Abstract
Thirty-nine Staphylococcus isolates with different mechanisms of resistance were recovered from the feces of 50 healthy children and tested for their susceptibilities to 17 antibiotics. The percentages of resistance of the staphylococci to some antibiotics were as follows: penicillin, 87%; erythromycin, 64%; tobramycin, 36%; tetracycline, 20.5%; kanamycin, 15%; and gentamicin, 13%. The mecA gene was detected in nine coagulase-negative staphylococci.
Staphylococci are important causes of human infections but are also found as nonpathogenic microorganisms in human fecal samples (3, 4). The use of antibiotics can be a selection factor for antibiotic resistance in the intestinal environment. To date, a number of studies have reported on the resistance mechanisms in clinical Staphylococcus isolates; nevertheless, very few data on the antibiotic resistance phenotypes or the mechanisms of resistance in nonpathogenic intestinal isolates are available (4, 17, 24). In the present study we analyzed the antibiotic resistance phenotypes and mechanisms of resistance in Staphylococcus isolates recovered from the feces of healthy children.
Fifty fecal samples were collected from 50 healthy children (ages, 7 to 23 months; 25 boys and 25 girls) in two nurseries in La Rioja, Spain, from November 2000 to January 2001. Diluted samples were inoculated onto mannitol salt agar plates, and for each sample one colony with a morphology typical of that of staphylococci was selected. The isolates were identified with the API 20 Staph system, and the identities of isolates identified as Staphylococcus aureus were confirmed by a PCR specific for the nuc gene (5). Thirty-nine Staphylococcus isolates (15 S. epidermidis, 8 S. simulans, 5 S. xylosus, 3 S. aureus, 3 S. warneri, 2 S. haemolyticus, 2 S. capitis, and 1 S. caprae isolates) were obtained from the 50 samples analyzed. Most of the staphylococcal isolates (92%) belonged to the coagulase-negative staphylococci (CoNS). The susceptibilities of the isolates were determined by the agar dilution method (19), and the results are shown in Table 1.
TABLE 1.
Resistance to 17 antibiotics in 39 Staphylococcus isolates recovered from feces of healthy children
| Antibiotic | No. of resistant isolates of the following species (no. of isolates):
|
Total isolates (n = 39)
|
|||
|---|---|---|---|---|---|
| S. aureus (3) | S. epidermidis (15) | Others CoNSa (21) | MIC range (μg/ml) | % Resistance | |
| Penicillin | 3 | 11 | 19 | —b | 87.2 |
| Oxacillinc | 7 | 3 | ≤0.125-4 | 25.6 | |
| Gentamicin | 5 | ≤0.25-64 | 12.8 | ||
| Streptomycin | ≤1-8 | 0 | |||
| Kanamycin | 6 | ≤0.25->64 | 15.4 | ||
| Tobramycin | 10 | 4 | ≤0.25-64 | 35.9 | |
| Amikacin | ≤0.5-64 | 0 | |||
| Erythromycin | 2 | 10 | 13 | ≤0.25->64 | 64.1 |
| Clindamycin | 2 | — | 5.1 | ||
| Spiramycin | 1 | 1->64 | 2.6 | ||
| Virginamycin | — | 0 | |||
| Tetracycline | 3 | 5 | ≤0.25->64 | 20.5 | |
| Chloramphenicol | 1 | ≤0.25->64 | 2.6 | ||
| Ciprofloxacin | 1 | ≤0.25-8 | 2.6 | ||
| Vancomycin | 0.25-4 | 0 | |||
| Teicoplanin | ≤0.25-4 | 0 | |||
S. simulans, n = 8; S. xylosus, n = 5; S. warneri, n = 3; S. haemolyticus, n = 2; S. capitis, n = 2; S. caprae, n = 1.
—, the disk diffusion method was used for this antibiotic.
Oxacillin was used for detection of methicillin resistance.
Beta-lactams.
A large proportion (87.2%) of the isolates were found to be penicillin resistant, and only 5 CoNS were penicillin susceptible. Oxacillin resistance was identified in 10 isolates, and the mecA gene was detected by PCR (18) in 9 of them (6 S. epidermidis isolates and 1 isolate each of S. haemolyticus, S. simulans, and S. warneri). The main mechanism of oxacillin (or methicillin) resistance involves the expression of a low-affinity penicillin-binding protein (PBP2a), encoded by the mecA gene (6, 9, 18, 21). Nevertheless, other mechanisms of resistance such as a decreased binding capacity of PBP 3 (22) or the production of methicillinase (16) have been described in staphylococci. The mecA gene has previously been reported in different species of CoNS (7, 10, 12, 21, 32). mecA-positive isolates of CoNS have frequently been detected in neonates with nosocomial infections in intensive care units (7), but they have also been isolated from the nares and skin of healthy chickens (12).
Aminoglycosides.
The rates of resistance to tobramycin, kanamycin, and gentamicin among our isolates were 35.9, 15.4, and 12.8%, respectively. Resistance to streptomycin and amikacin was not found in our series of isolates. The isolates were analyzed for the presence of the aminoglycoside-modifying enzyme genes aac(6′)-aph(2"), aph(3′)-IIIa, and ant(4′)-Ia by PCR (34). At least one aminoglycoside resistance gene was found in all aminoglycoside-resistant isolates (Table 2). The aac(6′)-aph(2") and ant(4′)-Ia genes were identified in all gentamicin- and tobramycin-resistant isolates, respectively. The aph(3′)-IIIa gene was detected in two of the six kanamycin-resistant isolates. The aac(6′)-aph(2"), aph(3′)-IIIa, and ant(4′)-Ia genes were found in one of the isolates (S. epidermidis Sa16), which was found to be resistant to gentamicin, kanamycin, and tobramycin. The ant(4′)-Ia gene was identified in 10 S. epidermidis isolates and 4 S. simulans isolates (Table 2). Similar percentages of tobramycin resistance were detected in another study performed with 249 clinical isolates of CoNS recovered in different European hospitals; nevertheless, higher values for kanamycin and gentamicin resistance (49 and 33%, respectively) were obtained (25). Those investigators reported that aac(6′)-aph(2") and ant(4′)-Ia were the predominant genes detected. Other groups have also proved that the ant(4′)-Ia gene is the predominant gene associated with aminoglycoside resistance in isolates of S. aureus and CoNS (11, 20, 33).
TABLE 2.
Aminoglycoside resistance genes in Staphylococcus isolates with different aminoglycoside resistance phenotypes
| Species | No. of isolates | MIC range (μg/ml)a
|
Genes detected by PCR
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| STR | GEN | KAN | TOB | AMK | aac(6′)-aph(2′′) | aph(3′)-IIIa | ant(4′)-Ia | ||
| S. epidermidis | 6 | 1-4 | 1-4 | 2-8 | 8-16 | ≤0.5-8 | − | − | + |
| S. epidermidis (Sal6) | 1 | 4 | 8 | >64 | >64 | 2 | + | + | + |
| S. epidermidis | 3 | 2 | 64 | >64 | 8-32 | ≤0.25-2 | + | − | + |
| S. epidermidis | 1 | 4 | 16 | >64 | 4 | 2 | + | − | − |
| S. epidermidis | 1 | 1 | 1 | >64 | ≤0.25 | 1 | − | + | − |
| S. simulans | 4 | ≤1-2 | 1 | 4-16 | 8-32 | ≤0.5-2 | − | − | + |
| Staphylococcus spp.b | 23 | ≤1-8 | ≤0.25-4 | ≤0.25-4 | ≤0.25-2 | ≤0.5-2 | − | − | − |
STR, streptomycin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; AMK, amikacin. The MICs included in the resistance category are indicated in boldface.
S. xylosus, n = 5; S. simulans, n = 4; S. epidermidis, n = 3; S. aureus, n = 3; S. warneri, n = 3; S. haemolyticus, n = 2; S. capitis, n = 2; S. caprae, n = 1.
MLS antibiotics.
Erythromycin resistance (MICs, ≥8 μg/ml) was found in 64.1% of the isolates (Table 1). Phenotypes for resistance to the macrolide, lincosamide, and streptogramin B (MLS) antibiotics were investigated as described previously (29). The MLS resistance phenotype (including resistance to the MLS antibiotics) was found in 72% of the erythromycin-resistant (Eryr) isolates tested. Inducible expression of the MLS phenotype (the iMLS phenotype) was observed in all except one of the isolates tested. S. epidermidis isolate Sa16 was the only one with a constitutive MLS phenotype (the cMLS phenotype). The M resistance phenotype (resistance to erythromycin but not to spiramycin, lincosamides, or streptogramins) was demonstrated in seven S. epidermidis isolates (Table 3). The isolates were analyzed by PCR for the presence of the methylase genes (ermA, ermB, and ermC) (30) and efflux genes mefA (30) and msrA (36). The ermA gene or the ermC gene was detected in 11 of the 24 Eryr isolates (all 11 isolates with the iMLS phenotype). Both the ermA and the ermB genes were identified in S. epidermidis Sa16 (which had the cMLS phenotype). ermA or ermC was also the gene most frequently found in Eryr staphylococcal isolates in other studies (13, 14, 35).
TABLE 3.
Resistance mechanisms in 39 Staphylococcus isolates linked to the erythromycin MIC
| Species | No. of isolates | Erythromycin MIC (μg/ml) | Phenotype | Gene detected by PCR
|
||||
|---|---|---|---|---|---|---|---|---|
| ermA | ermB | ermC | msrA | mefA | ||||
| S. epidermidis | 7 | 32->64 | M | − | − | − | + | − |
| S. epidermidis | 1 | >64 | iMLS | − | − | + | − | − |
| S. epidermidis (Sal6) | 1 | >64 | cMLS | + | + | − | − | − |
| S. aureus | 2 | >64 | iMLS | + | − | − | − | − |
| S. simulans | 3 | 32->64 | iMLS | − | − | − | − | − |
| S. simulans | 4 | >64 | iMLS | + | − | − | − | − |
| S. simulans | 1 | >64 | iMLS | − | − | + | − | − |
| S. xylosus | 2 | >64 | iMLS | − | − | − | − | − |
| S. warneri | 1 | >64 | iMLS | + | − | − | − | − |
| S. haemolyticus | 2 | >64 | iMLS | + | − | − | − | − |
| Staphylococcus spp.a | 15 | ≤0.25-12 | − | − | − | − | − | |
S. epidermidis, n = 6; S. xylosus, n = 3; S. warneri, n = 2; S. capitis, n = 2; S. aureus, n = 1; S. caprae, n = 1.
The msrA gene was detected in the seven Eryr S. epidermidis isolates with the M phenotype. In line with our results, Lina et al. (13) found that the msrA gene is more prevalent in isolates of CoNS than in S. aureus isolates. The mefA gene was not found in our isolates. No resistance genes were detected in five Eryr Staphylococcus isolates (all of them were CoNS with the iMLS phenotype) (Table 3). Strains with similar characteristics have been reported previously (15, 26). MLS resistance genes were not found in any of the 17 Staphylococcus isolates for which the erythromycin MICs were ≤2 μg/ml.
Tetracycline.
Eight tetracycline-resistant (Tetr) isolates were detected in this study; and the isolates were analyzed by PCR for the presence of the tetK, tetL, tetM, and tetO genes (2). The tetK gene was detected in five Tetr isolates, and the tetM and tetK genes were detected in the remaining three isolates (two S. epidermidis isolates and one S. xylosus isolate). Neither the tetL nor the tetO gene was detected in any of our isolates. The tetM and tetK genes were also the genes most frequently detected in Tetr staphylococcal isolates by other groups (23, 27, 28, 31). Diekema et al. (8) reported 15.8 and 22.2% rates of tetracycline resistance in methicillin-susceptible and methicillin-resistant clinical isolates of CoNS from European hospitals, respectively.
Other antibiotics.
Only one isolate (S. epidermidis Sa16) was found to be resistant to chloramphenicol and ciprofloxacin, and the catA gene was detected in that isolate by PCR (1). No staphylococcal isolates with diminished susceptibility to glycopeptides were found in this study.
Table 4 shows the phenotypes and the mechanisms of antibiotic resistance of the nine isolates in which the mecA gene was detected. The wide antibiotic resistance pattern shown by S. epidermidis isolate Sa16 is noteworthy.
TABLE 4.
Phenotypes of antibiotic resistance and genes detected in the nine mecA-positive Staphylococcus isolates recovered in the study
| Species | No. of isolates | Phenotype of antibiotic resistancea | Gene(s) detected by PCR |
|---|---|---|---|
| S. epidermidis (Sal6) | 1 | PEN OXA GEN KAN TOB ERY CLI SPI CHL CIP | aac(6′)-aph(2′′), aph(3′)-IIIa, ant(4′)-Ia, ermA, ermB, catA |
| S. epidermidis | 1 | PEN OXA GEN KAN TOB ERY CLI | aac(6′)-aph(2′′), ant(4′)-Ia, msrA |
| S. epidermidis | 1 | PEN OXA GEN KAN ERY TET | aac(6′)-aph(2′′), msrA, tetM, tetK |
| S. epidermidis | 1 | PEN OXA GEN KAN TOB ERY | aac(6′)-aph(2′′), ant(4′)-Ia, msrA |
| S. warneri | 1 | PEN OXA ERY TET | ermA, tetK |
| S. epidermidis | 2 | PEN OXA TOB | ant(4′)-Ia |
| S. simulans | 1 | PEN OXA TOB ERY | ant(4′)-Ia |
| S. haemolyticus | 1 | PEN OXA ERY | ermA |
PEN, penicillin; OXA, oxacillin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; ERY, erythromycin; CLI, clindamycin; SPI, spiramycin; CHL, chloramphenicol; CIP, ciprofloxacin.
We conclude that the rates of antibiotic resistance in staphylococcal isolates recovered from the feces of healthy children are high, especially in isolates of the CoNS. These isolates can act as reservoirs of antibiotic resistance genes that could be transferred to pathogenic bacteria. A follow-up study should be carried out to analyze the resistance mechanisms in fecal nonpathogenic bacteria more extensively. The results of such a study could help to establish more prudent measures for the use of antibiotics in humans.
Acknowledgments
This work was partly supported by grants from the Fondo de Investigaciones Sanitarias (grant FIS 00/0545) and the Consejería de Educación del Gobierno de La Rioja (grant ACPI2000/021) of Spain.
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