J Infect Chemother (2007) 13:63–66 DOI 10.1007/s10156-006-0492-5

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2007

NOTE Wataru Higuchi · Hirokazu Isobe · Yasuhisa Iwao Soshi Dohmae · Kohei Saito · Tomomi Takano Taketo Otsuka · Tatiana Baranovich · Chiho Endo Nobuaki Suzuki · Yasuyuki Tomiyama Tatsuo Yamamoto

Extensive multidrug resistance of coagulase-negative staphylococci in medical students

Received: August 4, 2006 / Accepted: October 24, 2006

Abstract Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from the nasal mucosa of medical students were examined for susceptibility to 16 antimicrobial agents. No methicillin-resistant S. aureus (MRSA) was isolated, while MRCNS was present in 23.5% of the medical students. CNS exhibited significantly more resistance to antimicrobial agents such as gentamicin, in addition to oxacillin, compared to S. aureus, and 13.1% of the CNS strains (mostly MRCNS) were multidrug-resistant (to five or more drugs). In contrast, ampicillin resistance was higher in S. aureus. The rate of hospitalization or of taking an antimicrobial agent within the past 1 year was lower in CNS+ students than in S. aureus+ students. The data suggest that CNS could serve as a reservoir of drug resistance by persistent colonization in the nasal mucosa. In this study, MRCNS with multidrug resistance was found in medical students. More attention should be given to nasal MRCNS in medical students as a possible spreader in hospitals. Key words Coagulase-negative staphylococci (CNS) · Staphylococcus aureus · Drug resistance · Nasal mucosa · Medical students

Staphylococci colonize skin and nasal mucosa as their normal habitat, with population in the anterior nares and axillae at 103 to 106 CFU/cm2.1 Among staphylococci, Staphylococcus aureus is the most virulent, and it is associated with a wide spectrum of diseases, including skin and soft tissue infections (SSTIs), systemic infections, and exotoxinrelated diseases.2 W. Higuchi · H. Isobe · Y. Iwao · S. Dohmae · K. Saito · T. Takano · T. Otsuka · T. Baranovich · C. Endo · N. Suzuki · Y. Tomiyama · T. Yamamoto (*) Division of Bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 757 Ichibanchou, Asahimachidori, Niigata 951-8510, Japan Tel. +81-25-227-2050; Fax +81-25-227-0762 e-mail: [email protected]

Methicillin-resistant S. aureus (MRSA) has been a major cause of nosocomial infections since the early 1960s,3 and since 1997, another type of MRSA, producing PantonValentine leucocidin (PVL), has emerged in the community.4,5 It is called community-acquired MRSA (CA-MRSA), and it is associated with SSTIs, as well as with necrotizing pneumonia, in children and adolescents.6 In Japan, our group has isolated CA-MRSA from an infant with bullous impetigo and a high school student with abscess.7 Other staphylococci, called coagulase-negative staphylococci (CNS), are also pathogenic, when the host is compromised. CNS are the most common pathogens in nosocomial bloodstream infections.1 Methicillin-resistant CNS (MRCNS) has also been noted worldwide.1,8,9 Moreover, CNS may donate its drug resistance to MRSA.10 The drug resistance of staphylococci, especially CNS, in medical students has not been studied. The aim of this study was to find the present drugresistance status of staphylococci colonizing the nasal mucosa of medical students and also to examine for the possible presence of PVL genes in these strains. For this purpose, we investigated the in vitro susceptibility of S. aureus and CNS, isolated from the nasal mucosa of medical students, to antimicrobial agents and we examined for PVL genes by polymerase chain reaction (PCR). S. aureus and CNS were isolated from healthy medical students prior to their beginning clinical practice (98 thirdyear students). All 98 students took part in the experiments for this study. Susceptibility testing of bacterial strains was done by the agar dilution method, with Mueller-Hinton agar (Difco, Sparks, MD) according to previous procedures.11 The final concentrations of antimicrobial agents ranged from 0.001 to 128 µg/ml. The antimicrobial agents were gifts from their manufacturers. Resistance was defined according to Clinical and Laboratory Standards Institute (CLSI).11 In case of arbekacin, fosomycin, and fusidic acid, resistance was defined as a peak with higher MIC values, distinct from a peak of susceptible strains, in MIC distribution curves. Statistical comparison of groups with S. aureus and CNS was performed using Pearson’s χ2 test or Fisher’s exact test. All P values were two-tailed, and P < 0.05 was

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considered significant. The mecA gene12,13 and PVL genes13 were detected by PCR, using previously reported primers. Of the 98 students, 96 (98.0%) were staphylococcipositive. S. aureus and CNS were isolated in 36 (37.5%) and 84 (87.5%) of these 96 students, respectively; S. aureus only was isolated from 12 students (12.5%); S. aureus and CNS were simultaneously isolated from 24 students (25.0%); and CNS only was isolated from 60 students (62.5%). All S. aureus and CNS strains were PVL-negative. Next, 36 S. aureus strains, isolated from 36 students, and 84 CNS strains, isolated from 84 students, were examined for in vitro susceptibility to 16 antimicrobial agents (Table 1). All the S. aureus strains were susceptible to oxacillin. In contrast, 27.4% of the CNS strains (23/84; i.e., 23.5% of the medical students; 23/98) were resistant to oxacillin; mecA gene carriage was found in 15/15, 3/4, and 0/4 strains with minimum inhibitory concentrations (MICs) of 4 µg/ml or higher, 2 µg/ml, and 0.5 or 1 µg/ml, respectively. The CNS strains exhibited significantly more resistance, compared with S. aureus, to ceftazidime, gentamicin, and kanamycin (P < 0.05; Table 1). The CNS strains also tended to be more resistant than the S. aureus to tetracycline, clindamycin, levofloxacin, fosfomycin, and fusidic acid, although the difference between S. aureus and CNS was not

significant (Table 1). For erythromycin, the resistance rate of CNS strains was similar to that of S. aureus. For ampicillin, the resistance rate of CNS strains was significantly lower than that of S. aureus (P < 0.05; Table 1). Drug resistance patterns are summarized in Table 2. In S. aureus, 47.2% of the strains (17/36) showed resistance. Of these 17 strains, 52.9% (9/17) were resistant to ampicillin alone, 29.4% (5/17) showed resistance to two drugs (ampicillin and erythromycin), 5.9% (1/17) showed resistance to three drugs (ampicillin, erythromycin, and clindamycin or fusidic acid), and 5.9% (1/17) showed resistance to four drugs (tetracycline, erythromycin, levofloxacin, and fosfomycin). In the CNS strains, half (48.8%; 41/84) showed drug resistance, similar to findings in S. aureus. However, resistance to ampicillin alone was shown in only 7.3% of the resistant CNS strains (3/41), although the rate of resistance to one drug was 34.1% (14/41). Moreover, CNS strains showed a wide variety of resistance patterns, ranging from one- to seven-drug resistance (Table 2). Multidrug-resistant strains showing resistance to five or more drugs were present in 26.8% of the resistant CNS strains (11/41); no such multidrug-resistant strains (showing resistance to five or more drugs) were detected in S. aureus

Table 1. Minimum inhibitory concentrations (MICs) of antimicrobial agents against Staphylococcus aureus or coagulase-negative staphylococci (CNS) isolated from the nasal mucosa of medical students Antimicrobial agents

S. aureus (n = 36)

CNS (n = 84)

MIC (µg/ml)

MIC (µg/ml)

MIC50 Penicillins Oxacillin Ampicillin Cephems Ceftazidime Glycopeptides Vancomycin Teicoplanin Oxazolidinones Linezolid Aminoglycosides Arbekacin Gentamicin Kanamycin Tetracyclines Tetracycline Minocycline Macrolides Erythromycin Lincosamides Clindamycin Quinolones Levofloxacin Others Fosfomycin Fusidic acid a

MIC90

Range

% Resistanta

MIC50

0 44.4

0.125 0.125

P value for % resistant (S. aureus versus CNS) Range

% Resistanta

64 2

0.032–>128 0.015–8

27.4 25.0

0.004 0.034

2–64

10.7

0.041

MIC90

0.25 0.25

1 2

0.063–1 0.063–2

8

8

8–16

0

4

16

1 1

1 2

1 0.5–2

0 0

2 2

2 8

0.063–2 0.5–16

0 0

NAb NAb

2

2

1–2

0

1

2

0.25–4

0

NAb

0.5 0.25 2

1 0.5 4

0.125–2 0.125–4 0.5–16

– 0 0

0.25 0.125 2

0.063–1 0.063–64 0.5–>128

– 10.7 13.1

NAb 0.041 0.022

0.25 0.032

0.5 0.032

0.125–64 0.032–8

2.8 0

0.25 0.032

0.032–128 0.008–0.5

11.9 0

NSc NAb

0.25–>128

22.2

0.25

0.125–>128

22.6

NSc

0.25

>128

0.5 16 >128 8 0.063 >128

0.125

0.063–128

2.8

0.063

0.125

0.032–>128

0.25

0.25

0.125–16

2.8

0.25

4

0.063–8

14.3

NSc

2 0.125

16 0.125

0.25–>128 0.063–2

4 0.125

128 0.25

0.25–>128 0.032–2

−(7.1)d −(9.5)d

NSc,e NSc,e

−(2.8)d −(2.8)d

7.14

NSc

0.125

% Resistant, based on CLSI breakpoints; (−), no CLSI breakpoints available NA, not applicable c NS, not significant (P ≥ 0.05) d Resistance was defined as a peak with higher MIC values, distinct from a peak of susceptible strains, in MIC distribution curves. Resistant: MICs ≥ 256 µg/ml for fosfomycin; MICs ≥ 1 µg/ml for fusidic acid e Statistral comparison of % resistant in parentheses (d) between S. aureus and CNS b

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(P = 0.018). A significant difference was also observed when resistance to three or more drugs was examined (53.7% [22/41] in CNS strains versus 17.6% [3/17] in S. aureus; P 0.012). Oxacillin-resistant strains (MRCNS) accounted for 56.1% of the resistant CNS strains (23/41), and 90.9% (10/11) of the multidrug-resistant CNS strains showing resistance to five or more drugs. The percentages of students who were hospitalized or took antimicrobial agents within the past 1 year were, for S. aureus: 26.3% (5/19) of the carriers of nonresistant strains and 29.4% (5/17) of the carriers of resistant strains; and for CNS, 9.3% (4/43) of the carriers of nonresistant strains and 17.1% (7/41) of the carriers of resistant strains (Table 2). Thus, the rate of students who were hospitalized or took antimicrobial agents within the past 1 year was lower in the CNS group than in the S. aureus group. However, drug resistance was more frequent in the CNS group. In this study, we isolated nasal S. aureus and CNS from medical students prior to their beginning clinical practice. Although MRSA was not detected, MRCNS was detected

in 23.5% of the medical students. A striking feature of the drug resistance we found in the CNS strains was that gentamicin (and kanamycin) resistance, in addition to oxacillin resistance, was present significantly more frequently than in S. aureus, and that drug resistance patterns were heterogenous, with multidrug resistance to five to seven drugs being seen in 26.8% of CNS resistant strains. Also, most strains exhibiting multidrug resistance were MRCNS, indicating that CNS (mostly MRCNS) serves as a reservoir of drug resistance in the nasal mucosa. It has been demonstrated that once macrolide-resistant S. epidermidis (a representative strain of CNS) is selected with the use of clarithromycin, the same resistant strain persists for at least up to 4 years without any further selection.14 Therefore, there is a possibility that, even in this study, CNS had persistently colonized the nasal mucosa for longer than S. aureus and had accumulated many resistance genes during a long period of time. In Europe, MRCNS also exhibits multidrug resistance, including gentamicin resistance.8 Further, it has been reported that CNS donates its drug resistance to MRSA.10

Table 2. Drug resistance patterns of Staphylococcus aureus and CNS isolated from the nasal mucosa Resistance patterna

S. aureus (n = 36)

CNS (n = 84) b

None A T E L (S)

No. of resistant strains (%)

No. of resistant strainsb (%)

19H1,A4 (52.8) 9H1,A1 (25.0)

43A4 (51.2) 3A1 (3.6) 3 (3.6) 3 (3.6) 2 (2.4) 3 (3.6) 3A1 (3.6)

O, A A, E E, I

A2

5

(13.8)

O, A, T O, A, E O, E, (F) A, E, I A, E, (F)

3 (3.6) 1 (1.2) 2 (2.4) 1 (2.8) 1 (2.8) 2A1 (2.4) 1 (1.2)

O, T, (F) O, E, I, (F) G, K, L, (S) T, E, L, (S) O, A, E, I O, A, G, K, L

1 (1.2) 1 (1.2)

1 (1.2) 1A1 (2.8) 1H1 (1.2) 2 (2.4)

G, K, E, L, (F)

1 (1.2)

O, A, E, I, L O, A, G, K, E, L O, A, G, K, T, E, L O, K, T, E, I, (F, S) O, A, G, K, E, L, (S) O, A, G, K, E, I, (F)

1A1 (1.2) 3A1 (3.6) 1 (1.2) 1 (1.2) 1A1 (1.2) 1 (1.2)

a O, Oxacillin; A, ampicillin; G, gentamicin; K, kanamycin; T, tetracycline; E, erythromycin; I, clindamycin; L, levofloxacin; F, fusidic acid; S, fosfomycin. Data for drug resistance are from Table 1; drug resistance (shown as percentages in parentheses) is based on the definition used in this study (Table 1 footnote). Resistance patterns with oxacillin resistance are shadowed b S. aureus and CNS strains were isolated from the nasal mucosa of medical students. H, number of students who were hospitalized within the past 1 year; A, number of students who took antimicrobial agents within the past 1 year (as outpatients)

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In contrast, ampicillin resistance, which is encoded by a penicillinase (PCase) plasmid,15 was significantly more prominent in S. aureus than in CNS. This may reflect the host range of PCase plasmids, preferring S. aureus to CNS. CNS is the most common pathogen in nosocomial bloodstream infections.1 In this study, MRCNS strains with multidrug resistance were found in medical students. More attention should be given to nasal MRCNS in medical students as a possible spreader in hospitals. No strains showing PVL positivity, which has been noted as a major characteristic of CA-MRSA,4,5 were demonstrated in nasal staphylococci in this study. Further studies are necessary to investigate the molecular nature of extensive multidrug resistance as well as persistent, nasal colonization of CNS, especially MRCNS. Acknowledgments We thank Masaaki Itoh for encouragement regarding medical practice in infectious diseases; Kohei Akazawa for help in statistical analyses; and the third-year students (2006) at Niigata University School of Medicine for their collaboration in this study.

5.

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11.

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