1. Background
The Staphylococcus genus includes at least 40 species. Of these, nine have two subspecies, one has three subspecies, and one has four subspecies (1). Most are harmless and reside normally on the skin and mucous membranes of humans and other organisms. Found worldwide, they are a small component of soil microbial flora (2). Coagulase-negative staphylococci (CoNS) species make up a large and heterogeneous group of Gram-positive bacteria, into which more than 30 species have been introduced (3, 4).
In recent years, the importance of CoNS has increased, especially because of their major causative role in the nosocomial infections in both developed and developing countries (5, 6). The widespread use of immunosuppressive drugs, indwelling intravascular catheters, artificial tools, patient- and personnel-related factors, and health-care settings have been reported to be associated with the spread of these infections (7). Premature infants are at a high risk of CoNS infections (6).
Among the CoNS, S. epidermidis, S. haemolyticus and S. saprophyticus are more common in nosocomial infections, most of which have gained resistance to methicillin, penicillin, and other antibiotics (8). The genes responsible for the multi-drug resistance (MDR) phenotype of CoNS are located on plasmids, which can be easily exchanged between the species. Indeed, despite continuing efforts, antibiotic resistance remains a major problem in controlling the CoNS infections (9). To achieve effective treatment, a variety of glycopeptide antibiotics, including vancomycin, teicoplanin, telavancin, ramoplanin, and decaplanin are primarily prescribed (10). Vancomycin is the most important drug for treatment of the MDR S. epidermidis (11). However, resistance to vancomycin has been frequently reported among the staphylococci (12, 13). In 1987, Schwalbe and colleagues reported the first clinical isolate of vancomycin-resistant CoNS (13). Since then, many vancomycin-resistant or other glycopeptide-resistant CoNS isolated from clinical samples have been documented in different countries (14-18). Additionally, the emergence of resistance to teicoplanin has been reported (15). Therefore, the use of vancomycin and teicoplanin as appropriate therapeutic agents against CoNS infections is under debate (19).
2. Objectives
Because of the importance of CoNS, especially in nosocomial and neonatal infections, and the worldwide emergence of vancomycin-resistant strains, we aimed to determine the prevalence of teicoplanin- and vancomycin-resistant CoNS isolates from patients in a hospital in Gorgan, northern Iran, using both phenotypic and molecular methods.
3. Materials and Methods
3.1. Clinical Samples and Bacterial Isolates
From January 2013 to May 2014, a total of 100 clinical CoNS isolates were collected from patients hospitalized in distinct wards of Sayyad Shirazi hospital in Gorgan, Iran. The origins of the isolates were different clinical specimens, including blood, urine, eyes, tracheal aspirates, wounds, and catheters. Each specimen was cultured on blood agar and incubated at 37°C for 24 h. Identification of CoNS to the species level was done with Gram staining, then standard biochemical tests were performed, including cultivation in mannitol salt agar; oxidation-fermentation (OF); the Voges-Proskauer (VP) test; hydrolysis of L-pyrrolidonyl-beta-naphthylamide (PYR); nitrate reduction; acid production; coagulase, catalase, phosphatase, ornithine decarboxylase, and urease enzymatic tests; and resistance to deferoxamine, bacitracin, albamycin, and polymyxin B antibiotics (20, 21). The isolates verified as CoNS were stored in trypticase soy broth with glycerol at -20°C until being subjected to further experiments.
3.2. Antimicrobial Susceptibility Testing
Kirby-Bauer’s disk agar diffusion method was carried out for the determination of CoNS susceptibility to 16 antimicrobial agents. Antibiotic discs were purchased from Rosco Diagnostica (Denmark) and included ampicillin (AMP, 10 μg), cephazolin (CFZ, 30 μg), cefotaxime (30 μg), ciprofloxacin (CIP, 5 μg), clindamycin (CLI, 2 μg), erythromycin (ERY, 15 μg), gentamicin (GM, 10 μg), nitrofurantoin (NIT, 300 μg), norfloxacin (NOR, 10 μg), oxacillin (OXA, 1 μg), penicillin (PEN, 5 μg), rifampin (RIF, 10 μg), cotrimoxazole (STX, 25 μg), teicoplanin (TEC, 30 μg), tetracycline (TE, 5 μg), and vancomycin (VAN, 30 μg). The test used a bacterial suspension with a turbidity adjusted equivalent to a 0.5 McFarland standard, and was performed on Mueller-Hinton agar plates (Merck, Germany) based on the Clinical & Laboratory Standards Institute (CLSI) guidelines (22, 23). S. aureus, strain COL, was used as a control. MDR of an isolate was defined as resistance at least to three different classes of antibiotics (24).
3.3. Detection of the vanA Gene
PCR assays were performed on CoNS isolates for detection of the gene encoding vancomycin resistance, vanA. Total DNA was extracted using the phenol/chloroform/isoamyl alcohol method as previously described (25). The primer pair, 5'-GGCAAGTCAGGTGAAGATG-3' and 5'-ATCAAGCGGTCAATCAGTTC-3', was used for amplification of fragments with lengths of 713 bp (26). The PCR thermal profile was comprised of enzyme activation at 94°C for 5 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 60 seconds, and a final extension at 72°C for 5 minutes. The PCR products were subjected to electrophoresis in a 1.5% agarose gel containing ethidium bromide, then photographed with a UV transilluminator. Enterococcus faecalis PTCC 1237 was used as a positive control.
3.4. Vancomycin E-Test
The minimum inhibitory concentration (MIC) of vancomycin was determined using E-test strips, according to the manufacturer’s guidelines (Bioanalyse, Turkey). Briefly, the bacterial suspension with a turbidity adjusted equivalent to a 0.5 McFarland standard was swabbed on Mueller-Hinton agar medium supplemented with 2% NaCl, then an E-test strip containing vancomycin-concentration gradient was placed on the medium and incubated at 37°C for 24 hours. Finally, the MIC was calculated as previously described (26).
3.5. Statistical Analysis
The data were analyzed using the statistical package for the social sciences (SPSS) software, version 16 (IBM Co., IL, USA). The chi-square test was performed for the categorical variables and ANOVA was used for numerical ones, and the results were expressed as frequency percentage and mean ± standard deviation (SD), respectively. P values of < 0.05 were considered statistically significant.
4. Results
As shown in Table 1, S. haemolyticus and S. epidermidis were the most prevalent CoNS isolates from different wards of the hospital (34% and 27%, respectively). S. hominis (12%), S. saprophyticus (9%), and other species (18%) appeared with less frequency. Of the CoNS isolates, 37% were detected in patients hospitalized in the internal medicine ward, 14% in the ICU, 21% in the pediatric ward, and 28% in other wards. The majority of isolates were recovered from blood and urine specimens (43% and 33%, respectively). Moreover, 69% of CoNS were isolated from females and 31% from males. The highest percentage of isolates (35%) was from patients aged < 20 years old.
Table 1. Prevalence of Coagulase-Negative Staphylococci According to Species, Patient Gender and Age, Hospital Ward, and Isolation Source
| Variable | Frequency (%) |
|---|---|
| Bacterial species | |
| S. epidermidis | 24 (24) |
| S. haemolyticus | 37 (37) |
| S. hominis | 12 (12) |
| S. saprophyticus | 9 (9) |
| Other | 18 (18) |
| Patient gender | |
| Female | 69 (69) |
| Male | 31 (31) |
| Patient age | |
| < 20 | 35 (35) |
| 20 - 45 | 32 (32) |
| > 45 | 33 (33) |
| Hospital ward | |
| Internal medicine | 37 (37) |
| ICU | 14 (14) |
| Pediatrics | 21 (21) |
| Unspecified | 28 (28) |
| Isolation source | |
| Blood | 43 (43) |
| Urine | 33 (33) |
| Eye | 8 (8) |
| Tracheal aspirate | 8 (8) |
| Wound/abscess | 2 (2) |
| Catheter | 6 (6) |
| Total | 100 |
4.1. Multi-Drug Resistance (MDR)
Out of 100 CoNS, 62 (62%) strains exhibited MDR and 38 (38%) were identified as non-MDR. As depicted in Table 2, there was a significant difference in MDR distribution among various CoNS species (P = 0.002); S. haemolyticus showed higher rates of MDR compared to the other CoNS, and S. saprophyticus had the lowest frequency among the MDR CoNS. There was no significant difference in frequency distribution of MDR CoNS isolated from different hospital wards or from different specimens (P > 0.05). However, MDR was seen at higher rates among the CoNS isolated from blood and urine specimens (42% and 30%, respectively).
Table 2. Prevalence of Multidrug Resistant (MDR) Coagulase-Negative Staphylococci According to Species, Hospital Ward, and Isolation Sourcea
| Variable | Non-MDR | MDR | P Value |
|---|---|---|---|
| Bacterial species | 0.002 | ||
| S. epidermidis | 9 (24) | 15 (24) | |
| S. haemolyticus | 6 (16) | 31 (50) | |
| S. hominis | 5 (13) | 7 (11) | |
| S. saprophyticus | 6 (16) | 3 (5) | |
| Other | 12 (31) | 6 (10) | |
| Hospital ward | 0.122 | ||
| Internal medicine | 18 (47) | 19 (31) | |
| ICU | 4 (11) | 10 (16) | |
| Pediatrics | 4 (11) | 17 (27) | |
| Unspecified | 12 (31) | 16 (26) | |
| Isolation source | 0.317 | ||
| Blood | 17 (45) | 26 (42) | |
| Urine | 14 (37) | 19 (30) | |
| Eye | 2 (5) | 6 (10) | |
| Tracheal aspirate | 2 (5) | 6 (10) | |
| Wound/abscess | 2 (5) | 0 (0) | |
| Catheter | 1 (3) | 5 (8) | |
| Total | 38 (38) | 62 (62) |
aValues are expressed as No. (%).
4.2. Glycopeptide Susceptibility Pattern
Out of 100 CoNS, 99 (99%) strains were susceptible to vancomycin and one (1%) strain (S. hominis) showed resistance to this antibiotic, according the antibiogram test. However, all strains (100%) were revealed to be vanA-negative on PCR. In addition, out of 100 CoNS, 85 (85%) strains demonstrated complete susceptibility and 15 (15%) showed intermediate susceptibility to teicoplanin; none of the CoNS strains were resistant to teicoplanin. The mean inhibition zones of vancomycin and teicoplanin are presented in Table 3 in terms of species, patient gender and age, hospitalization ward, and CoNS isolation source. There is a significant difference between CoNS species regarding the mean inhibition zones of vancomycin (P < 0.001) and teicoplanin (P < 0.001); S. epidermidis exhibited the highest susceptibility to both antibiotics, and S. saprophyticus appeared to have the least. However, no significant differences were found in the inhibition zones of vancomycin and teicoplanin against CoNS based on gender, age group, hospital ward, and isolation source (P > 0.05). In addition, the overall mean inhibition zone of vancomycin was significantly higher than that of teicoplanin (P < 0.001), which indicates that CoNS might be more sensitive to vancomycin than to teicoplanin.
Table 3. Inhibition Zone (mm) of Vancomycin and Teicoplanin Against Coagulase-Negative Staphylococcia
| Variable | Vancomycin | P Value | Teicoplanin | P Value |
|---|---|---|---|---|
| Bacteria species | 0.001 | 0.001 | ||
| S. epidermidis | 19.29 ± 1.55 | 18.04 ± 1.78 | ||
| S. haemolyticus | 18.24 ± 1.14 | 15.38 ± 2.43 | ||
| S. hominis | 18.00 ± 1.60 | 15.50 ± 2.32 | ||
| S. saprophyticus | 17.56 ± 1.24 | 15.33 ± 2.29 | ||
| Others | 19.28 ± 0.96 | 16.39 ± 2.14 | ||
| Patient gender | 0.472 | 0.183 | ||
| Female | 18.52 ± 1.53 | 15.99 ± 2.54 | ||
| Male | 18.74 ± 1.09 | 16.69 ± 2.11 | ||
| Patient age | 0.528 | 0.117 | ||
| 1 - 20 | 18.49 ± 1.67 | 16.47 ± 2.47 | ||
| 20 - 45 | 18.47 ± 1.39 | 15.48 ± 2.37 | ||
| > 45 | 18.82 ± 1.10 | 16.64 ± 2.36 | ||
| Hospital ward | 0.295 | 0.095 | ||
| Internal medicine | 18.27 ± 1.59 | 15.50 ± 2.48 | ||
| ICU | 18.93 ± 1.14 | 16.36 ± 2.95 | ||
| Pediatrics | 18.57 ± 1.66 | 17.12 ± 2.90 | ||
| Unspecified | 18.86 ± 0.97 | 16.39 ± 2.28 | ||
| Isolation source | 0.424 | 0.083 | ||
| Blood | 18.74 ± 1.16 | 16.61 ± 2.51 | ||
| Urine | 18.18 ± 1.55 | 15.24 ± 2.32 | ||
| Eye | 19.00 ± 2.27 | 16.13 ± 1.36 | ||
| Tracheal aspirate | 18.63 ± 1.30 | 16.88 ± 1.73 | ||
| Wound/abscess | 19.50 ± 0.71 | 18.50 ± 2.12 | ||
| Catheter | 18.83 ± 0.98 | 17.08 ± 3.23 | ||
| Total | 18.59 ± 1.41 | 16.21 ± 2.43 |
aValues are expressed as mean ± SD.
4.3. Vancomycin MIC Values
The MIC values for vancomycin were determined with the E-test as follows: 0.75 µg/mL in 3 strains (3.03%), 1 µg/mL in 36 strains (36.36%), 1.5 µg/mL in 56 strains (56.57%), and 2 µg/mL in 4 strains (4.04%). In fact, the MIC values for all CoNS were calculated to be ≤ 2 µg/mL, confirming 100% susceptibility of these bacteria to vancomycin. As shown in Table 4, there was a significant difference in vancomycin MIC values among the CoNS species (P = 0.001) and the isolation sources (P = 0.007), while no significant differences were found between MIC values based on patient gender and age or on hospital ward (P > 0.05).
Table 4. Vancomycin MIC Values Among the Coagulase-Negative Staphylococcus Isolates
| Variable | MIC (mean ± SD) | P Value |
|---|---|---|
| Bacterial species | 0.001 | |
| S. epidermidis | 1.48 ± 0.23 | |
| S. haemolyticus | 1.19 ± 0.27 | |
| S. hominis | 1.42 ± 0.29 | |
| S. saprophyticus | 1.19 ± 0.30 | |
| Others | 1.35 ± 0.29 | |
| Patient gender | 0.434 | |
| Female | 1.30 ± 0.30 | |
| Male | 1.36 ± 0.28 | |
| Patient age | 0.425 | |
| 1 - 20 | 1.33 ± 0.26 | |
| 20 - 45 | 1.26 ± 0.35 | |
| > 45 | 1.36 ± 0.26 | |
| Hospital ward | 0.832 | |
| Internal medicine | 1.29 ± 0.32 | |
| ICU | 1.30 ± 0.28 | |
| Pediatrics | 1.36 ± 0.23 | |
| Unspecified | 1.32 ± 0.31 | |
| Isolation source | 0.007 | |
| Blood | 1.39 ± 0.29 | |
| Urine | 1.17 ± 0.26 | |
| Eye | 1.38 ± 0.23 | |
| Tracheal aspirate | 1.50 ± 0.27 | |
| Wound/abscess | 1.50 ± 0.22 | |
| Catheter | 1.25 ± 0.27 | |
| Total | 1.35 ± 0.29 |
5. Discussion
CoNS are considered a major cause of nosocomial infections, mainly occurring in immunocompromised patients and premature newborns, and are mostly associated with healthcare settings. Antibiotic resistance has been a growing issue in the treatment of CoNS infections (23, 27). The current study revealed that S. haemolyticus and S. epidermidis were the most prevalent CoNS from clinical isolates. This result, in line with many previous studies, confirms the predominance of these two species in various clinical samples compared to other CoNS species (28-31). In addition, S. haemolyticus and S. epidermidis have been reported to be among the most frequent CoNS species acquiring resistance to multiple antibiotics, which is in agreement with the results of the current study (3, 8). Indeed, we found S. haemolyticus and S. epidermidis among the strains with higher rates of MDR compared to the other CoNS. Furthermore, nearly consistent with Singh and colleagues (32) and Ghadiri and colleagues (33), 43% and 33% of the CoNS were from blood and urine specimens in our study, among which higher rates of MDR were detected.
Among all CoNS species, S. haemolyticus, S. epidermidis, S. hominis, and S. warneri have more common shown resistance to vancomycin (23). However, in the current study, only one CoNS isolate, belonging to S. hominis, exhibited resistance to vancomycin using the disk diffusion test, which was revealed on PCR not to possess the vanA gene. This could be indicative of the higher sensitivity of the latter method compared to the former, or may be due to technical errors related to either of these methods. Furthermore, 99% of the CoNS demonstrated susceptibility to vancomycin, and this finding is in accordance with previous investigations among other populations (29, 34-36). Although in our study, none of the CoNS showed resistance to teicoplanin, 15% had intermediate resistance to it. Moreover, similar to our findings, resistance to teicoplanin has been more frequently reported than to vancomycin (14, 15). Tacconelli et al. found that among 535 CoNS isolates, 20 (4%) and 1 (0.2%) strains were resistant to teicoplanin and vancomycin, respectively (15). These findings suggest vancomycin as an antibiotic of choice in cases of MDR to non-glycopeptide antibiotics.
The MIC value refers to the minimum concentration of an antibiotic that inhibits the growth of a certain microorganism (37). Vancomycin has been suggested as a treatment of choice for MDR CoNS (38). The CoNS with MIC values of ≤ 1 have been suggested to be highly susceptible to vancomycin, while those presenting with MIC values of ≥ 2 necessitate the use of non-vancomycin alternatives (such as daptomycin) in order to be efficiently eradicated (39-41). The mean MIC value for CoNS isolates in the current study was calculated as 1.35 ± 0.29 µg/mL, establishing sufficient susceptibility to vancomycin. S. haemolyticus and S. saprophyticus had lower MIC values for vancomycin compared to S. epidermidis and S. hominis, and therefore demonstrated a higher susceptibility to this antibiotic. In addition, we found that these MIC values were independent of patient gender and age and of hospital ward, while they significantly differed between CoNS from different specimens.
In conclusion, S. haemolyticus and S. epidermidis were the most prevalent among the CoNS in patients in Gorgan. In addition, it seems that CoNS isolates from Gorgan are fully susceptible to vancomycin, despite presenting a higher resistance to multiple non-glycopeptide antibiotics that are routinely prescribed. Thus, vancomycin can be considered an effective antibiotic of choice for controlling infections caused by CoNS depending on the species, but regardless of patient gender and age, type of disease, isolation source, and hospital ward. However, much more research is required to support this suggestion.
