3%) were susceptible to oxacillin. The induced nitrocefin test was 100% sensitive and specific for the detection of β-lactamase compared to the blaZ PCR results, whereas the penicillin disk zone edge and cloverleaf tests showed sensitivities of 100% but specificities of only 9.1% and 89.1%, respectively. The penicillin MIC test had 100% categorical agreement with blaZ PCR, while penicillin disk diffusion yielded one major error. Only 3/31 patients with penicillin-susceptible isolates were treated with a penicillin family antimicrobial. The majority of cases were treated with other β-lactams, trimethoprim-sulfamethoxazole, or vancomycin. These data indicate that nearly all isolates of S. lugdunensis are susceptible to narrow-spectrum antimicrobial agents. Clinical laboratories in areas with resistance levels similar to those described here can help promote the use of these agents versus vancomycin by effectively designing their antimicrobial susceptibility reports to convey this message.

KEYWORDS: Staphylococcus, antimicrobial resistance, beta-lactams, lugdunensis


Staphylococcus spp. are normal skin flora inhabitants and, in some cases, opportunistic human pathogens (1). The coagulase-negative staphylococci (CoNS) are typically less virulent than Staphylococcus aureus in healthy human hosts. One exception is Staphylococcus lugdunensis, which may exhibit increased virulence compared to that of other CoNS (2). Infections associated with S. lugdunensis can include skin and soft tissue infections, native valve endocarditis, urinary tract infections, and prosthetic device infections (2).

Isolates of CoNS, in particular Staphylococcus epidermidis, that harbor the mecA gene can have oxacillin MICs in the 0.5- to 2.0-μg/ml range, which is much lower than the oxacillin MICs observed in mecA-positive isolates of S. lugdunensis and S. aureus (3,7). As such, the Clinical and Laboratory Standards Institute (CLSI) recommends that S. lugdunensis MICs be interpreted using S. aureus oxacillin and cefoxitin breakpoints rather than those used for other CoNS (8). Accurate detection of penicillin resistance for Staphylococcus spp. is also somewhat complicated. Penicillin resistance among the staphylococci can be mediated by either altered penicillin binding proteins or a blaZ-encoded β-lactamase (7, 9,12). Standard disk diffusion and MIC tests performed and interpreted according to CLSI recommendations do not always detect β-lactamase-mediated penicillin resistance in the staphylococci. Because of this, CLSI recommends the use of the penicillin disk zone edge test to test penicillin-susceptible isolates of S. aureus for the presence of β-lactamases (8, 13). This test involves examining the edge of the zone of inhibition surrounding a penicillin disk; a fuzzy zone edge indicates the absence of β-lactamase, whereas a sharp zone edge indicates the presence of β-lactamase (8). For staphylococci other than S. aureus, a nitrocefin-based test is recommended to identify the presence of β-lactamase in penicillin-susceptible isolates. However, several reports document failure of phenotypic tests, including the nitrocefin disk, to detect β-lactamase-mediated penicillin resistance in some isolates of CoNS other than S. lugdunensis (14, 15). This finding, coupled with the infrequent incidence of penicillin susceptibility among CoNS in the United States (16), has precluded the use of the penicillins for the treatment of infections caused by most CoNS. However, S. lugdunensis is unique among the CoNS, as up to 75.0% of isolates have been reported to be penicillin susceptible, though susceptibility varies between reports (17,19). The Sanford guide recommends penicillin as a primary agent for the treatment of S. lugdunensis infections, provided the isolate does not harbor a β-lactamase (17). Few data exist in the literature regarding the optimal method for laboratories to detect the presence of β-lactamase in isolates of S. lugdunensis.

The purpose of this study was to evaluate clinical and microbiological aspects of S. lugdunensis isolates at a tertiary care center. The aims were to (i) evaluate the susceptibilities of 993 S. lugdunensis isolates to 13 antimicrobial agents over an 8-year period, (ii) evaluate the accuracy of β-lactamase testing methods, including penicillin MIC, penicillin disk diffusion, induced nitrocefin, cloverleaf, and penicillin zone edge tests, for 92 oxacillin-susceptible S. lugdunensis isolates, and (iii) review the clinical significance, antimicrobial therapy, and outcome for 62 patients from whom S. lugdunensis was isolated.

(This work was presented in part at IDWeek 2014, Infectious Diseases Society of America, 8 to 12 October 2014, Pittsburgh, PA, and at the American Society for Microbiology General Meeting, 30 May to 2 June 2015, New Orleans, LA.)


S. lugdunensis MIC distributions over an 8-year period.

In 2008, the clinical laboratory began to routinely perform tube coagulase, pyrrolidonyl arylamidase (PYR), and ornithine decarboxylase (ODC) tests on CoNS isolated from wound and sterile body site cultures to identify S. lugdunensis. Between January 2008 and December 2015, 993 S. lugdunensis isolates were identified and subjected to antimicrobial susceptibility testing (AST). With the exception of 2012, both the yearly ratio of S. lugdunensis to other CoNS and the absolute number of S. lugdunensis isolates increased (Fig. 1). Roughly half (50.4%) of the isolates originated from skin, wounds, or abscesses, while 14.7% of isolates originated from sterile body sources, including bone, blood, tissue, or other sterile fluids (Table 1). The remaining 34.8% of isolates were recovered from various specimen types, including respiratory secretions, fluids, eyes, and genitals.

The number of isolates reported as S. lugdunensis are shown for each year between 2008 and 2015. Below each year, the yearly percentage of CoNS isolates that were identified as S. lugdunensis is shown. With the exception of 2012, both the percentage and ...
Specimen sources for 993 isolates of S. lugdunensis

The penicillin susceptibility of all 993 isolates was evaluated. As shown by the results in Fig. 2A, the penicillin susceptibility rates (Fig. 2A, white) ranged from 48.5% to 63.2%, much lower than the published estimates of 75.0% (17). No temporal trend was found for penicillin susceptibility rates between 2008 and 2015. Among the 476 penicillin-resistant isolates, the majority (65.0%) had penicillin MICs of ≥2 μg/ml (Fig. 2B), whereas only 1.3% had a MIC of 0.25 μg/ml and 259 (26.1%) isolates had a MIC of 0.12 μg/ml. Conversely, oxacillin susceptibility, as defined by MICs showing susceptibility to both oxacillin and cefoxitin, remained high, ranging from 78.9% to 97.4% over the study period (Fig. 2C). For 10 isolates (1.0%), the oxacillin MICs were ≤2 μg/ml (susceptible), but the cefoxitin MICs were ≥8 μg/ml (resistant) (Fig. 2D) (20). The mechanism of oxacillin resistance was not evaluated for these isolates, but all tests with discordant results were repeated and confirmed at the time of isolation.

Antimicrobial susceptibilities of S. lugdunensis isolates. (A to D) White areas indicate susceptibility to penicillin or oxacillin, and gray or black areas indicate resistance. (A and C) The percentage of susceptible S. lugdunensis isolates is shown by ...

Susceptibilities to several additional antimicrobial agents were evaluated (Table 2). All isolates were susceptible to vancomycin (MIC90, 1 μg/ml), daptomycin (MIC90, 0.25 μg/ml), linezolid (MIC90, 1 μg/ml), and quinupristin-dalfopristin (MIC90, 0.5 μg/ml). Most isolates (99.9%) were susceptible to rifampin (MIC90, 0.5 μg/ml), 97.2% were susceptible to ciprofloxacin (MIC90, 0.5 μg/ml), 98.6% were susceptible to trimethoprim-sulfamethoxazole (MIC90, 0.5 μg/ml), and 99.1% were susceptible to doxycycline (MIC90, 1 μg/ml) (Table 2). Eight hundred (80.5%) isolates were susceptible to erythromycin and 879 (88.5%) were clindamycin susceptible.

Susceptibilities to antimicrobials tested against isolates of S. lugdunensis

Overall, 407 (41.0%) isolates were susceptible to all antimicrobials tested (Fig. 2E). Conversely, 90 (9.1%) isolates were resistant to three or more antimicrobial classes.

β-Lactamase detection.

Among 92 oxacillin-susceptible S. lugdunensis isolates, blaZ was detected by PCR in 36 (39.1%) and 56 (60.9%) were blaZ negative (Table 3). The penicillin MIC was 100% accurate at differentiating isolates with blaZ (MIC range, 1 to 2 μg/ml) from those without blaZ (MIC range, 0.03 to 0.12 μg/ml); however, there were no isolates with penicillin MICs of 0.25 to 0.5 μg/ml. Penicillin disk diffusion was similarly 100% sensitive but slightly less specific, at 98.2% (95% confidence interval [CI], 90.3% to 100%) (Table 4). Disk diffusion exhibited one major error (false resistance), which was repeated, for an isolate lacking a detectable β-lactamase by blaZ PCR but with a measured penicillin zone of 28 mm, just below the susceptible breakpoint. This isolate was negative by the induced nitrocefin test and had a penicillin-susceptible MIC (0.12 μg/ml). The penicillin zones ranged from 28 to 45 mm for blaZ-negative isolates and 15 to 21 for blaZ-positive isolates (Table 3).

Penicillin disk diffusion and β-lactamase results related to penicillin MIC for 92 oxacillin-susceptible isolates of S. lugdunensis
Performance of phenotypic β-lactamase tests versus blaZ PCR in 92 oxacillin-susceptible isolates

Nearly all (86/92, 93.5%) S. lugdunensis isolates exhibited sharp penicillin zone edges, regardless of the presence or absence of the blaZ gene (Fig. 3A). Overall, compared to the results of blaZ PCR, 100% sensitivity and 9.1% specificity (95% CI, 3.0% to 20.0%) were observed for the penicillin zone edge test. The cloverleaf test (Fig. 3B) was superior to the penicillin zone edge test, with a sensitivity of 94.6% (95% CI, 81.8% to 99.3%) and specificity of 89.1% (95% CI, 77.8% to 95.9%) (Table 4) compared to the results of blaZ PCR. However, we found this test difficult to interpret, due to subtle variations in zone edge shapes adjacent to the test isolates (not shown). The induced nitrocefin test was positive for 36/36 (100%) blaZ-positive isolates and negative for 56/56 blaZ-negative isolates, yielding 100% sensitivity (95% CI, 90.5% to 100%) and 100% specificity (95% CI, 93.5% to 100%) (Table 4).

Examples of phenotypic β-lactamase tests evaluated. Plates shown contain two isolates each. (A) Fuzzy zone edges (left isolate) of Staphylococcus isolates around penicillin disks correlate with the absence of β-lactamases, while sharp ...

All isolates with discordant results were retested to confirm results. Only one isolate yielded a corrected result; it had a MIC of 0.25 μg/ml initially but was susceptible by penicillin disk diffusion, negative by blaZ PCR, and negative by induced nitrocefin. This isolate had a penicillin MIC of 0.12 μg/ml on repeat, and all other test results were in line with the previous readings. This false-susceptible MIC was not included in the calculations above and attributed to random error.

Clinical and microbiological features of selected patient population.

To investigate the clinical features and antimicrobial choices for the treatment of S. lugdunensis at our center, 62 patients were selected for retrospective chart review (Table 5). Isolates from sterile body sites were preferentially selected for this analysis and included 22 isolated from blood, 1 from bone, and 1 from aortic tissue. The remainder included 36 isolates from skin and soft tissue sources and 2 from corneal scrapings. The average patient age was 52 years, and 41.9% were female. Some level of immunosuppression was demonstrated by 43.5% patients, including 9 with diabetes mellitus, 5 with solid organ transplant, 10 with cancer, 1 who was alcoholic, and 1 who was on immune suppression for treatment of Crohn's disease.

Patient and culture characteristics from chart reviewa

Overall, 15 patients (24.2%) met clinical criteria for bacteremia or osteomyelitis, 29 (46.8%) for skin and soft tissue infections (SSTI), and 1 (1.6%) for prosthetic joint infection, while 17 (27.4%) did not exhibit any clinical criteria consistent with infection. In the latter patients, the isolation of S. lugdunensis was presumed to represent contamination of the culture by colonizing flora. For those patients from whom S. lugdunensis was isolated from normally sterile body site cultures, 66.7% (16/24) had clinical signs of infection. Of the 22 blood isolates, 13 (59%) were considered clinically significant. Similarly, 76.3% (29/38) of patients with S. lugdunensis isolates from nonsterile body sites had clinical signs of infection. Overall, S. lugdunensis was isolated in pure culture from 61.2% (38/62) of patients, whereas 38.9% were present in cultures with >1 organism isolated. Notably, culture purity did not always indicate clinical significance, as 9/38 (23.7%) patients with S. lugdunensis isolated in pure culture did not meet clinical criteria for infection. Similarly, 6/24 (25.0%) patients with mixed cultures did not meet clinical criteria for infection.

The antimicrobial prescriptions were evaluated for the 62 patients. Overall, 91.2% (52/57) of patients with documented antimicrobial treatment available in the electronic medical record were started on empirical treatment to cover staphylococcal infections, including 8 (15.4%) patients who lacked clinical signs or symptoms of infection. For 6 patients (9.7%), antimicrobials were discontinued following receipt of culture and susceptibility results, including 4 for whom the isolate was considered clinically significant on retrospective review. All 4 patients had SSTIs and debridement. For 1 patient, antimicrobials were started following receipt of culture data; this patient had bacteremia that was considered clinically significant. For 50.0% (26/52) of patients, the antimicrobials prescribed were changed upon receipt of culture and susceptibility data, including the one for whom treatment was started.

Two of 8 patients with S. lugdunensis recovered in blood samples received a penicillin family antibiotic (ampicillin). One of these patients also had enterococcal endocarditis, and the decision to use ampicillin was likely not based on the clinician's knowledge of recommended treatment for S. lugdunensis but, rather, its use in combination with an aminoglycoside to treat the Enterococcus. Among 14 patients with oxacillin-susceptible S. lugdunensis bacteremia or osteomyelitis, only 5 (35.7%) were deescalated to a β-lactam. For the remaining patients, 3 were treated with vancomycin, 2 with ciprofloxacin, and 2 with a carbapenem, while 2 expired before treatment could be adjusted. Among 16 patients with SSTIs caused by penicillin-susceptible isolates, only three (18.8%) were narrowed to a penicillin family antimicrobial upon receipt of AST results. Among 29 patients with oxacillin-susceptible SSTIs, 13 were narrowed or maintained on a β-lactam (including nafcillin, oxacillin, cephalexin, ceftriaxone, and ertapenem), 7 were treated with trimethoprim-sulfamethoxazole, 2 with both cephalexin and trimethoprim-sulfamethoxazole, 2 with doxycycline, 1 with mupirocin ointment, and 1 with clindamycin. Three patients had no documented consolidative therapy.

Clinical outcomes were measured for the 45 patients with S. lugdunensis infections. Of 29 patients with SSTIs, 15/29 (51.7%) were cured, 3/29 (10.3%) had recurrence of infection with S. lugdunensis, and 11/29 (37.9%) had unknown outcomes. Of 16 patients with bacteremia, osteomyelitis, or prosthetic joint infections, 9/16 (56%) were cured, 1/16 (6.3%) was maintained on suppression therapy, 1/16 (6.3%) died from complications associated with the infection, and 5/16 (31.3%) had unknown outcomes. Narrowed β-lactam antimicrobial therapy did not result in significantly different outcomes compared with those of broad-spectrum therapy as determined by Fisher's exact test (P = 0.79), albeit the sample size was very small.

Notably, penicillin-resistant isolates were observed significantly (Fisher's exact test, P = 0.03) more frequently among patients with bacteremia or osteomyelitis (11/15 penicillin resistant, 73.3%) than among patients with SSTIs (12/30 penicillin resistant, 40.0%). A similar trend (Fisher's exact test, P = 0.002) was observed across the 574 isolates analyzed, which consisted of 334 isolates from SSTI-related specimen sources (137/334 [41.0%] penicillin resistant) and 46 patients with S. lugdunensis isolate from blood or bone specimens (30/46 [65.2%] penicillin resistant). These findings are summarized in Table S2 in the supplemental material.


Previous studies indicate that S. lugdunensis is relatively uncommon in clinical specimens but is unique from the perspectives of virulence and antimicrobial susceptibility (2, 21). The present retrospective analysis identified 993 isolates of S. lugdunensis with antimicrobial susceptibility data between 2008 and 2015. As shown by the results in Fig. 2A, the proportions of CoNS that were S. lugdunensis and the isolation frequency of S. lugdunensis have steadily increased at our tertiary care center over the last 8 years. This increase may reflect improved proficiency by technologists at identifying the organism and/or a true increase in the incidence of this organism, as well as the addition of S. lugdunensis to the M100 AST standard by the CLSI in 2005. Concordant with previous reports (22), S. lugdunensis was found in multiple specimen types but was mostly isolated from wounds, abscesses, and skin and skin structure specimens. In addition, 1.7% of isolates originated from ocular specimens, supporting previously identified roles in postoperative endophthalmitis and suppurative keratitis (23,25). The finding that 14.7% of isolates originated from normally sterile body sites is noteworthy, and based on the chart review presented herein, 16/24 (66.7%) isolates from sterile body sites were deemed clinically significant. In contrast to our finding, a previous study found that only 6/20 (30.0%) blood isolates were clinically significant (26). In addition, we demonstrated that nearly a quarter of patients from whom S. lugdunensis was the only potential pathogen isolated in culture did not have clinical signs of infection, and yet, 47% of patients without signs of infection were treated. It is unclear whether the laboratory report of S. lugdunensis with susceptibility results affected the decision to treat in these cases. However, laboratories should carefully consider what scenarios warrant CoNS identification to the species level and susceptibility testing. Further data are required to determine the optimal practice for testing and reporting S. lugdunensis in clinical laboratories. While we documented clinical outcomes, one limitation of our study was that we did not specifically evaluate the adequacy of therapy in relation to outcomes. Larger studies are required to evaluate the clinical impact of the use of beta-lactam therapy for S. lugdunensis infections.

S. lugdunensis penicillin susceptibility was much lower than previous observations in the United States (27, 28). In addition, increased penicillin resistance was observed in isolates derived from blood and bone specimens compared to the penicillin resistance in isolates from skin, wound, abscess, and tissue specimens. Importantly, despite the 52.1% penicillin susceptibility rate of S. lugdunensis isolates, very few patients received a penicillin class antimicrobial. Similarly, although nearly all isolates were oxacillin susceptible, only 44.4% of patients with a clinically significant, oxacillin-susceptible isolate were narrowed to a β-lactam. Other studies have documented higher rates of penicillin prescription, perhaps highlighting differences in regional/institutional practices or drug availability/pricing (26, 29, 30). In addition, resistance to non-β-lactam drugs was observed, including erythromycin and clindamycin, which has also been noted previously (18, 31). Among the 190 erythromycin-resistant isolates, 113 (59.5%) were also penicillin resistant (chi square test, P = 3 × 10−4), indicating that penicillin susceptibility in erythromycin-resistant isolates is not common and should be carefully evaluated.

The results presented herein support the CLSI recommendation that induced nitrocefin testing is preferable to penicillin zone edge testing for S. lugdunensis, as nearly all isolates exhibited sharp penicillin inhibition zones, regardless of the presence of β-lactamase. While the reliability of the penicillin disk diffusion test was slightly marred by a single false-resistant error, a penicillin zone of ≤28 mm was otherwise concordant with blaZ PCR results. The induced nitrocefin and penicillin MIC tests had equally high sensitivities and specificities. While the reference broth microdilution (BMD) penicillin MIC results correlated 100% with those of blaZ PCR, 259 (26.1%) isolates had penicillin MICs of 0.12 μg/ml, near the susceptible breakpoint, which could prove problematic for automated antimicrobial susceptibility platforms that have a history of imperfect penicillin susceptibility detection (32,35). However, no β-lactamase was detectable in tested isolates with penicillin MICs of ≤0.12 μg/ml by reference broth microdilution, unlike S. aureus strains, which may harbor β-lactamase despite a susceptible penicillin MIC and thus require confirmation using the penicillin zone edge test (8). Our study was performed using CLSI reference methods (broth microdilution and disk diffusion) and did not evaluate the performance of automated antimicrobial susceptibility platforms. Laboratories using these platforms may consider following M100S guidance (8) and perform a nitrocefin test before reporting these as susceptible to penicillin, if performance has not specifically been evaluated by the laboratory for contemporary isolates, particularly for serious infections caused by S. lugdunensis when penicillin is considered as a treatment option. If the nitrocefin test is negative, CLSI recommends an induced nitrocefin test, to ensure detection of resistance. Alternatively, laboratories may consider performing a penicillin disk diffusion test on Mueller-Hinton agar (MHA), which yielded excellent results in the present study.

In conclusion, the data presented provide updates on antimicrobial susceptibility, implemented antimicrobial treatment strategies, and β-lactamase detection for S. lugdunensis isolates. Importantly, the observed β-lactamase positivity rate exceeds previous estimates, possibly reflecting broader trends toward resistance. Despite this, the discordance between penicillin susceptibility and penicillin utilization is vast, and patient care may be improved in areas with resistance rates similar to those reported here if penicillin family antimicrobial susceptibilities are routinely reported for S. lugdunensis isolates and physicians are reminded of treatment recommendations.



From 2008 through 2015, all records indicating that S. lugdunensis (n = 993) was identified as part of the standard of care for specimens submitted to the UCLA Clinical Microbiology Laboratory were reviewed. During this time, S. lugdunensis was identified by positive pyrrolidonyl arylamidase and ornithine decarboxylase reactions. Antimicrobial susceptibility testing (AST) was performed using the CLSI broth microdilution MIC reference method (8).

A subset of 92 oxacillin-susceptible isolates was selected for further study, stored at −70°C in brucella broth plus 20% glycerol (BBL; BD, Sparks, NJ), and subcultured twice onto tryptic soy agar supplemented with 5% sheep blood (BBL; BD) plates prior to testing. Identification of these isolates as S. lugdunensis was confirmed by using the bioMérieux Vitek MS instrument (bioMérieux, Durham, North Carolina). Genus and species calls were generated based on a weighted similarity metric. Isolates that were not confirmed as S. lugdunensis by the Vitek MS or that yielded identification with poor confidence by the Vitek MS were subjected to 16S rRNA gene PCR and sequencing as described previously (36).

Broth microdilution MIC testing.

MICs for penicillin, oxacillin, cefoxitin, vancomycin, daptomycin, linezolid, quinupristin-dalfopristin, erythromycin, clindamycin, doxycycline, rifampin, ciprofloxacin, and trimethoprim-sulfamethoxazole were determined by reference BMD according to CLSI standards (8), using panels prepared in-house. Quality control was performed using both CLSI-recommended quality-control strains and in-house isolates with in-range MICs for each antimicrobial that were confirmed by external laboratories. MICs were interpreted using CLSI breakpoints (8), and the MIC90 was defined as the lowest MIC for each agent that inhibited 90% of the 993 isolates tested. Isolates were considered oxacillin susceptible if the oxacillin MIC was ≤2 μg/ml and the cefoxitin MIC was ≤4 μg/ml. Isolates were considered penicillin susceptible if the penicillin MIC was ≤0.12 μg/ml and the isolate was β-lactamase negative by blaZ PCR. Penicillin-resistant (S. aureus ATCC 29213) and -susceptible (S. aureus ATCC 25923) quality-control strains were included in each day of testing. Inducible clindamycin resistance was evaluated by performing the D zone test, following CLSI recommendations, on purity plates from MIC testing (8).

Penicillin and cefoxitin disk diffusion testing.

Disk diffusion was performed according to CLSI recommendations using 30-μg cefoxitin and 10-μg penicillin disks (BBL; BD, Sparks, NJ) (8) and unsupplemented Mueller-Hinton agar (MHA) (BBL; BD, Sparks, NJ). Oxacillin susceptibility was confirmed using a cefoxitin disk as a surrogate (zones of ≥22 mm were considered susceptible). For isolates with penicillin-susceptible zones (≥29 mm), the edge of the zone was examined to assess β-lactamase production, again according to CLSI recommendations, to confirm the reliability of the current penicillin breakpoint. Isolates showing zone edges with a fuzzy appearance were considered β-lactamase negative, and those showing zone edges with a sharp appearance were considered β-lactamase positive (Fig. 3A). Penicillin-resistant (S. aureus ATCC 29213) and -susceptible (S. aureus ATCC 25923) quality-control strains were included on each day of testing.

Induced nitrocefin disk test.

Isolates were tested for β-lactamase production using nitrocefin disks (BD) according to the manufacturer's recommendations, using cells that were subjected to cefoxitin induction after overnight incubation. Briefly, growth from the periphery of the cefoxitin disk diffusion zone was applied to a premoistened nitrocefin disk. Following 1 h of incubation at room temperature, a red color at the site of inoculation was considered positive and no color change was considered negative for β-lactamase production. β-Lactamase-positive (S. aureus ATCC 29213) and -negative (S. aureus ATCC 25923) quality-control strains were included on each day of testing.

Cloverleaf β-lactamase test.

The cloverleaf test was performed as described previously (37,40). Using a sterile swab, a sample from a 0.5-McFarland standard suspension of penicillin-susceptible S. aureus ATCC 25923 was spread over the surface of an MHA plate (100-mm diameter) as done for routine disk diffusion testing. Next, a 10-μg penicillin disk was applied to the center of the plate. S. lugdunensis isolates were then streaked in straight lines out from the disk to the edge of the plate (Fig. 3B), allowing two isolates to be tested in duplicate per plate. Following 16 to 20 h of incubation at 35°C, any encroachment by the indicator strain (S. aureus ATCC 25923) into the zone of inhibition was considered to show it as positive for β-lactamase. β-Lactamase-positive (S. aureus ATCC 29213) and -negative (S. aureus ATCC 25923) quality-control strains were included on each day of testing.

Molecular detection of blaZ.

Ten to 20 colonies of each isolate were transferred to 0.5 ml deionized water, incubated at 95°C for 10 min to lyse cells, and then centrifuged at 13,000 × g for 1 min. Two microliters of each supernatant was used in each individual downstream PCR on a GeneAmp PCR system 9700 (Applied Biosystems, NY). Detection of blaZ was performed using primers described elsewhere and listed in Table S1 in the supplemental material (12, 41). To maximize the detection of blaZ variants while minimizing false positivity due to nonspecific reactions, isolates were considered positive for blaZ if a PCR product was detectable by at least two of the primer sets used. A positive control (S. aureus 29213) and a negative control (S. aureus 25923) were included with each PCR run. PCRs were performed using Platinum Taq (Invitrogen, NY), with preincubation at 95°C for 10 min and then 40 cycles of the following: 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Completed PCRs were analyzed for appropriately sized DNA bands on 2% agarose gels.

Resolution of discrepancies and data analysis.

All β-lactamase detection tests (penicillin MIC, penicillin disk diffusion, induced nitrocefin test, cloverleaf test, and blaZ PCR) were performed using the same bacterial suspension. Any isolate demonstrating discordant results between any combination of penicillin MIC, penicillin disk diffusion, induced nitrocefin test, cloverleaf test, and/or blaZ PCR was retested by all 5 methods. If the discordant result persisted after repeat testing, it was included in the calculations. If the discordant result resolved after repeat testing, it was not counted as an error and the initial result was disregarded. The results of penicillin MIC, penicillin disk diffusion, penicillin disk zone edge test, induced nitrocefin test, and cloverleaf test were compared to the result of blaZ PCR as the gold standard. The percentages of agreement, sensitivity, and specificity and the PPV and NPV values were calculated. All statistical, mathematical, and graphical analysis was performed in R using custom scripts (42).

Chart review.

Medical records from 62 patients from whom S. lugdunensis was isolated from a normally sterile body site were chosen for in-depth review by an infectious disease clinician (J.V.). These included 31 patients with penicillin-resistant and 31 patients with penicillin-susceptible isolates. The following data were extracted from electronic medical records: age, sex, underlying medical conditions that might predispose for infection (diabetes, rheumatological disease, cancer, or other immune deficiencies), clinical diagnosis, and interventions such as removal of hardware, incision and drainage or surgical debridement, empirical antimicrobials, and consolidative therapy (i.e., antimicrobials chosen upon receipt of culture and susceptibility results).

Clinical diagnoses assigned by the treating physician were corroborated or modified through chart review. The criteria used to define clinical significance of an isolate included fever, white blood cell count, white blood cell differential, elevated C-reactive protein, increased erythrocyte sedimentation rate, physical exam findings consistent with bacterial skin or soft tissue infection (SSTI), radiographic findings consistent with infection, or blood cultures from different days on the same hospital admission growing the same organism. Patients with isolates meeting the criteria for clinical significance were further classified by infection type based on isolation source and clinical course. All study protocols were reviewed and approved by the UCLA Institutional Review Board (UCLA IRB 14-000304).

Supplementary Material

Supplemental material:


The authors declare that they have no competing interests.

I.H.M., J.H., J.V., and R.M.H. planned the study. J.V. performed chart review. I.H.M. and J.H. collected data. I.H.M. and K.B. performed data analysis. I.H.M., J.H., J.V., and R.M.H. wrote the paper.


Supplemental material for this article may be found at


1. Human Microbiome Project Consortium. 2012. Structure, function and diversity of the healthy human microbiome. Nature 486:207–214. doi:.10.1038/nature11234 [PMC free article] [PubMed] [Cross Ref]
2. Frank KL, Del Pozo JL, Patel R 2008. From clinical microbiology to infection pathogenesis: how daring to be different works for Staphylococcus lugdunensis. Clin Microbiol Rev 21:111–133. doi:.10.1128/CMR.00036-07 [PMC free article] [PubMed] [Cross Ref]
3. Wu AB, Wang MC, Tseng CC, Lin WH, Teng CH, Huang AH, Hung KH, Chiang-Ni C, Wu JJ 2011. Clinical and microbiological characteristics of community-acquired Staphylococcus lugdunensis infections in southern Taiwan. J Clin Microbiol 49:3015–3018. doi:.10.1128/JCM.01138-11 [PMC free article] [PubMed] [Cross Ref]
4. Lin JF, Cheng CW, Kuo AJ, Liu TP, Yang CC, Huang CT, Lee MH, Lu JJ 2015. Clinical experience and microbiologic characteristics of invasive Staphylococcus lugdunensis infection in a tertiary center in northern Taiwan. J Microbiol Immunol Infect 48:406–412. doi:.10.1016/j.jmii.2013.12.010 [PubMed] [Cross Ref]
5. Pereira EM, Schuenck RP, Nouer SA, Santos KR 2011. Methicillin-resistant Staphylococcus lugdunensis carrying SCCmec type V misidentified as MRSA. Braz J Infect Dis 15:293–295. doi:.10.1016/S1413-8670(11)70192-1 [PubMed] [Cross Ref]
6. Tee WS, Soh SY, Lin R, Loo LH 2003. Staphylococcus lugdunensis carrying the mecA gene causes catheter-associated bloodstream infection in premature neonate. J Clin Microbiol 41:519–520. doi:.10.1128/JCM.41.1.519-520.2003 [PMC free article] [PubMed] [Cross Ref]
7. Tenover FC, Jones RN, Swenson JM, Zimmer B, McAllister S, Jorgensen JH 1999. Methods for improved detection of oxacillin resistance in coagulase-negative staphylococci: results of a multicenter study. J Clin Microbiol 37:4051–4058. [PMC free article] [PubMed]
8. CLSI. 2016. Performance standards for antimicrobial susceptibility testing; 26th informational supplement. CLSI M100-S26. Clinical And Laboratory Standards Institute, Wayne, PA.
9. Chambers HF. 1988. Methicillin-resistant staphylococci. Clin Microbiol Rev 1:173–186. doi:.10.1128/CMR.1.2.173 [PMC free article] [PubMed] [Cross Ref]
10. Livermore DM. 2000. Antibiotic resistance in staphylococci. Int J Antimicrob Agents 16(Suppl 1):S3–S10. doi:.10.1016/S0924-8579(00)00299-5 [PubMed] [Cross Ref]
11. Lowy FD. 2003. Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest 111:1265–1273. doi:.10.1172/JCI18535 [PMC free article] [PubMed] [Cross Ref]
12. Olsen JE, Christensen H, Aarestrup FM 2006. Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci. J Antimicrob Chemother 57:450–460. doi:.10.1093/jac/dki492 [PubMed] [Cross Ref]
13. Gill VJ, Manning CB, Ingalls CM 1981. Correlation of penicillin minimum inhibitory concentrations and penicillin zone edge appearance with staphylococcal beta-lactamase production. J Clin Microbiol 14:437–440. [PMC free article] [PubMed]
14. Fass RJ, Helsel VL, Barnishan J, Ayers LW 1986. In vitro susceptibilities of four species of coagulase-negative staphylococci. Antimicrob Agents Chemother 30:545–552. doi:.10.1128/AAC.30.4.545 [PMC free article] [PubMed] [Cross Ref]
15. Ferreira AM, Bonesso MF, Mondelli AL, Camargo CH, Cunha MLRS 2012. Oxacillin resistance and antimicrobial susceptibility profile of Staphylococcus saprophyticus and other staphylococci isolated from patients with urinary tract infection. Chemotherapy 58:482–491. doi:.10.1159/000346529 [PubMed] [Cross Ref]
16. Becker K, Heilmann C, Peters G 2014. Coagulase-negative staphylococci. Clin Microbiol Rev 27:870–926. doi:.10.1128/CMR.00109-13 [PMC free article] [PubMed] [Cross Ref]
17. Gilbert DN, Chambers HF, Eliopoulos GM, Saag MM, Black D, Freedman DO, Pavia AT, Schwartz BS 2014. The Sanford guide to antimicrobial therapy 2014. Antimicrobial Therapy, Inc., Sperryville, VA.
18. Herchline TE, Barnishan J, Ayers LW, Fass RJ 1990. Penicillinase production and in vitro susceptibilities of Staphylococcus lugdunensis. Antimicrob Agents Chemother 34:2434–2435. doi:.10.1128/AAC.34.12.2434 [PMC free article] [PubMed] [Cross Ref]
19. Hellbacher C, Tornqvist E, Soderquist B 2006. Staphylococcus lugdunensis: clinical spectrum, antibiotic susceptibility, and phenotypic and genotypic patterns of 39 isolates. Clin Microbiol Infect 12:43–49. doi:.10.1111/j.1469-0691.2005.01296.x [PubMed] [Cross Ref]
20. Pottumarthy S, Fritsche TR, Jones RN 2005. Evaluation of alternative disk diffusion methods for detecting mecA-mediated oxacillin resistance in an international collection of staphylococci: validation report from the SENTRY Antimicrobial Surveillance Program. Diagn Microbiol Infect Dis 51:57–62. doi:.10.1016/j.diagmicrobio.2004.08.002 [PubMed] [Cross Ref]
21. De Paulis AN, Predari SC, Chazarreta CD, Santoianni JE 2003. Five-test simple scheme for species-level identification of clinically significant coagulase-negative staphylococci. J Clin Microbiol 41:1219–1224. doi:.10.1128/JCM.41.3.1219-1224.2003 [PMC free article] [PubMed] [Cross Ref]
22. Herchline TE, Ayers LW 1991. Occurrence of Staphylococcus lugdunensis in consecutive clinical cultures and relationship of isolation to infection. J Clin Microbiol 29:419–421. [PMC free article] [PubMed]
23. Chiquet C, Pechinot A, Creuzot-Garcher C, Benito Y, Croize J, Boisset S, Romanet JP, Lina G, Vandenesch F 2007. Acute postoperative endophthalmitis caused by Staphylococcus lugdunensis. J Clin Microbiol 45:1673–1678. doi:.10.1128/JCM.02499-06 [PMC free article] [PubMed] [Cross Ref]
24. Bannerman TL, Rhoden DL, McAllister SK, Miller JM, Wilson LA 1997. The source of coagulase-negative staphylococci in the Endophthalmitis Vitrectomy Study. A comparison of eyelid and intraocular isolates using pulsed-field gel electrophoresis. Arch Ophthalmol 115:357–361. [PubMed]
25. Pinna A, Zanetti S, Sotgiu M, Sechi LA, Fadda G, Carta F 1999. Identification and antibiotic susceptibility of coagulase negative staphylococci isolated in corneal/external infections. Br J Ophthalmol 83:771–773. doi:.10.1136/bjo.83.7.771 [PMC free article] [PubMed] [Cross Ref]
26. Ebright JR, Penugonda N, Brown W 2004. Clinical experience with Staphylococcus lugdunensis bacteremia: a retrospective analysis. Diagn Microbiol Infect Dis 48:17–21. doi:.10.1016/j.diagmicrobio.2003.08.008 [PubMed] [Cross Ref]
27. Frank KL, Reichert EJ, Piper KE, Patel R 2007. In vitro effects of antimicrobial agents on planktonic and biofilm forms of Staphylococcus lugdunensis clinical isolates. Antimicrob Agents Chemother 51:888–895. doi:.10.1128/AAC.01052-06 [PMC free article] [PubMed] [Cross Ref]
28. Hebert GA. 1990. Hemolysins and other characteristics that help differentiate and biotype Staphylococcus lugdunensis and Staphylococcus schleiferi. J Clin Microbiol 28:2425–2431. [PMC free article] [PubMed]
29. Zinkernagel AS, Zinkernagel MS, Elzi MV, Genoni M, Gubler J, Zbinden R, Mueller NJ 2008. Significance of Staphylococcus lugdunensis bacteremia: report of 28 cases and review of the literature. Infection 36:314–321. doi:.10.1007/s15010-008-7287-9 [PubMed] [Cross Ref]
30. Papapetropoulos N, Papapetropoulou M, Vantarakis A 2013. Abscesses and wound infections due to Staphylococcus lugdunensis: report of 16 cases. Infection 41:525–528. doi:.10.1007/s15010-012-0381-z [PubMed] [Cross Ref]
31. You YO, Kim KJ, Min BM, Chung CP 1999. Staphylococcus lugdunensis—a potential pathogen in oral infection. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 88:297–302. doi:.10.1016/S1079-2104(99)70031-4 [PubMed] [Cross Ref]
32. Goessens WH, Lemmens-den Toom N, Hageman J, Hermans PW, Sluijter M, de Groot R, Verbrugh HA 2000. Evaluation of the Vitek 2 system for susceptibility testing of Streptococcus pneumoniae isolates. Eur J Clin Microbiol Infect Dis 19:618–622. doi:.10.1007/s100960000332 [PubMed] [Cross Ref]
33. Batista N, Fernandez MP, Lara M, Laich F, Mendez S 2009. Evaluation of methods for studying susceptibility to oxacillin and penicillin in 60 Staphylococcus lugdunensis isolates. Enferm Infecc Microbiol Clin 27:148–152. (In Spanish.) doi:.10.1016/j.eimc.2008.04.005 [PubMed] [Cross Ref]
34. Nolte FS, Metchock B, Williams T, Diem L, Bressler A, Tenover FC 1995. Detection of penicillin-resistant Streptococcus pneumoniae with commercially available broth microdilution panels. J Clin Microbiol 33:1804–1806. [PMC free article] [PubMed]
35. Kiska DL, Kerr A, Jones MC, Chazotte NN, Eskridge B, Miller S, Jordan M, Sheaffer C, Gilligan PH 1995. Comparison of antimicrobial susceptibility methods for detection of penicillin-resistant Streptococcus pneumoniae. J Clin Microbiol 33:229–232. [PMC free article] [PubMed]
36. Deak E, Charlton CL, Bobenchik AM, Miller SA, Pollett S, McHardy IH, Wu MT, Garner OB 2015. Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory. Diagn Microbiol Infect Dis 81:27–33. doi:.10.1016/j.diagmicrobio.2014.09.018 [PubMed] [Cross Ref]
37. El Feghaly RE, Stamm JE, Fritz SA, Burnham C-AD 2012. Presence of the blaZ beta-lactamase gene in isolates of Staphylococcus aureus that appear penicillin susceptible by conventional phenotypic methods. Diagn Microbiol Infect Dis 74:388–393. doi:.10.1016/j.diagmicrobio.2012.07.013 [PubMed] [Cross Ref]
38. Jarlov JO, Rosdahl VT 1986. Quantitative determination of beta-lactamase production in Staphylococcus aureus strains compared to qualitative testing by a microbiological clover leaf test, a chromogenic cephalosporin test and a iodometric test. Acta Pathol Microbiol Immunol Scand B 94:415–421. [PubMed]
39. Ørstavik I, Ødegaard K 1971. A simple test for penicillinase production in Staphylococcus aureus. Acta Pathol Microbiol Scand B Microbiol Immunol 79:855–856. [PubMed]
40. Kaase M, Lenga S, Friedrich S, Szabados F, Sakinc T, Kleine B, Gatermann SG 2008. Comparison of phenotypic methods for penicillinase detection in Staphylococcus aureus. Clin Microbiol Infect 14:614–616. doi:.10.1111/j.1469-0691.2008.01997.x [PubMed] [Cross Ref]
41. Martineau F, Picard FJ, Grenier L, Roy PH, Ouellette M, Bergeron MG 2000. Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial. J Antimicrob Chemother 46:527–534. doi:.10.1093/jac/46.4.527 [PubMed] [Cross Ref]
42. R Development Core Team. 2016. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria:

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