Research Article
 

Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches



Thongchai Taechowisan, Nutthapol Mungchukeatsakul and Waya S. Phutdhawong
 
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ABSTRACT

Background and Objective: Staphylococcus aureus has long been recognized as one of the most important causes of bacterial disease in humans. This study investigates the prevalence of Staphylococcus aureus in clinical and environmental samples and their susceptibility patterns to antibiotics. Methodology: A total of 69 samples, 36 clinical specimens obtained from patients and 33 environmental samples from hospital facilities, were screened for S. aureus using standard microbiological and biochemical methods and PCR-based assay. Isolates resistant to both cefoxitin and oxacillin were considered to be Methicillin Resistant S. aureus (MRSA). Results: Of the two categories of samples screened, 31 (86.1%) and 23 (69.7%), respectively tested positive for S. aureus. The highest prevalence of MRSA from clinical samples (28.6%) was found in sputum and wounds and the highest (25%) from environmental samples was found in corridor, door handle and patient bed samples. Conclusion: In suggesting that healthcare personnel and hospital environments serve as potential reservoirs of S. aureus, these findings have practical, clinical and epidemiological importance. Ten of 16 of multi-drug resistant S. aureus isolates were MRSA, suggesting a correlation between the results of PCR patterns and their phenotypic multi-drug resistance testing.

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Thongchai Taechowisan, Nutthapol Mungchukeatsakul and Waya S. Phutdhawong, 2018. Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches. Research Journal of Microbiology, 13: 100-118.

URL: https://scialert.net/abstract/?doi=jm.2018.100.118
 

INTRODUCTION

Staphylococcus aureus has long been recognized as one of the most important causes of bacterial disease in humans. It is the leading cause of skin and soft tissue infections such as abscesses, furuncles and cellulitis and causes serious infections such as bloodstream infections, pneumonia, or bone and joint infections1. The continuing emergence of methicillin resistant S. aureus (MRSA), including hospital acquired (HA)-MRSA and community acquired (CA)-MRSA, is a major and increasing threat to public health. Most MRSA isolates are resistant to multiple antibiotics and effective antibiotic treatment of MRSA infections are consequently limited2. Clinically, infections caused by HA-MRSA strains are also associated with higher mortality and morbidity3 and some CA-MRSA strains express additional virulence factors that enable them to cause more serious diseases4.

Environmental surfaces in communities carry the least risk of disease transmission and can be safely decontaminated using less rigorous methods than those used on medical instruments and devices, especially in public hospital areas. Microbiologically contaminated surfaces can serve as reservoirs of potential pathogens. Hospital environments play an important role in nosocomial infection in that healthcare environments contain a diverse population of microorganisms. Transfer of microorganisms from environment surfaces to hosts can occur indirectly, for example, by hand contact with other surfaces5. Jalalpoor has also shown that 54.7% of S. aureus isolates among hospital environment specimens were antibiotic resistant strains6. This is unacceptably high.

The aims of this study were threefold. Firstly, it aimed to determine the prevalence and antibiotic susceptibility of S. aureus and MRSA in clinical and environmental specimens. It also aimed to determine the antibiotic susceptibility levels of isolated S. aureus and to evaluate the antimicrobial resistance profiles of S. aureus strains isolated from clinical and environmental specimens. Finally, it sought to compare the phenotypic and genotypic methods for detection of S. aureus and suggests how the research findings may suggest practical imperatives for infection reduction and control by healthcare professionals.

MATERIALS AND METHODS

Ethics statement: The study was approved by the Committee of the Scientific Study of Humane Technique in Laboratory Animal Experiments and Human Ethics, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand. No written informed consent was required because all the patients were anonymous and no other personal information was used in this study.

Bacterial isolates: From May to August, 2017, a total of 36 specimens from blood, sputum, pus, wounds, urine and nasopharyngeal swab samples were collected from patients admitted to Nakorn Pathom Hospital and 33 swab samples were also collected from door handles, stair railings, stair floors, corridors, toilet floors and patient beds at the same hospital. Staphylococcus aureus isolates were identified in 31 (86.1%) of the clinical specimens obtained from patients and in 23 (69.7%) of the environmental samples from hospital facilities, isolates were examined by conventional methods such as colony morphology on blood agar and mannitol salt agar, Gram stain, catalase production and mannitol utilization and coagulase test. Identified strains were stored at -20°C in Nutrient broth (Oxoid) containing 20% glycerol.

Determination of methicillin resistance: Methicillin resistance was evaluated using three methods: (1) A disk diffusion test using 30 μg cefoxitin disk (<21 mm indicated MRSA), (2) An oxacillin MIC (Minimum Inhibitory Concentration) test (>4 μg mL1 indicated MRSA) and (3) A polymerase chain reaction (PCR) for the detection of mecA gene (positive indicated MRSA)7. Antibiotic disks and Oxacillin powder were obtained from Himedia (Himedia Laboratories, Pvt. Ltd., Mumbai, India). All tests were compared for sensitivity and specificity with PCR for mecA gene as a reference method. Sensitivity was calculated by dividing the number of mecA-positive isolates detected as resistant using phenotypic methods by the total number of mecA-positive strains (ether susceptible or resistant). Specificity was calculated through dividing the number of mecA-negative isolates classified as sensitive based on phenotypic criteria by the total number of mecA-negative isolates8.

Antibiotic sensitivity test: Antibiotic susceptibility of the bacteria isolates was assayed according to the Kirby-Bauer disk diffusion method9. All the plates were incubated for 20 min before inoculation and placement of antibiotic discs to allow excess moisture to dry. After drying, a single loop of each isolate was inoculated into sterile normal saline and compared with the 0.5 McFarland standard. The suspension was aseptically swabbed on the surface of Mueller-Hinton plates and antibiotic sensitivity disks that contained penicillin (10 units), cefoxitin (30 μg), chloramphenicol (30 μg), tetracycline (30 μg), erythromycin (15 μg), trimethoprim/sulfamethoxazole (25 μg), gentamicin (10 μg), clindamycin (2 μg), rifampicin (30 μg), linezolid (30 μg) and ciprofloxacin (25 μg). The MICs of oxacillin and vancomycin in both MRSA and MSSA isolates were determined by the broth dilution method. All procedures were carried out and interpreted according to Clinical and Laboratory Standards Institute guidelines (CLSI)10. Staphylococcus aureus ATCC25923 was used as a control strain in both the disk diffusion and broth dilution methods.

PCR amplification: Total bacterial DNA was extracted from S. aureus using a modified phenol-chloroform method. Briefly, S. aureus was cultured on 5 mL brain heart infusion broth and incubated at 37°C for 24 h. Afterwards, 1.5 mL of the culture was centrifuged for 5 min at a velocity of 14,000 rpm. Each pellet was resuspended in 600 μL Tris-EDTA buffer by repeated pipetting or vortexing. Then 3 μL of 10% SDS and 3 μL of 20 mg mL1 proteinase K were added, mixed and incubated for 30 min at 65°C in a water bath, 600 μL of phenol/chloroform/isoamyl alcohol was added, mixed, centrifuged for 5 min at 14,000 rpm and the supernatant was transferred to a fresh tube. An equal volume of ethanol was then added and mixed gently until DNA precipitated. Each pellet was then centrifuged for 5 min and the supernatant was discarded. The pellet was washed with 1 mL of 70% ethanol, mixed, centrifuged for 5 min and the supernatant was discarded and then dried for 10 min at a velocity vac/45°C. Finally, it was resuspended in 30 μL TE buffer. The DNA concentration was read using 2 μL in a Nanodrop machine using TE buffer as blank. The concentration was made up to 100 ng mL1 in each sample and stored at -20°C.

Primers used for detection of the femA gene were primers FemA1 and FemA2, leading to an S. aureus-specific 450 bp PCR product11. The mecA gene was detected with the primers MecA1 and MecA2, yielding a 519 bp PCR product for methicillin and oxacillin resistance12. The aac(6')/aph(2'') gene was detected with the primers aac(6')/aph(2'')1 and aac(6')/aph(2'')2, yielding a 407-bp PCR product for gentamicin resistance13. The blaZ gene was detected with the primers blaZ1 and blaZ2, yielding a 774 bp PCR product for penicillin resistance12. The ermA gene was detected with the primers ermA1 and ermA2, yielding a 190 bp PCR product for erythromycin resistance. The tetK and tetM genes were detected with the primers tetK1 and tetK2, yielding a 360 bp PCR product and primers tetM1 and tetM2, yielding a 158 bp PCR product, respectively14. The following reaction mixture was added to each sample: 2 μL DNA (100 ng), 2 μL primer (100 pmol), PCR mixture (1.5 μL MgSO4, 2.5 μL 10xPCR buffer, 0.5 μL dNTPs, 0.2 μL Taq polymerase) and completed to 25 μL volume by H2O. The primer sequences and the PCR conditions were showed in Table 1. For the visualization of the product, 10 μL of the each PCR reaction was mixed with 5 μL 6x loading dye and loaded on 1.5% agarose gel for electrophoresis and visualization of the amplified PCR products. A 100 bp molecular weight DNA ladder was used to validate the length of the amplified products (Vivantis Technologies).

Table 1: Primer sequences and PCR conditions used
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches

RESULTS

A total of 54 S. aureus isolates including 31 isolates from clinical specimens and 23 isolates from environmental specimens were cultured on nutrient agar (NA) and mannitol salt agar (MSA) medium. All isolates showed yellow-colonies on NA medium. They fermented mannitol in MSA medium and produced yellow-colored colonies surrounded by yellow. These isolates were identified as Gram positive cocci in grape like clusters under microscope with coagulase, catalase and β-hemolysis positive. The PCR amplification of femA gene was done for all isolates to detect the species of S. aureus. In this study the PCR product appeared as a single band DNA with a size equal to 450 bp fragment corresponding to the femA amplicon (Fig. 1).

A total of 31 S. aureus isolates including 7 (22.6%) MRSA and 24 (77.4%) MSSA were isolated from different clinical specimens. The prevalence of MRSA was higher in sputum (28.6%) and wound (28.6%) than other specimens (Table 2). As the similar results in MSSA, the majority of isolates were isolated from sputum specimens (n = 11, 45.8%). A total of 23 S. aureus isolates including 8 (34.8%) MRSA and 15 (65.2%) MSSA were isolated from different environmental specimens. The prevalence of MRSA was higher in corridor (25%), door handle (25%) and patient bed (25%) than other specimens (Table 3). As the similar results in MSSA, the majority of isolates were isolated from corridor (20%), patient bed (20%) and stair railing (20%).

Table 2: Prevalence of S. aureus among clinical specimens
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches

Table 3: Prevalence of S. aureus among environmental specimens
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches

Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
Fig. 1:
Amplicon of femA gene; lane M: 100 bp DNA marker; Lane 1: S. aureus ATCC25923 (positive control), Lane 2-6 are tested isolates with positively amplified femA as indicated by 450 bp PCR amplicon, Lane 7 is femA negative (Escherichia coli ATCC 25922)

Table 4: Frequency and range of oxacillin and vancomycin MICs of S. aureus (MRSA and MSSA) isolated from clinical specimens by broth dilution method
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches

Table 5: Frequency and range of oxacillin and vancomycin MICs of S. aureus (MRSA and MSSA) isolated from environmental specimens by broth dilution metho
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches

The MICs for oxacillin and vancomycin from the clinical isolates are listed in Table 4. The MICs for oxacillin were between 0.5 to >64 μg mL1 for MRSA and 0.125-1 μg mL1 (except only one isolate was >64 μg mL1) for MSSA isolates. The MICs for vancomycin were between 0.5-2 μg mL1 for MRSA and 0.25-0.5 μg mL1 (except 2 isolates were 1-2 μg mL1) for MSSA isolates.

For the MICs for oxacillin and vancomycin from the environmental isolates are listed in Table 5. The MICs for oxacillin were between 0.25 to >64 μg mL1 for MRSA and 0.125-32 μg mL1 (except only one isolate was 64 μg mL1) for MSSA isolates. The MICs for vancomycin were between 0.25-1 μg mL1 for MRSA and 0.25-2 μg mL1 for MSSA isolates. Since no isolate had the MIC value for vancomycin greater than 2 μg mL1 that mean these isolates did not fall into vancomycin resistant category according to CLSI.

Table 6 and 7 represent the resistance pattern of S. aureus isolates (MRSA and MSSA) from clinical specimens and environmental specimens to the tested antibiotics, respectively. In this study the entire S. aureus isolates were susceptible to vancomycin, chloramphenicol, linezolid and rifampicin for the clinical specimens and vancomycin, chloramphenicol, gentamicin, linezolid, ciprofloxacin and rifampicin for the environmental specimens. Among other antibiotics trimethoprim/sulfamethoxazole showed to be the most effective antibiotics against MSSA isolates. Seven of MRSA isolates (100%) and 23 of the MSSA isolates from clinical specimens were resistant to penicillin, while seven of MRSA isolates (87.5%) and 8 of MSSA isolates from environmental specimens were resistant to penicillin. The PCR testing revealed the presence of mecA gene in all isolates (Fig. 2) which were determined as methicillin resistant by the phenotypic methods. The sensitivity of oxacillin MIC test and cefoxitin disk diffusion test was 53.3%, whereas, the sensitivity of penicillin disk diffusion test was 93.3% and the specificity of oxacillin MIC test and cefoxitin disk diffusion test was 12.8% and 15.4%, respectively, whereas the specificity of penicillin disk diffusion test was 79.5% (Table 8).

From the 54 positive S. aureus isolates, 16 (29.6%) were multi-drug resistant (resistant to three or more antibiotics), 12 (22.2%) were resistant to only two antibiotics, 21 (38.9%) were resistant to only one antibiotic and the remaining five (9.3%) showed no resistance to any of the antibiotics. Nine (56.25%) and 7 (43.75%) out of the sixteen multi-drug resistant S. aureus isolates were from clinical and environmental specimens, respectively.

Table 6: Antibiotic susceptibility profiles of S. aureus strains isolated from clinical specimens by disk diffusion method
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aOxacillin and Vancomycin susceptibility profiles were determined using broth dilution method

Table 7: Antibiotic susceptibility profiles of S. aureus strains isolated from environmental specimens by disk diffusion method
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
aOxacillin and Vancomycin susceptibility profiles were determined using broth dilution method

Table 8: Determination of methicillin resistant sensitivities of clinical and environmental isolates
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
aOxacillin susceptibility test was determined using broth dilution method, bCefoxitin and Penicillin susceptibility test was determined using disk diffusion method

Nine (75.0%) of those resistant to only two antibiotics were from clinical specimens while the remaining three (25.0%) were from environmental specimens. Twelve (57.1%) of the isolates resistant to only one antibiotic were from clinical specimens, nine (42.9%) were from environmental specimens. Out of the five that were not resistant to any antibiotic, only one (20.0%) was from clinical specimen and four (80.0%) were from environmental specimens (Table 9). Six (66.7%) of multi-drug resistant S. aureus from clinical specimens were MRSA and other three (33.3%) were MSSA, while four (57.1%) of multi-drug resistant S. aureus from environmental specimens were MRSA and other three (42.9%) were MSSA (Table 9). Of the total 16 (29.6%) multi-drug resistant isolates, two (12.5%) were resistant to six antibiotics, five (31.25%) were resistant to five and four antibiotics and the remaining isolates, four (25.0%) were resistant to three antibiotics.

Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
Fig. 2:
Amplicon of blaZ and mecA genes by Multiplex-PCR; M: 100 bp DNA marker, Lane 1-6: blaZ (774 bp), Lane 2, 3 and 5: mecA (519 bp), Lane 7: S. aureus ATCC25923 (negative control)

Table 9: Antibiotic resistant pattern of S. aureus isolates from clinical and environmental specimens
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
aPEN: Penicillin, ERY: Erythromycin, TE: Tetracycline, FOX: Cefoxitin, GEN: Gentamicin, CIP: Ciprofloxacin, SXT: Trimethoprim-sulfamethoxazole, OXA: Oxacillin, DA: Clindamycin. bsp: Sputum, pu: Pus, bl: Blood, wo: Wound, co: Corridor, tf: Toilet floor, sf: Stair floor, sr: Stair railing

Among the multi-drug resistant MRSA from clinical specimens were from sputum, two isolates (33.3%), wound, two isolates (33.3%); pus, one isolate (16.7%) and blood, one isolate (16.7%), while the multi-drug resistant MSSA from clinical specimens were from sputum, one isolate (33.3%); wound, one isolate (33.3%) and blood, one isolate (33.3%). And among the multi-drug resistant MRSA from environmental specimens were from corridor, two isolates (50.0%); toilet floor, one isolate (25.0%); and stair railing, one isolate (25.0%), while the multi-drug resistant MSSA from environmental specimens were from corridor, one isolates (33.3%); toilet floor, one isolates (33.3%); and stair floor, one isolate (33.3%).

Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
Fig. 3: Amplicon of aac(6)/aph(2) gene, Lane M: 100 bp DNA marker, Lane 1, 2, 5 and 6: aac(6)/aph(2) (407 bp), Lane 7: S. aureus ATCC25923 (negative control)

Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
Fig. 4:
Amplicon of ermA, tetK tetM genes by Multiplex-PCR, M: 100 bp DNA marker, Lane 1, 2, 4 and 6: ermA (190 bp), Lane 1, 4, 5 and 6: tetK (360 bp), Lane 1, 3 and 6: tetM (158 bp), lane 7: S. aureus ATCC25923 (negative control)

Of the six multi-drug resistant MRSA isolates from clinical specimens, the four that were resistant to five to six antibiotics were from sputum, 2 isolates (33.3%); pus, 1 isolate (16.7%) and wound, 1 isolate (16.7%), while the total four isolates of multi-drug resistant MRSA from environmental specimens, only one isolate (25.0%) that was resistant to five antibiotics was from toilet floor (Table 9). For 16 phenotypic multi-drug resistance S. aureus isolates determined by disk diffusion and broth microdilution assay, we compared with the results of PCR-based assay for the simultaneous detection of antibiotic resistance genes (Fig. 1-4). These isolates, we found a correlation between the results of the PCR patterns and those of classical resistance testing (Table 10).

Table 10: Correlation between multi-drug resistant pattern of S. aureus isolates and PCR results
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
aSw: Swab specimen from environmental samples, Sp: Specimen from clinical samples, bPEN: Penicillin, ERY: Erythromycin, TE: Tetracycline, FOX: Cefoxitin, GEN: Gentamicin, CIP: Ciprofloxacin, SXT: Trimethoprim-sulfamethoxazole, OXA: Oxacillin, DA: Clindamycin

Table 11: Correlation between phenotypic cefoxitin and oxacillin susceptibility tests and PCR results of mecA gene
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
R: Resistant, S: Susceptible

Table 12: Correlation between phenotypic penicillin susceptibility test and PCR results of blaZ gene
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
R: Resistant, S: Susceptible

Correlation between phenotypic antibiotic resistance and PCR results. For 54 S. aureus isolates, we compared susceptibility results determined by disk diffusion and broth microdilution assay with the results of PCR-based assay for the simultaneous detection of antibiotic resistance genes. Of 13 oxacillin-resistant isolates, 8 isolates carried a mecA gene, while 14 cefoxitin-resistant isolates, 8 isolates carried a mecA gene. And of 41 oxacillin-sensitive isolates, 7 isolates carried a mecA gene, while 40 cefoxitin-sensitive isolates, 7 isolates carried a mecA gene (Table 11). All penicillin-resistant isolates were shown to have the blaZ gene, while 2 of 9 penicillin-sensitive isolates were shown to have this gene (Table 12). A total of 16 isolates were resistant to erythromycin, 14 of these isolates, the ermA gene was present, while 5 of 38 erythromycin-sensitive isolates were shown to have this gene (Table 13). All two gentamicin-resistant isolates were shown to have the aac(6')/aph(2'') gene, while 5 of 52 gentamicin-sensitive isolates were shown to have this gene (Table 14). A total of 7 isolates were resistant to tetracycline, 6 and 5 isolates carried tetK gene and tetM gene, respectively, while a total of 47 isolates were sensitive to tetracycline, 5 and 4 isolates were carried tetK gene and tetM gene, respectively (Table 15). Thus, the results of the PCR-based assay did not completely correlate with the results of the phenotypic antibiotic resistance determination.

Table 13: Correlation between phenotypic erythromycin susceptibility test and PCR results of ermA gene
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
R: Resistant, S: Susceptible

Table 14: Correlation between phenotypic gentamicin susceptibility test and PCR results of aac(6')/aph(2'') gene
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
R: Resistant, S: Susceptible

Table 15: Correlation between phenotypic tetracycline susceptibility test and PCR results of tetK and tetM genes
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
R: Resistant, S: Susceptible

Table 16: Cefoxitin and oxacillin susceptibility test for nmrMSSA, mrMSSA, nmrMRSA and mrMRSA
Image for - Antimicrobial Resistance Pattern of Staphylococcus aureus Strains Isolated from Clinical and Hospital Environment specimens and Their Correlation with PCR-based Approaches
R: Resistant, S: Susceptible

Correlation of oxacillin and cefoxitin susceptibility results and multi-drug and nonmulti-drug resistant S. aureus is presented in Table 16. Most of nonmulti-drug resistant MSSA (nmrMSSA) isolates were sensitive to both oxacillin and cefoxitin (87.7%), three isolates (9.1%) were sensitive to oxacillin but resistant to cefoxitin and only one isolates (3.0%) was resistant to both oxacillin and cefoxitin, while most of multi-drug resistant MSSA (mrMSSA) isolates were resistant to both oxacillin and cefoxitin (50.0%), two isolates were sensitive to both oxacillin and cefoxitin (33.3%) and only one isolate (16.7%) was resistant to oxacillin but sensitive to cefoxitin. These results were similar to those MRSA isolates which most nonmulti-drug resistant MRSA (nmrMRSA) isolates were sensitive to both oxacillin and cefoxitin (80.0%), only one isolate was sensitive to oxacillin but resistant to cefoxitin (20.0%), while most of multi-drug resistant MRSA (mrMRSA) isolates were resistant to both oxacillin and cefoxitin (70.0%), two isolates were sensitive to both oxacillin and cefoxitin (20.0%) and only one isolate (10.0%) was resistant to oxacillin but sensitive to cefoxitin.

DISCUSSION

Staphylococcus aureus is major causes of community-acquired and nosocomial infection, it has spread throughout the world and has become highly endemic in many geographical areas15. This study was carried out to determine the prevalence and antibiotic resistance of S. aureus isolated from clinical and hospital environmental samples. In this study the results of bacterial culture for a total 54 isolates on BA and MSA revealed S. aureus with all isolates appeared as Gram-positive. The frequency of femA in S. aureus was 100%, but neither of this gene was found in other bacteria. Thus, there was 100% agreement between the conventional identification results and the amplification of the 450 bp fragment of the species-specific gene, femA. The gene product of femA has been suggested to have a role in cell wall metabolism and is reported to be present in all S. aureus species during the active growth phase16. There was perfect correlation between the conventional phenotypic tests and molecular technique results for identification of S. aureus17. The prevalence of S. aureus isolation from clinical and hospital environmental samples was 86.1 and 69.7%, respectively. The prevalence of S. aureus isolation from hospital environment in this study is higher than those of Saba et al.18 for the public hospitals in Ghana where the S. aureus prevalence was 39%, Newman19 for a hospital in Accra, Ghana was 44% and Hammuel et al.20 for a hospital in Zaria, Nigeria was 50.8%. The highest frequency of MRSA isolation from clinical specimens was noted from sputum and wound samples (28.6%). This study’s findings are in agreement with a study done in Windhoek, Namibia which found from sputum (41.3%) and pus swab (35.0%)21. Contamination of corridors, door handles and patient beds with MRSA occurred more than the other of the surface samples. Although routine cleaning procedures are undertaken in these areas, they are not completely effective and improved methods of disinfecting these environments are recommended22. During the course of this study, it was observed that more attention was paid to cleaning floors rather than corridors, door handles or knobs and patient beds, which may have allowed a build-up of S. aureus on these surfaces. Subsequently, the high contamination rate of door handles and surfaces of patient bed by S. aureus in hospitals is likely to be a contributing factor for these bacteria being implicated in blood stream infections23,24. Preventive measures such as improved personal hygiene and the regular cleaning and disinfection of hospital corridors, door handles, patient beds, stair railings, floors and other points of contact are highly recommended, which recorded the highest S. aureus contamination rate. The MRSA prevalence of this study are similar to those of Carvalho et al.25 for a Brazilian university hospital which was 33.3% (versus 34.8% in our study). In a similar study by Oie et al.26 for 27% (versus 17.4% in this study) of the door handles in a University hospital in Japan were contaminated with S. aureus of which 20.9% (versus 65.2% in this study) were MSSA and 8.7% (34.8% in this study) were MRSA. In this study, oxacillin susceptibility by broth dilution method and cefoxitin disk diffusion test could not detect all MRSA isolates. The oxacillin and cefoxitin susceptible mecA-positive S. aureus are on the rise. Because of their susceptibility to oxacillin and cefoxitin, it is very difficult to detect them by using phenotypic methods. This finding is in agreement with Chambers27, who found that among the MRSA isolates, only few express homogeneous oxacillin resistance while the majority show heterogeneous drug resistance. Phenotypically oxacillin and cefoxitin susceptible and mecA-positive S. aureus clinical isolates are being increasingly reported28-35. Dependence on growth conditions like temperature and osmolarity of the medium for phenotypic expression of resistance further complicates susceptibility testing of MRSA by standard microbiological methods27. On the basis of the Clinical and Laboratory Standards Institute (CLSI) guidelines, a method based on agar containing 6 μg mL1 of oxacillin was developed to screen S. aureus isolates36. Though it can detect true MRSA effectively, it is likely to miss mecA-positive S. aureus having an oxacillin MIC of <2 μg mL1. Such isolates have been considered to be extremely hetero resistant (<1 in 108 of the population is highly resistant to methicillin), but there are also reports documenting the existence of nonheterogeneous phenotypically oxacillin-susceptible mecA-positive S. aureus31. The use of β-lactams to treat such isolates may cause an increase in the MIC of oxacillin well above the established breakpoint for resistance (oxacillin MIC, >4 μg mL1), ultimately leading to failure of therapy37. Using PCR for mecA gene detection for MRSA as reference method, the sensitivity for disk diffusion test using cefoxitin and oxacillin MIC test was lower than penicillin disk diffusion test. These results are difference to those previous reports by Dibah et al.38 and Farahani et al.39 that cefoxitin disk diffusion and oxacillin MIC tests showed the sensitivity equal to PCR for MRSA detection. However, the emergence of mecA-positive oxacillin susceptible and mecA-negative oxacillin resistant S. aureus strains reduces the sensitivities of both the phenotypic and genotypic methods 30,40,41. Thus, combination of genotypic and phenotypic tests is necessary to detect the methicillin resistance in S. aureus accurately. In this study, the incidence rate of MRSA detection in clinical and environmental specimens was similar, with an estimated prevalence 22.6 and 34.8%, respectively. Previously, it has been documented that MRSA accounted for 25.5% of total isolates of CA S. aureus infections and 67.4% of HA infections in Asian countries, whereas, 2.5% of CA S. aureus and 57.0% of HA infections in Thailand42. However, recent reports indicate declining clinical acquired MRSA infection with applying appropriate infection control measures, rapid and reliable detection of methicillin resistance and effective antibiotic therapy43,44. In this study, all isolates were susceptible to vancomycin, chloramphenicol, linezolid and rifampicin. The absence of resistance to these antibiotics may be related to the low usage of these antibiotics in the study setting. The vancomycin is the drug of choice for the treatment of infections due to MRSA45. Several studies reported emergence of vancomycin resistant clinical MRSA isolates around the world46-48. In our study all of the isolates displayed MICs of <2 μg mL1 to vancomycin and were susceptible to vancomycin. This is in agreement with previous studies22,49. And all isolates of S. aureus were sensitive to rifampicin and the rifampicin was an important antibiotic in treatment S. aureus infection and tuberculosis50,51. We speculate that the effectiveness observed with the drug might be due to its high cost in our environment making, it less readily available and hence less misused. Most of the reports suggested vancomycin as a credible drug for treating S. aureus infection52,53. However, regular monitoring of the drug's sensitivity is of importance because resistance has been reported in the USA, Japan and Korea49,54. The other reports indicated that most clinical samples of S. aureus were resistant to vancomycin55,56, while the 28.57% of MRSA from clinical samples were rifampicin-resistant57. Multi-drug resistant characteristics of MRSA and emergence of glycopeptide resistant strains have been frequently caused treatment failure of MRSA infections58. These findings have promoted researchers to seek new antibiotics for the treatment of MRSA infection59. Linezolid showed good activity in vitro and in vivo and are promising therapeutic options against staphylococcal infections60. In this study all isolates were susceptible to linezolid. Similar to those previous study on S. aureus strains isolated from clinical specimens in Aralabil, Iran by Dibah et al.38, that all of MRSA and MSSA isolates were found to be susceptible to linezolid. This antibiotic is not generally used in Thailand for treatment of staphylococcal infections. Therefore, emergence of linezolid susceptible S. aureus strains in this finding could be reasoning. There is also a similar finding that has been reported from Iran38. In this study, 0% of the tested isolates were resistant to chloramphenicol which is comparable to China and Iran where 0.8 and 0% of the isolates were resistant, respectively61,62. The studies carried out in European countries have revealed that in Greece 100% of the MRSA isolates from community acquired infections were susceptible to chloramphenicol63 while in UK almost 92.3% of such isolates recovered from patients of otitis externa were sensitive to this antimicrobial64. The results from studies carried out in Japan and Korea have also revealed similar pattern as 91.6 and 100% of MRSA isolates were susceptible to this compound, respectively65,66. The published literature from USA has revealed that although in vitro efficacy of chloramphenicol against MRSA is still very encouraging but there is declining trend noted from different regions of the country67,68. Only one study was found from African country of Uganda where 88.2% of the isolates were susceptible69. The most significant finding of our results was 100% of the isolates susceptible to chloramphenicol. This result could be attributed to the fact that chloramphenicol is not being routinely used in clinical practice to treat majority of infections caused by Gram positive and Gram negative bacteria. Bone marrow toxicity is the major complication of chloramphenicol. This side effect may occur as either due to dose related bone marrow suppression or idiosyncratic aplastic anaemia. Keeping in view the low cost and oral preparation of chloramphenicol coupled with very high rate of in vitro susceptibility makes this antimicrobial an ideal choice for wide variety of infections caused by MRSA. Further studies focusing more on the clinical outcome of patients of MRSA treated with chloramphenicol would definitely give icing on cake for in vitro results achieved for this compound. It is also imperative that since this compound has shown very promising results against MRSA isolates, the availability of this antibiotic must be ensured in the market for the benefit of patients.

The high susceptibility rate for most commonly used antibiotics was observed among MSSA isolates in comparison to MRSA. Most MSSA isolates were susceptible to nearly all antimicrobial agents used in this study. In contrast, in the case of MRSA, multiple-drug resistance was common and only few antibiotics were active against these isolates. Except for above mentioned antibiotics, ciprofloxacin showed the lowest resistance rate for MRSA isolates, tetracycline and oxacillin showed the lowest resistance rate for MSSA isolates. However, MRSA strains from clinical specimens should be considered as resistant to all β-lactam agents other than the cephalosporins with anti-MRSA activity as stated by CLSI10. Resistance of MRSA to ciprofloxacin in general is low. According to a study published by Zabielinski et al.70, MRSA became more sensitive to ciprofloxacin, while MSSA demonstrated increased antibiotic resistance to ciprofloxacin.

The pattern of resistance observed might be due to the fact that antibiotics are used in auto-therapy in this locality, which may result in a multitude of antibiotics used at sub-therapeutic levels heralding the emergence of resistant strains. Antimicrobial resistance patterns revealed a total of twenty-one phenotype patterns of which the most prevalent was penicillin which accounted for 19 (35.2%) of the isolates. The second resistance pattern was noted for the resistance indices designated pattern 3 (which penicillin and erythromycin) exhibited by 9 (16.6%) of the isolates. Approximately 16(29.6%) of the isolates were resistant to three or more antibiotics (multi-drug resistant). The majority of these multi-drug resistant isolates originated from clinical samples which was 9 (56.25%) and most of them were MRSA, while 7 (43.75%) of the isolates were of environmental samples. The multi-drug resistant strain may occur when the uncontrolled disposing of antibiotics and chemicals into the hospital, which turns to create selective pressure on the drugs. Pellegrino et al.71 reported that the uncontrolled use of antibiotics in hospital and the community created a reservoir of bacteria that could become resistant. Therefore, we are constrained to hypothesize that this situation may be a reason for the multi-drug resistance MRSA was observed in this study.

Resistance determinants may be included the clinical and technical considerations. Since the PCR amplification of the gene fragments is limited, we chose genes most frequently associated with resistance of S. aureus to clinically relevant anitbiotics72-75.

In this study, all of the isolates were identified as S. aureus based on morphological and biochemical characteristics and all S. aureus isolates were positive by PCR for the species specific femA gene. Methicillin resistance in S. aureus has been reported to be associated with the presence of penicillin-binding proteins, encoded by the mecA gene. The heterogeneous expression of mecA gene may not be the completed and, therefore, these strains may not be detectable with phenotypical methods76-79. Although, methicillin resistance was observed in 27.8% of total isolates when tested by oxacillin and vancomycin MIC methods which was equal to 27.8% of total isolates had mecA gene by PCR assay. Phenotypically methicillin susceptible 7 isolates also carried the mecA gene. Comparison of conventional method and PCR assay did not show a good agreement. Additionally, the absence of mecA gene within resistant staphylococcal isolates was presented in the various studies80-82. Furthermore, moderate methicillin resistance was observed in isolates that lacked the mecA gene mutation83,84. A previous report in Nigeria found that the complete absence of five major SCCmec types, mecA gene and the gene product of PBP2a in the isolates of MRSA, suggested that a probability of hyper production of β-lactamase as a cause of the phenomenon85. Moreover, Ba et al.86 pointed out specific mutations of amino acids in penicillin binding proteins (PBP1, 2 and 3) which may play a role in antibiotic resistance. These mutations were found to include three amino acid substitutions which were identical and were present in PBPs 1, 2 and 386. In additionally, the same amino acid was found to have two other different substitutions in PBP1. Both the identical and different amino acid substitutions were observed in isolates from different multilocus types. These findings distributed that there are other mechanisms responsible for β-lactam resistance of MRSA than the presence of mecA gene that molecular methods alone are not enough for confirmed characterization of MRSA isolates.

The blaZ gene encoding β-lactamase that inactivates the β-lactam antibiotics by hydrolysis of the β-lactam ring87. In this study, all the penicillin-resistant S. aureus isolates exhibited genotypic resistance to penicillin, but in various studies, no resistance genes could be detected in some resistant isolates88-90.

In some isolates, phenotypic resistance may be caused by point mutations rather than gene acquisition. Additionally, except for the general resistance mechanisms91,other pathways such as biofilm formation may play a major role in the resistance mechanisms92. These results suggest that the blaZ gene carried by penicillin-resistant S. aureus may play a major role in the penicillin-resistant phenotype, but the resistance gene cannot be used alone as a diagnostic indicator for penicillin resistance. Mechanisms of resistance to antibacterials are so complex that the presence or absence of a certain resistance gene does no certainly indicate that the particular isolate is resistant or sensitive to the corresponding antimicrobial agent93.

The aac(6')/aph(2'') gene encodes the AAC(6')-APH(2'') enzyme, a bifunctional enzyme with kinase activity, that inactivates a broad range of aminoglycosides and confers concomitant resistance to gentamicin and the majority of aminoglycosides commonly used in medical practice94,95. As also reported in other studies, the aac(6')/aph(2'') gene was the most common amino glycoside modifying enzyme (AME) genes among the S. aureus isolates and was found either alone or with other AME for example; aph(2'')-Ib, aph(2'')-Ic, aph(2'')-Id, aph(3')-IIIa, ant(4')-Ia and ant(6)-Ia genes95,96. In this study, drug-susceptibility testing showed rates of gentamicin-resistant S. aureus strains of 3.7% (all staphylococcal strains resistant to gentamicin in the present study carried the aac(6')/aph(2'') gene), surprisingly, this gene was detected in five gentamicin susceptible isolates. In some studies have also reported similar findings78,97,98. The detection of resistance genes in antibiotic susceptible isolates may be due to the amplification of repressed antibiotic resistance gene97 or AME of these strains display lower enzymatic activity, detected also in other study of S. aureus78. Vanhoof et al.78 also showed that the aac(6')/aph(2'') gene is the most prevalent gene encoding AME enzymes among clinical MRSA isolates in European countries and Choi et al.77 obtained similar results in South Korea. They present study's results were similar to the predominant gene. In this case, 100% of gentamicin-resistant S. aureus isolates carried the aac(6')/aph(2'') gene.

Erythromycin resistance in staphylococci is predominantly mediated by erythromycin resistance methylase encoded by erm genes99. In human infections caused by staphylococci ermA and ermC are the most common methylase genes100. In the present study, the incidence of ermA was 35.2% for S. aureus. A similar incidence for ermA has been reported for S. aureus isolated from various sites and clinical specimens56 and 87.5% of erythromycin-resistant S. aureus from the present study carried ermA. Similar results have also observed high incidences (82-94%) for ermA in erythromycin-resistant S. aureus isolated from blood100,101. However, two of erythromycin-resistant S. aureus strains were absent ermA gene which have the other erm or msrA genes99,102,103. Lim et al.104 reported that the ermA gene was more prevalent than the other erythromycin resistance genes in S. aureus isolates and ermC gene was found mostly in coagulase-negative staphylococci (CoNS)104. Similarly, in a study performed by Martineau et al. 32 the ermC gene has been reported to be more prevalent in CoNS.

Tetracycline resistance is the common resistance phenotype in MRSA strains isolated worldwide. Two main mechanisms of resistance to tetracycline have been described in S. aureus; active efflux, resulting from the acquisition of the plasmid-located tetK and tetL genes and ribosomal protection by elongation factor-like proteins that are encoded by chromosomal or transposonal tetM or tetO determinants105,106. In the present study, the phenotypic resistance to tetracycline in S. aureus was observed as 12.9%. Whereas 9.3% S. aureus isolates carried the tetK and tetM genes. Tetracycline resistance genes tetK and tetM were found positive by PCR method in four isolates which were phenotypically sensitive to tetracycline. A majority of tetracycline-resistant strains harboured tetK gene followed by tetM gene. This is similar to those reports by Trzcinski et al.107, Jones et al.108 and El-Mahdy et al.109 where tetK gene was the predominant gene in tetracycline-resistant strains. In addition, this study showed that a strain which harboured tetK gene also harboured tetM gene. This is in agreement with the report of Schmitz et al.74 as their MRSA strains also harbour both tetK and tetM genes.

CONCLUSION

This is the first study conducted on S. aureus on clinical and hospital environment samples in Thailand. There were high levels of contamination of S. aureus and MRSA on corridor, door handle and patient bed. Isolates of S. aureus and MRSA had high rates of resistance to the antibiotics used in this study. There is a need for periodic surveillance and monitoring of S. aureus and MRSA in clinical and hospital environment samples as well as regular and effective cleaning of contact surfaces in hospital. Vancomycin, chloramphenicol, linezolid and rifampicin have shown very good in vitro susceptibility against MRSA and MSSA and are likely to have a key role in the treatment of infections caused by S. aureus. These antimicrobials can serve as an alternative to new expensive antimicrobials in resource poor countries. There is need to further evaluate these antimicrobials for determining the in vitro as well as in vivo efficacy before broad usage of this compound can be undertaken. And this study has shown that some of S. aureus isolates recovered from clinical and environmental specimens contain a variety of β-lactam, erythromycin, tetracycline and aminoglycoside resistance genes were common blaZ, ermA, tet and AME genes among the S. aureus isolates, respectively.

SIGNIFICANCE STATEMENT

Staphylococcus aureus is one of the important bacteria that cause infections acquired in the community and hospital, its ability to develop resistance to many antibiotics. The high prevalence of MRSA was found in hospital environment. It is suggested that improved personal hygiene, effective cleaning of contact surfaces and disinfection in hospital environment are highly recommended. Since, The phenotypic antibiotic susceptibility patterns of S. aureus isolates were not similar to those obtained by genotyping done by PCR. Rapid reliable methods for antibiotic susceptibility are important to determine the appropriate prophylaxis and therapy. The PCR can be used for confirmation of the results obtained by conventional phenotypic methods.

ACKNOWLEDGMENTS

This study was supported by Department of Microbiology, Faculty of Science, Silpakorn University, Thailand. We gratefully acknowledge Department of Pathology, Nakorn Pathom Hospital, Nakorn Pathom, Thailand for providing clinical and environmental specimens for this study.

REFERENCES

  1. Talan, D.A., A. Krishnadasan, R.J. Gorwitz, G.E. Fosheim, B. Limbago, V. Albrecht and G.J. Moran, 2011. Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency department patients, 2004 and 2008. Clin. Infect. Dis., 53: 144-149.
    CrossRef  |  Direct Link  |  


  2. Gleghorn, K., E. Grimshaw and E. Kelly, 2015. New antibiotics in the management of acute bacterial skin and skin structure infections. Skin Ther. Lett., 20: 7-9.
    Direct Link  |  


  3. Cosgrove, S.E., G. Sakoulas, E.N. Perencevich, M.J. Schwaber, A.W. Karchmer and Y. Carmeli, 2003. Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: A meta-analysis. Clin. Infect. Dis., 36: 53-59.
    CrossRef  |  PubMed  |  Direct Link  |  


  4. Gordon, R.J. and F.D. Lowy, 2008. Pathogenesis of methicillin-resistant Staphylococcus aureus infection. Clin. Infect. Dis., 46: S350-S359.
    CrossRef  |  PubMed  |  Direct Link  |  


  5. Jalalpoor, S., R. Kermanshahi, A. Noohi and S.H.Z. Isfahani, 2010. The comparative frequency of β-lactamase production and antibiotic susceptibility pattern of bacterial strains isolated from staff hands and hospital surfaces in Alzahra Hospital–Isfahan. Iran. J. Med. Microbiol., 3: 37-45.
    Direct Link  |  


  6. Jalalpoor, S., 2011. Study of the antibiotic resistance pattern among the bacterial isolated from the hospital environment of Azzahra Hospital, Isfahan, Iran. Afr. J. Microbiol. Res., 5: 3317-3320.
    CrossRef  |  Direct Link  |  


  7. Murakami, K., W. Minamide, K. Wada, E. Nakamura, H. Teraoka and S. Watanabe, 1991. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J. Clin. Microbiol., 29: 2240-2244.
    PubMed  |  Direct Link  |  


  8. Mulder, J.G., 1996. Comparison of disk diffusion, the E test and detection of mecA for determination of methicillin resistance in coagulase-negative staphylococci. Eur. J. Clin. Microbiol. Infect. Dis., 15: 567-573.
    CrossRef  |  Direct Link  |  


  9. Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Turck, 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 45: 493-496.
    CrossRef  |  PubMed  |  Direct Link  |  


  10. CLSI., 2011. Performance standards for antimicrobial susceptibility testing; twenty first informational supplement. CLSI Document M100-S21, Clinical and Laboratory Standards Institute, Wayne, PA., USA.


  11. Davoodi, N.R., S.D. Siadat, F. Vaziri, J.V. Yousefi and N. Harzandi et al., 2015. Identification of Staphylococcus aureus and coagulase-negative Staphylococcus (CoNS) as well as detection of methicillin resistance and panton-valentine leucocidin by multiplex PCR. J. Pure Applied Microbiol., 9: 467-471.
    Direct Link  |  


  12. Wilailuckana, C., C. Tribuddharat, C. Tiensasitorn, P. Pongpech and P. Naenna et al., 2006. Discriminatory Powers of Molecular Typing Techniques for Methicillin-Resistant Staphylococcus aureus in a University Hospital, Thailand. Southeast Asian Trop. Med. Public Health, 37: 327-334.
    PubMed  |  Direct Link  |  


  13. Tsuchizaki, N., K. Ishino, F. Saito, J. Ishikawa, M. Nakajima and K. Hotta, 2006. Trends of arbekacin-resistant MRSA strains in Japanese hospitals (1979 to 2000). J. Antibiot., 59: 229-233.
    CrossRef  |  PubMed  |  Direct Link  |  


  14. Strommenger, B., C. Kettlitz, G. Werner and W. Witte, 2003. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J. Clin. Microbiol., 41: 4089-4094.
    CrossRef  |  Direct Link  |  


  15. Khadri, H. and M. Alzohairy, 2010. Prevalence and antibiotic susceptibility pattern of methicillin-resistant and coagulase-negative staphylococci in a tertiary care hospital in India. Int. J. Med. Med. Sci., 2: 116-120.
    Direct Link  |  


  16. Vannuffel, P., J. Gigi, H. Ezzedine, B. Vandercam, M. Delmee, G. Wauters and J.L. Gala, 1995. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J. Clin. Microbiol., 33: 2864-2867.
    Direct Link  |  


  17. Braoios, A., A. Fluminhan Junior and A.C. Pizzolitto, 2009. Multiplex PCR Use for Staphylococcus aureus identification and oxacillin and mupirocin resistance evaluation. Rev. Cienc. Farm. Basica Apl., 30: 303-307.


  18. Saba, C.K.S., J.K. Amenyona and S.W. Kpordze, 2017. Prevalence and pattern of antibiotic resistance of Staphylococcus aureus isolated from door handles and other points of contact in public hospitals in Ghana. Antimicrob. Resistance Infect. Control, Vol. 6.
    CrossRef  |  Direct Link  |  


  19. Newman, M.J., 2002. Neonatal intensive care Unit: Reservoirs of nosocomial pathogens. West Afr. J. Med., 21: 310-312.
    PubMed  |  Direct Link  |  


  20. Hammuel, C., E.D. Jatau and C.M.Z. Whong, 2014. Prevalence and antibiogram pattern of some nosocomial pathogens isolated from hospital environment in Zaria, Nigeria. Aceh Int. J. Sci. Technol., 3: 131-139.
    CrossRef  |  Direct Link  |  


  21. Festus, T., M.M. Mukesi and S.R. Moyo, 2016. The distribution of methicillin resistant Staphylococcus aureus isolated at the Namibia Institute of Pathology in WindHoek, Namibia. Indian J. Med. Res. Pharm. Sci., 3: 1-8.
    CrossRef  |  Direct Link  |  


  22. Boyce, J.M., 2016. Modern technologies for improving cleaning and disinfection of environmental surfaces in hospitals. Antimicrob. Resistance Infect. Control,
    CrossRef  |  Direct Link  |  


  23. Opoku-Okrah, C., P. Feglo, N. Amidu and M.P. Dakorah, 2009. Bacterial contamination of donor blood at the Tamale Teaching Hospital, Ghana. Afr. Health Sci., 9: 13-18.
    Direct Link  |  


  24. Newman, M.J., E. Frimpong, E.S. Donkor, J.A. Opintan and A. Asamoah-Adu, 2011. Resistance to antimicrobial drugs in Ghana. J. Infect. Drug Resistance, 49: 215-220.
    CrossRef  |  Direct Link  |  


  25. Carvalho, K.S., M.C. Melo, G.B. Melo and P.P. Gontijo-Filho, 2007. Hospital surface contamination in wards occupied by patients infected with MRSA or MSSA in a Brazilian university hospital. Rev. Cienc. Farm. Basica Apl., 28: 159-163.


  26. Oie, S., I. Hosokawa and A. Kamiya, 2002. Contamination of room door handles by methicillin-sensitive/methicillin-resistant Staphylococcus aureus. J. Hospital Infect., 51: 140-143.
    CrossRef  |  PubMed  |  Direct Link  |  


  27. Chambers, H.F., 1997. Methicillin resistance in staphylococci: Molecular and biochemical basis and clinical implications. Clin. Microbiol. Rev., 10: 781-791.
    PubMed  |  Direct Link  |  


  28. Kumar, V.A., K. Steffy, M. Chatterjee, M. Sugumar and K.R. Dinesh et al., 2013. Detection of oxacillin-susceptible mecA-positive Staphylococcus aureus isolates by use of chromogenic medium MRSA ID. J. Clin. Microbiol., 51: 318-319.
    CrossRef  |  Direct Link  |  


  29. Cuirolo, A., L.F. Canigia, N. Gardella, S. Fernández, G. Gutkind, A. Rosato and M. Mollerach, 2011. Oxacillin- and cefoxitin-susceptible meticillin-resistant Staphylococcus aureus (MRSA). Int. J. Antimicrob. Agent, 37: 178-179.
    CrossRef  |  Direct Link  |  


  30. Hososaka, Y., H. Hanaki, H. Endo, Y. Suzuki and Z. Nagasawa et al., 2007. Characterization of oxacillin-susceptible mecA-positive Staphylococcus aureus: A new type of MRSA. J. Infect. Chemother., 13: 79-86.
    CrossRef  |  Direct Link  |  


  31. Ikonomidis, A., G. Michail, A. Vasdeki, M. Labrou and V. Karavasilis et al., 2008. In vitro and in vivo evaluations of oxacillin efficiency against mecA-positive oxacillin-susceptible Staphylococcus aureus. Antimicrob. Agents Chemother., 52: 3905-3908.
    CrossRef  |  Direct Link  |  


  32. Martineau, F., F.J. Picard, J.N. Lansac, C. Menard, P.H. Roy, M. Ouellette and M.G. Bergeron, 2000. Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob. Agents Chemother., 44: 231-238.
    CrossRef  |  Direct Link  |  


  33. Oliveira, D.C. and H. de Lencastre, 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother., 46: 2155-2161.
    CrossRef  |  PubMed  |  Direct Link  |  


  34. Petinaki, E., F. Kontos and A.N. Maniatis, 2002. Emergence of two oxacillin-susceptible mecA-positive Staphylococcus aureus clones in a greek hospital. J. Antimicrob. Chemother., 50: 1090-1091.
    CrossRef  |  Direct Link  |  


  35. Saeed, K., M. Dryden and R. Parnaby, 2010. Oxacillin-susceptible MRSA, the emerging MRSA clone in the UK? J. Hospital Infect., 76: 267-268.
    CrossRef  |  Direct Link  |  


  36. Chambers, H.F. and M. Sachdeva, 1990. Binding of β-lactam antibiotics to penicillin-binding proteins in methicillin-resistant Staphylococcus aureus. J. Infect. Dis., 161: 1170-1176.
    CrossRef  |  Direct Link  |  


  37. Sakoulas, G., H.S. Gold1, L. Venkataraman, P.C. DeGirolami, G.M. Eliopoulos and Q. Qian, 2001. Methicillin-resistant Staphylococcus aureus: Comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains. J. Clin. Microbiol., 39: 3946-3951.
    CrossRef  |  Direct Link  |  


  38. Dibah, S., M. Arzanlou, E. Jannati and R. Shapouri, 2014. Prevalence and antimicrobial resistance pattern of methicillin resistant Staphylococcus aureus (MRSA) strains isolated from clinical specimens in Ardabil, Iran. Iran J. Microbiol., 6: 163-168.
    PubMed  |  Direct Link  |  


  39. Farahani, A., P. Mohajeri, B. Gholamine, M. Rezaei and H. Abbasi, 2013. Comparison of different phenotypic and genotypic methods for the detection of methicillin-resistant Staphylococcus aureus. North Am. J. Med. Sci., 5: 637-640.
    CrossRef  |  PubMed  |  Direct Link  |  


  40. Jannati, E., M. Arzanlou, S. Habibzadeh, S. Mohammadi and P. Ahadi et al., 2013. Nasal colonization of mecA-positive, oxacillin-susceptible, methicillin-resistant Staphylococcus aureus isolates among nursing staff in an Iranian Teaching Hospital. Am. J. Infect. Control, 41: 1122-1124.
    CrossRef  |  Direct Link  |  


  41. Vincent, J.L., D. Bihari, P.M. Suter, H.A. Bruining and J. White et al., 1995. The prevalence of nosocomial infection in intensive care units in Europe: Results of the European Prevalence of Infection in Intensive Care (EPIC) study.. J. Am. Med. Assoc., 274: 639-644.
    CrossRef  |  PubMed  |  Direct Link  |  


  42. Song, J.H., P.R. Hsueh, D.R. Chung, K.S. Ko and C.I. Kang et al., 2011. Spread of methicillin-resistant Staphylococcus aureus between the community and the hospitals in Asian countries: An ANSORP study. J. Antimicrob. Chemother., 66: 1061-1069.
    CrossRef  |  Direct Link  |  


  43. Johnson, A.P., 2011. Methicillin-resistant Staphylococcus aureus: The European landscape. J. Antimicrob. Chemother., 66: iv43-iv48.
    CrossRef  |  Direct Link  |  


  44. Erdem, H., M. Dizbay, S. Karabey, S. Kaya and T. Demirdal et al., 2013. Withdrawal of Staphylococcus aureus from intensive care units in Turkey. Am. J. Infect. Control, 41: 1053-1058.
    CrossRef  |  Direct Link  |  


  45. Schmitz, F.J. and M.E. Jones, 1997. Antibiotics for treatment of infections caused by MRSA and elimination of MRSA carriage. What are the choices? Int. J. Antimicrob. Agents, 9: 1-19.
    CrossRef  |  Direct Link  |  


  46. Aligholi, M., M. Emaneini, F. Jabalameli, S. Shahsavan, H. Dabiri and H. Sedaght, 2008. Emergence of high-level vancomycin-resistant Staphylococcus aureus in the Imam Khomeini Hospital in Tehran. Med. Principles Pract., 17: 432-434.
    CrossRef  |  Direct Link  |  


  47. Askari, E., S.M. Tabatabai, A. Arianpoor and M.N. Nasab, 2013. VanA-positive vancomycin-resistant Staphylococcus aureus: Systematic search and review of reported cases. Infect. Dis. Clin. Pract., 21: 91-93.
    CrossRef  |  Direct Link  |  


  48. Azimian, A., S.A. Havaei, H. Fazeli, M. Naderi and K. Ghazvini et al., 2012. Genetic characterization of a vancomycin-resistant Staphylococcus aureus isolate from the respiratory tract of a patient in a university hospital in Northeastern Iran. J. Clin. Microbiol., 50: 3581-3585.
    CrossRef  |  Direct Link  |  


  49. Shittu, A.O. and J. Lin, 2006. Antimicrobial susceptibility patterns and characterization of clinical isolates of Staphylococcus aureus in Kwa Zulu-Natal province, South Africa. BMC Infec. Dis., Vol. 6.
    Direct Link  |  


  50. Zhou, F. and Y. Wang, 2013. Characteristics of antibiotic resistance of airborne Staphylococcus isolated from metro stations. Int. J. Environ. Res. Public Health, 10: 2412-2426.
    CrossRef  |  Direct Link  |  


  51. Lim, K.T., C.S.J. Teh, M.Y.M. Yusof and K.L. Thong, 2013. Mutations in rpoB and fusA cause resistance to rifampicin and fusidic acid in methicillin-resistant Staphylococcus aureus strains from a tertiary hospital in Malaysia. Trans. R. Soc. Trop. Med. Hyg., 108: 112-118.
    CrossRef  |  Direct Link  |  


  52. Kruzel, M.C., C.T. Lewis, K.J. Welsh, E.M. Lewis and N.E. Dundas et al., 2011. Determination of vancomycin and daptomycin MICs by different testing methods for methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol., 49: 2272-2273.
    CrossRef  |  Direct Link  |  


  53. Holland, T.L. and V.G. Fowler Jr., 2011. Vancomycin minimum inhibitory concentration and outcome in patients with Staphylococcus aureus bacteremia: Pearl or pellet? J. Infect. Dis., 204: 329-331.
    CrossRef  |  Direct Link  |  


  54. Claassen, M., J. Nouwen, Y. Fang, A. Ott and H. Verbrugh et al., 2005. Staphylococcus aureus nasal carriage is not associated with known polymorphism in the vitamin D receptor gene. FEMS Immunol. Med. Microbiol., 43: 173-176.
    CrossRef  |  Direct Link  |  


  55. Brakstad, O.G., K. Aasbakk and J.A. Maeland, 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol., 30: 1654-1660.
    PubMed  |  Direct Link  |  


  56. Duran, N., B. Ozer, G.G. Duran, Y. Onlen and C. Demir, 2012. Antibiotic resistance genes and susceptibility patterns in staphylococci. Indian J. Med. Res., 135: 389-396.
    PubMed  |  Direct Link  |  


  57. Murugan, K., K. Kavitha and S. Al-Sohaibani, 2015. Rifampicin resistance among multi-resistant MRSA clinical isolates from Chennai, India and their molecular characterization. Genet. Mol. Res., 14: 2716-2725.
    CrossRef  |  


  58. Ruef, C., 2004. Epidemiology and clinical impact of glycopeptide resistance in Staphylococcus aureus. Infection, 32: 315-327.
    CrossRef  |  Direct Link  |  


  59. Lentino, J.R., M. Narita and V.L. Yu, 2008. New antimicrobial agents as therapy for resistant gram-positive cocci. Eur. J. Clin. Microbiol. Infect. Dis., 27: 3-15.
    CrossRef  |  Direct Link  |  


  60. Lyseng-Williamson, K.A. and K.L. Goa, 2003. Linezolid: In infants and children with severe gram-positive infections. Paediatr. Drugs, 5: 419-429.
    CrossRef  |  Direct Link  |  


  61. Wang, Z., B. Cao, Y.M. Liu, L. Gu and C. Wang, 2009. Investigation of the prevalence of patients co-colonized or infected with methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci in China: A hospital-based study. Chin. Med. J., 122: 1283-1288.
    PubMed  |  Direct Link  |  


  62. Shahsavan, S., L. Jabalameli, P. Maleknejad, M. Aligholi and H. Imaneini et al., 2011. Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran university of medical sciences: High prevalence of sequence type 239 (ST239) clone. Acta Microbiol. Immunol. Hung., 58: 31-39.
    CrossRef  |  PubMed  |  Direct Link  |  


  63. Vourli, S., H. Vagiakou, G. Ganteris, M. Orfanidou, M. Polemis, A. Vatopoulos and H. Malamou-Ladas, 2009. High rates of community-acquired, Panton-Valentine leukocidin (PVL)-positive methicillin-resistant S. aureus (MRSA) infections in adult outpatients in Greece. Eurosurveillance, Vol. 14.
    CrossRef  |  Direct Link  |  


  64. Ninkovic, G., V. Dullo and N.C. Saunders, 2008. Microbiology of otitis externa in the secondary care in United Kingdom and antimicrobial sensitivity. Auris Nasus Larynx, 35: 480-484.
    CrossRef  |  Direct Link  |  


  65. Hata, E., K. Katsuda, H. Kobayashi, K. Nishimori and I. Uchida et al., 2008. Bacteriological characteristics of Staphylococcus aureus isolates from humans and bulk milk. J. Dairy Sci., 91: 564-569.
    CrossRef  |  Direct Link  |  


  66. Chung, H.J., H.S. Jeon, H. Sung, M.N. Kim and S.J. Hong, 2008. Epidemiological characteristics of methicillin-resistant Staphylococcus aureus isolates from children with eczematous atopic dermatitis lesions. J. Clin. Microbiol., 46: 991-995.
    CrossRef  |  Direct Link  |  


  67. Tenover, F.C., S. McAllister, G. Fosheim, L.K. McDougal and R.B. Carey et al., 2008. Characterization of Staphylococcus aureus isolates from nasal cultures collected from individuals in the United States in 2001 to 2004. J. Clin. Microbiol., 46: 2837-2841.
    CrossRef  |  Direct Link  |  


  68. Mendes, R.E., H.S. Sader, L. Deshpande and R.N. Jones, 2008. Antimicrobial activity of tigecycline against community-acquired methicillin-resistant Staphylococcus aureus isolates recovered from North American medical centers. Diagn. Microbiol. Infect. Dis., 60: 433-436.
    CrossRef  |  Direct Link  |  


  69. Ojulong, J., T.P. Mwambu, M. Joloba, F. Bwanga and D.H. Kaddu-Mulindwa, 2009. Relative prevalence of methicilline resistant Staphylococcus aureus and its susceptibility pattern in Mulago Hospital, Kampala, Uganda. Tanzania J. Health Res., 11: 149-153.
    CrossRef  |  Direct Link  |  


  70. Zabielinski, M., M.P. McLeod, C. Aber, J. Izakovic and L.A. Schachner, 2013. Trends and antibiotic susceptibility patterns of methicillin-resistant and methicillin-sensitive Staphylococcus aureus in an outpatient dermatology facility. JAMA Dermatol., 149: 427-432.
    CrossRef  |  Direct Link  |  


  71. Pellegrino, F.L.P.C., L.M. Teixeira, M.D.G.S. Carvalho, S.A. Nouer and M.P. de Oliveira et al., 2002. Occurrence of a multidrug-resistant Pseudomonas aeruginosa clone in different hospitals in Rio de Janeiro, Brazil. J. Clin. Microbiol., 40: 2420-2424.
    CrossRef  |  Direct Link  |  


  72. Lina, G., A. Quaglia, M.E. Reverdy, R. Leclercq, F. Vandenesch and J. Etienne, 1999. Distribution of genes encoding resistance to macrolides, lincosamides and streptogramins among staphylococci. Antimicrob. Agents Chemother., 43: 1062-1066.
    Direct Link  |  


  73. Schmitz, F.J., A.C. Fluit, M. Gondolf, R. Beyrau and E. Lindenlauf et al., 1999. The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals. J. Antimicrob. Chemother., 43: 253-259.
    CrossRef  |  Direct Link  |  


  74. Schmitz, F.J., A. Krey, R. Sadurski, J. Verhoef, D. Milatovic and A.C. Fluit, 2001. Resistance to tetracycline and distribution of tetracycline resistance genes in European Staphylococcus aureus isolates. J. Antimicrob. Chemother., 47: 239-240.
    CrossRef  |  Direct Link  |  


  75. Schmitz, F.J., J. Petridou, A.C. Fluit, U. Hadding, G. Peters and C. von Eiff, 2000. Distribution of macrolide-resistance genes in Staphylococcus aureus blood-culture isolates from fifteen German university hospitals. Eur. J. Clin. Microbiol. Infect. Dis., 19: 385-387.
    CrossRef  |  Direct Link  |  


  76. Zapun, A., C. Contreras-Martel and T. Vernet, 2008. Penicillin-binding proteins and β-lactam resistance. FEMS Microbiol. Rev., 32: 361-385.
    CrossRef  |  Direct Link  |  


  77. Choi, S.M., S.H. Kim, H.J. Kim, D.G. Lee and J.H. Choi etal., 2003. Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species. J. Korean Med. Sci., 18: 631-636.
    CrossRef  |  PubMed  |  Direct Link  |  


  78. Vanhoof, R., C. Godard, J. Content and H.J. Nyssen, 1994. Detection by polymerase chain reaction of genes encoding aminoglycoside-modifying enzymes in methicillin-resistant Staphylococcus aureus isolates of epidemic phage types. J. Med. Microbiol., 41: 282-290.
    CrossRef  |  Direct Link  |  


  79. Yadegar, A., M. Sattari, N.A. Mozafari and G.R. Goudarzi, 2009. Prevalence of the genes encoding aminoglycoside-modifying enzymes and methicillin resistance among clinical isolates of Staphylococcus aureus in Tehran, Iran. Microb. Drug Resist., 15: 109-113.
    CrossRef  |  Direct Link  |  


  80. Aziz, H.W., T.H. Al-Dulaimi, A.H. Al-Marzoqi and N.K. Ahmed, 2014. Phenotypic detection of resistance in Staphylococcus aureus isolates: Detection of (mec A and fem A) gene in methicillin resistant Staphylococcus aureus (MRSA) by polymerase chain reaction. J. Nat. Sci. Res., 4: 112-118.
    Direct Link  |  


  81. Bignardi, G.E., N. Woodford, A. Chapman, A.P. Johnson and D.C.E. Speller, 1996. Detection of the mec-A gene and phenotypic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin resistance. J. Antimicrob. Chemother., 37: 53-63.
    CrossRef  |  Direct Link  |  


  82. Chambers, H.F., G. Archer and M. Matsuhashi, 1989. Low-level methicillin resistance in strains of Staphylococcus aureus. Antimicrob. Agents Chemother., 33: 424-428.
    CrossRef  |  Direct Link  |  


  83. Ligozzi, M., G.M. Rossolini, E.A. Tonin and R. Fontana, 1991. Nonradioactive DNA probe for detection of gene for methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother., 35: 575-578.
    CrossRef  |  Direct Link  |  


  84. Hiramatsu, K., H. Kihara and T. Yokota, 1992. Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction. Microbiol. Immunol., 36: 445-453.
    Direct Link  |  


  85. Olayinka, B.O., A.T. Olayinka, A.F. Obajuluwa, J.A. Onaolapo and P.F. Olurinola, 2009. Absence of mecA gene in methicillin-resistant Staphylococcus aureus isolates. Afr. J. Infect. Dis., 3: 49-56.
    CrossRef  |  Direct Link  |  


  86. Ba, X., E.M. Harrison, G.F. Edwards, M.T.G. Holden and A.R. Larsen et al., 2013. Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene. J. Antimicrob. Chemother., 69: 594-597.
    CrossRef  |  Direct Link  |  


  87. Jensen, S.O. and B.R. Lyon, 2009. Genetics of antimicrobial resistance in Staphylococcus aureus. Future Microbiol., 4: 565-582.
    CrossRef  |  Direct Link  |  


  88. Frey, Y., J.P. Rodriguez, A. Thomann, S. Schwendener and V. Perreten, 2013. Genetic characterization of antimicrobial resistance in coagulase-negative Staphylococci from bovine mastitis milk. J. Dairy Sci., 96: 2247-2257.
    CrossRef  |  Direct Link  |  


  89. Gao, J., F.Q. Yu, L.P. Luo, J.Z. He and R.G. Hou et al., 2012. Antibiotic resistance of Streptococcus agalactiae from cows with mastitis. Vet. J., 194: 423-424.
    CrossRef  |  Direct Link  |  


  90. Yang, F., Q. Wang, X. Wang, L. Wang and M. Xiao et al., 2015. Prevalence of blaZ gene and other virulence genes in penicillin-resistant Staphylococcus aureus isolated from bovine mastitis cases in Gansu, China. Turk. J. Vet. Anim. Sci., 39: 634-636.
    CrossRef  |  Direct Link  |  


  91. Pantosti, A., A. Sanchini and M. Monaco, 2007. Mechanisms of antibiotic resistance in Staphylococcus aureus. Future Microbiol., 2: 323-334.
    CrossRef  |  PubMed  |  Direct Link  |  


  92. Croes, S., R.H. Deurenberg, M.L.L. Boumans, P.S. Beisser, C. Neef and E.E. Stobberingh, 2009. Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage. BMC Microbiol., Vol. 9.
    CrossRef  |  


  93. Gow, S.P., C.L. Waldner, J. Harel and P. Boerlin, 2008. Associations between antimicrobial resistance genes in fecal generic Escherichia coli isolates from cow-calf herds in western Canada. Applied Environ. Microbiol., 74: 3658-3666.
    CrossRef  |  Direct Link  |  


  94. Ramirez, M.S. and M.E. Tolmasky, 2010. Aminoglycoside modifying enzymes. Drug Resistance Updates, 13: 151-171.
    CrossRef  |  Direct Link  |  


  95. Fatholahzadeh, B., M. Emaneini, M.M. Feizabadi, H. Sedaghat, M. Aligholi, M. Taherikalani and F. Jabalameli, 2009. Characterisation of genes encoding aminoglycoside-modifying enzymes among meticillin-resistant Staphylococcus aureus isolated from two hospitals in Tehran, Iran. Int. J. Antimicrob. Agents, 33: 264-265.
    CrossRef  |  Direct Link  |  


  96. Emaneini, M., R. Bigverdi, D. Kalantar, S. Soroush and F. Jabalameli et al., 2013. Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center. Ann. Burns Fire Dis., 26: 76-80.
    PubMed  |  Direct Link  |  


  97. Ounissi, H., E. Derlot, C. Carlier and P. Courvalin, 1990. Gene homogeneity for aminoglycoside-modifying enzymes in gram-positive cocci. Antimicrob. Agents Chemother., 34: 2164-2168.
    CrossRef  |  Direct Link  |  


  98. Udo, E.E. and A.A. Dashti, 2000. Detection of genes encoding aminoglycoside-modifying enzymes in staphylococci by polymerase chain reaction and dot blot hybridization. Int. J. Antimicrob. Agents, 13: 273-279.
    CrossRef  |  Direct Link  |  


  99. Weisblum, B., 1995. Erythromycin resistance by ribosome modification. Antimicrob. Agents Chemother., 39: 577-585.
    PubMed  |  Direct Link  |  


  100. Nicola, F.G., L.K. McDougal, J.W. Biddle and F.C. Tenover, 1998. Characterization of erythromycin-resistant isolates of Staphylococcus aureus recovered in the united states from 1958 through 1969. Antimicrob. Agents Chemother., 42: 3024-3027.
    Direct Link  |  


  101. Westh, H., D.M. Hougaard, J. Vuust and V.T. Rosdahl, 1995. Erm genes in erythromycin‐resistant Staphylococcus aureus and coagulase‐negative staphylococci. APMIS, 103: 225-232.
    CrossRef  |  Direct Link  |  


  102. Westh, H., D.M. Hougaard, J. Vuust and V.T. Rosdahl, 1995. Prevalence of erm gene classes in erythromycin-resistant Staphylococcus aureus strains isolated between 1959 and 1988. Antimicrob. Agents Chemother., 39: 369-373.
    CrossRef  |  Direct Link  |  


  103. Eady, E.A., J.I. Ross, J.L. Tipper, C.E. Walters, J.H. Cove and W.C. Noble, 1993. Distribution of genes encoding erythromycin ribosomal methylases and an erythromycin efflux pump in epidemiologically distinct groups of staphylococci. J. Antimicrob. Chemother., 31: 211-217.
    CrossRef  |  Direct Link  |  


  104. Lim, J.A., A.R. Kwon, S.K. Kim, Y. Chong, K. Lee and E.C. Choi, 2002. Prevalence of resistance to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in a Korean hospital. J. Antimicrob. Chemother., 49: 489-495.
    CrossRef  |  Direct Link  |  


  105. Esposito, S., S. Leone, E. Petta, S. Noviello and F. Ianniello, 2009. Treatment options for skin and soft tissue infections caused by meticillin-resistant Staphylococcus aureus: Oral vs. parenteral; home vs. hospital. Int. J. Antimicrob. Agents, 34: S30-S35.
    CrossRef  |  Direct Link  |  


  106. McCallum, N., B. Berger-Bachi and M.M. Senn, 2010. Regulation of antibiotic resistance in Staphylococcus aureus. Int. J. Med. Microbiol., 300: 118-129.
    CrossRef  |  PubMed  |  Direct Link  |  


  107. Trzcinski, K., B.S. Cooper, W. Hryniewicz and C.G. Dowson, 2000. Expression of resistance to tetracyclines in strains of methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother., 45: 763-770.
    CrossRef  |  Direct Link  |  


  108. Jones, C.H., M. Tuckman, A.Y.M. Howe, M. Orlowski, S. Mullen, K. Chan and P.A. Bradford, 2006. Diagnostic PCR analysis of the occurrence of methicillin and tetracycline resistance genes among Staphylococcus aureus isolates from phase 3 clinical trials of tigecycline for complicated skin and skin structure infections. Antimicrob. Agents Chemother., 50: 505-510.
    CrossRef  |  Direct Link  |  


  109. El-Mahdy, T.S., S. Abdalla, R. El-Domany and A.M. Snelling, 2010. Investigation of MLSB and tetracycline resistance in coagulase-negative staphylococci isolated from the skin of Egyptian acne patients and controls. J. Am. Sci., 6: 880-888.
    Direct Link  |  


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