Introduction

Staphylococcus is Gram positive bacterium that constitutes a major normal flora on the skin and mucous membrane. The virulent species is Staphylococcus aureus (S. aureus) that is associated with major hospital acquired infections such as surgical wound infections, sepsis and pneumonia (1).

Antibiotics therapy for S. aureus started with methicillin, which is formed of the phenol group of benzylpenicillin provided with two methoxy groups,  that was active toward β-lactamase produced by S. aureus to penicillin. However, inappropriate use of antibiotics leads to emergence of methicillin-resistant S. aureus (MRSA) strains. The resistance is mainly related to the expression of penicillin-binding protein (PBP2a). The use of several classes of antibiotics resulted in the emergence of multi-resistant MRSA strains through the mutations in genes coding for target proteins and the acquisition and accumulation of antibiotic resistance genes (2). The gene associated with methicillin resistance is a mobile heterogenous gene, mecA, encoded by a Staphylococcal Cassette Chromosome mec (SCCmec) (3).  SCCmec is formed of mecA gene with regulatory elements, a cassette chromosome recombinases (ccr) complex and the flanking direct repeat sequences that contain the integration site sequence for SCC. The classification of SCCmec is based upon the combination of ccr gene complex and the class of the mec gene (4-7).

Other antibiotics that are used for treatment of S. aureus especially in skin infections are tetracyclines. Nevertheless, there is an emergence of tetracycline resistance through the acquisition of tetracycline resistance (tet) genes and oxytetracycline resistance (otr) genes (8). Tetracycline resistance genes lead to resistance through active efflux pumps, ribosomal protection, and enzyme inactivation with 40 types of different resistant genes (9, 10).

Nowadays MRSA with resistant phenotypes to many antibiotics has emerged in healthcare associated infections. Understanding the molecular resistance mechanisms for these antibiotics is needed to develop active preventive measures for their spread.

The aim of the present study was to investigate the prevalence of tetracycline resistance genes among isolated S. aureus from healthcare associated surgical site infections at Mansoura University Hospitals.

Materials and Method

This study was a cross-sectional study that was carried out in Mansoura University hospitals from January 2017 till March 2018.

Specimens Collection

The wound swabs which were submitted to the microbiological laboratory from patients with healthcare associated infections according to CDC definition were included in the study (11). The study was approved by Mansoura Ethical committee and approvals were obtained from the participant patients.

Wound swabs were obtained from the patients by rolling the swabs over 1cm of the wound for 5 seconds then the swabs were transported in the sterile containers rapidly to the laboratory.

Specimens Processing

Swabs were cultured under aerobic conditions for 24 hours at 37°C. Colonies were identified by Gram stain then Gram positive cocci were identified by catalase, coagulase, and fermentation of mannitol tests (12). Identified S. aureus strains were subjected to antimicrobial susceptibility tests, detection of methicillin resistance by cefoxitin disc and molecular study of mecA and tet genes.

Antibiotics Susceptibility Testing by Discs Diffusion Method

The antibiotics susceptibility test was performed by discs diffusion method according to the Clinical Laboratory Standards Institute (CLSI) (13). The used discs were oxacillin (1 μg), gentamicin (10 μg), amikacin (30 μg), kanamycin (30 μg), erythromycin (15 μg), cefoxitin (5 μg), ceftazidime (30 μg), tetracycline (30 μg), doxycycline (30 μg), and minocycline (30 μg) (Oxoid-Thermal fisher). S. aureus ATCC 25923 was used as the control strain.

DNA Extraction

Pure colonies of S. aureus were prepared for DNA extraction by boiling method. Briefly, pure colonies were suspended in 100μl of sterile phosphate buffer and boiled at 100°C for 10 min in water bath then cooled. The suspension was kept frozen at -20°C till amplification procedures (14).

Polymerase Chain Reaction (PCR) for mecA Gene

The primers used for mecA amplification were listed in table 1.  Five microns of the DNA extracted by the boiling method were added to total volume 100μl of amplification mixtures from Qiagen (Qiagen-Germany) with 1μM of mecA primers. The amplification conditions were as the following; an initial denaturation step at 94°C for 5 min followed by 40 cycles of amplification formed of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 60 sec ending with a final extension step at 72°C for 5 min. After PCR amplification, 5μl of amplicon were removed and subjected to agarose gel electrophoresis (1.5% agarose, 1× Tris-borate-EDTA buffer, 100 V, 40 min) to estimate the sizes of the amplification products by comparison with molecular marker 1kbp (15).

Multiplex PCR for Amplification of tetK, tetL, tetM Genes

Amplification of tet genes (tetK, tetL, tetM ) were performed using Qiagen PCR master kit (Qiagen). Multiplex PCR was prepared in a final volume of 25µl containing 0.5µM of each primer (Table 1). The PCR protocol consisted of an initial denaturation step at 94°C for 5 min then 30 amplification cycles at 94°C for 1 min, 51°C for 1 min and 72°C for 1 min, followed by a final extension step at 72°C for 5 min. Amplified products were analyzed by electrophoresis on 1.5% agarose gel containing 0.5 μg/ml ethidium bromide and photographed under UV illumination (16).

PCR for tetO

The primers used for amplification of tetO were listed in table 1. Amplification procedures were similar to the above except that the amplification cycles were 30 cycles at 94°C for 1 min, 57°C for 1min, 72°C for 1 min and a final extension for 72°C for 5 min. The amplified products were visualized as mentioned previously (16).

Table 1: Oligonucleotides sequences and sizes of different primers used in the amplification of mecA and tet genes.

Statistical Analysis

The statistical analysis was performed using SPSS 22.0 (SPSS Inc., Chicago, Illinois, USA). Descriptive data was expressed as number and percentage. Comparison was performed by Qi-square and P was considered significant if P was < 0.05.

Results

This study recovered 170 isolates of S. aureus from 350 wound swabs culture (48.6%) collected during the period of the study (15 months) (Figure 1).

Antibiotics sensitivity testing revealed high resistance rates of isolated S. aureus to gentamicin (71.2%), kanamycin (66.5%), Amikacin (52.3%) and ceftazidime (41.8%). Resistances to tetracycline, doxycycline and minocycline were 60.6%, 56.5% and 45.3%, respectively (Table 2).

Antibiotics resistance pattern of the isolates showed that 90 isolates (52.9%) were MRSA according to resistance to cefoxitin and/or oxacillin discs.

In the comparison between MRSA and methicillin sensitive S. aureus (MSSA) as regards antibiotics resistance, there was a significant increase in resistance to tetracycline, doxycycline, minocycline (P=0.0001) and erythromycin (P=0.01) among MRSA strains compared to MSSA (Table 3).

The tetracycline resistant genes detected were tetK (92.3%) (Figure 2) and tetM (25.2%). Combined tetK and tetM genes were detected in 22.3% of S. aureus resistant to tetracycline (Table 4). None of tetracycline resistant isolates had tetL or tetO gene. There was a significant higher frequency of either tetK, tetM or both genes among MRSA compared to MSSA (P=0.0001) (Table 5).

Figure 1: Frequency of S. aureus strains isolated from healthcare associated surgical site infections.   Click here to view figure

Table 2: Antibiotics resistance pattern of the isolated S. aureus from surgical site infections.

Table 3: Comparison of antibiotics resistance pattern between MRSA and MSSA strains.

Table 4: Frequency of tet genes among S. aureus strains resistant to tetracycline as detected by multiplex PCR.

Table 5: Distribution of tet genes among MRSA and MSSA strains as detected by multiplex PCR.

Figure 2: Positive samples for tetK gene as detected by multiplex PCR. M: Molecular marker, 1-5: Positive samples.   Click here to view figure

Discussion

Staphylococcus aureus represents a major pathogen that is related to healthcare associated infections. S. aureus, especially MRSA and multidrug resistant strains, is a common cause of surgical site infections (17).

In the present study, the etiology of about half of the isolates from healthcare acquired surgical site infections were due to S. aureus. Previous studies determined the high prevalence rate of S. aureus in surgical site infections up to 57% (17, 18). In several studies from Egypt, S. aureus was considered a common pathogen (61%) causing hospital acquired infections (71%) (19, 20).

MRSA represented 52.9% of the isolated S. aureus from surgical site infections in the present study. This represents a lower prevalence rate of MRSA to previous studies from Nepal, Egypt, Libya and Iran (17, 20-22).  This may be attributed to the implantation of active surveillance and monitoring of patients and the rapid diagnostic tests, in addition to high orientation to preventive and control measures of MRSA during the period of the study in the hospital (23, 24). Moreover, the difference may be related to different clinical samples obtained from the patients, the duration of hospital stay and previous antimicrobial use.

MRSA strains in the present study were identified by phenotypic characters and molecular methods for detection of mecA gene. In agreement with our study, the molecular method for detection of mecA gene is an accurate and gold standard tool (25, 26). MRSA strains have resistance to methicillin and other semi-synthetic penicillinase resistant β-lactam antibiotics. The mecA gene is transferred horizontally and known to encode for a specific binding protein termed penicillin-binding protein 2a that has low affinity for β-lactam antibiotics which leads the complete synthesis of the bacterial cell wall without interruption by the β-lactam antibiotics (27, 28).

There were high resistance rates of isolated S. aureus to aminoglycosides and ceftazidime. Previous studies demonstrated multiple drug resistance among isolated S. aureus from healthcare associated infections (20). The high resistance to aminoglycosides among isolated S. aureus was similar to previous reports (20, 26, 29). The resistance pattern of isolated S. aureus depends upon the antibiotics policy in different institutions and infection control practices that lead to distribution diversity of antibiotics resistance genes.

In the present study, resistances to tetracycline, doxycycline and minocycline were 60.6%, 56.5% and 45.3%, respectively with significant increase in resistance to tetracycline, doxycycline, minocycline (P=0.0001) and erythromycin (P=0.01) in MRSA isolates compared to MSSA. These findings are in agreement with previous reports (16, 19). However, the resistance rate for tetracycline is higher than rates reported from the United States and Canada which have been reported to be 15.6% and 14.8%, respectively (30). Again, these findings reflect the difference in antibiotics policy between different geographic countries.

There are several mechanisms that lead to the development of tetracyclines resistance. One of these mechanisms is by the acquisition of tetK gene that leads to active efflux of divalent metal ion tetracycline from the bacterial cells (31).  Another mechanism for tetracyclines resistance is mediated by tetM gene. This gene leads to tetracycline resistance through protection of the ribosome by the production of a ribosome protection protein that dislodges tetracycline from the ribosome freeing it from the inhibition by the drug (32).

In the present study, the tetracycline resistant genes detected were tetK (92.3%) and tetM (25.2%). Combined genes were detected in 22.3% of S. aureus, while none of the clinical isolates was positive for tetL and tetO genes. Similar previous studies have reported that tetK gene is the predominant gene of tetracycline resistance in clinical isolates of S. aureus followed by tetM (16, 33). However, other studies have documented that tetM is the predominant gene followed by tetK gene (8, 34). Similar to many reports, tetL and tetO were not detected in any of the isolates in the present study (16, 34).

Both tetK and tetM genes in the present work predominated in MRSA compared to MSSA. This is in accordance to previous findings (16, 33). On contrary, a literature has reported that tetM is more frequent in MRSA isolates (8).

These findings demonstrated that both tetM and tetK genes either single or combined have an important role in tetracycline resistance among MRSA strains in agreement with the previous report of Schmitz et al. (35).  Similar to many reports, tetL and tetO were not detected in any of the isolates in the present study, indicating that these genes have no role in tetracycline resistance in our isolates (34, 36).

The predominance of tetM in tetracycline remittance MRSA may be attributed to previous exposure to tetracycline that leads to excision and transfer of the genetic element Tn916 carrying tetM gene (37). This theory may explain the relative less frequency of this gene among our isolates as the prescription of tetracycline is not common in our hospitals to S. aureus. On the other hand, tetK is transmitted through a small 4.4 kb plasmid known as pT181 from resistant strains in the absence of selective pressure (38).

Conclusion

The present study highlights the prevalence of multiple antibiotics resistance among clinical isolates of S. aureus associated with healthcare associated infections. The resistance increases among methicillin resistant S. aureus. The resistance to tetracycline, minocycline and doxycycline were common. The common genetic basis of the resistance to tetracyclines was the tetK and tetM genes.

Studying the mechanisms of antibiotics resistance is a mandatory tool for implementation of preventive and control measures to combat the resistant bacteria and reduce the risk of healthcare associated infections.

Conflicts of Interests

There is no conflicts of interests.

Funding Source

There is no funding source.

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