Biofilm formation and methicillin resistance of Staphylococcus aureus isolated from clinical samples

Background: Staphylococcus aureus including methicillin resistant S. aureus (MRSA) is one of the most effective biofilm-forming organisms. Biofilm formation contributes in protecting the microorganism from host defenses and prevent the effective penetration of antimicrobial agents. Also, it is considered as an important contributing factor for the initiation and establishment of chronic infection by S. aureus and its a major obstacle in the treatment of S. aureus infections. Aims: To screen clinical Staphylococcus aureus for their biofilm forming abilities and their association with antimicrobial resistance. Methods: A total of 196 clinical isolates of S. aureus were obtained from different sample sources using standard microbiological techniques from three major hospitals in Gaza strip. Biofilm formation of these isolates was determined by tissue culture plate (TCP) method and tube adherence method (TM). Antimicrobial susceptibility test was performed using the modified Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute guidelines. MRSA isolates was detected using the cefoxitin disk test. Results: Biofilm formation was observed in 174 (88.8 %) and in 145 (74.0%) isolates of S. aureus via TCP method and TM, respectively. The highest resistance percent was for penicillin (92.9%), followed by cefoxitin (80.6%) and oxacillin (67.9%), while the lowest resistance percent was for linezolid and ceftaroline (1%). Among the total 196 isolates, 140 (71.4%) were classified as MDR with a MAR index (≥ 0.2). A total of 158 isolates (80.6%) were identified as MRSA distributed among hospitals as the following; 90.4%, 79.4% and 70.9% from Al-Shifa, Al-Nasser pediatrics and Al-Aqsa hospitals respectively. Large proportions (82.1%) of biofilm producers were identified among MRSA isolates. Biofilm-producing MRSA exhibited a higher percent Biofilm formation and methicillin resistance of Staphylococcus aureus isolated from clinical samples Abdelraouf A. Elmanama1, Islam M. Al-Aydi2, Mariam R. Al-Reefi3 1 Medical laboratory Sciences Department, Islamic university-Gaza, P.O Box, 108, Gaza, Palestine. 2 Ministry of Health, Gaza, Palestine. 3 Medical Laboratory Sciences Department, Faculty of Health Sciences, Israa University of Gaza. Contact information:


Introduction
Staphylococcus aureus is described as Gram-positive cocci, facultative anaerobic microorganisms [1]. Cells ranging in size from 0.5 to 1.7 μm in diameter. It is immotile and is not capable of forming spores. Cells are aggregated in clusters that are often described as grape-like cluster [2]. Staphylococci are widely spread colonizers of human epithelia and are opportunistic pathogens implicated with various health care associated diseases [3][4].
Several predisposing factors to S. aureus infection including but not limited to; immunocompromised host, invasive procedure that cause damage to the skin or mucosal surface such as indwelling medical devices, surgery, hemodialysis. In addition, homosexual men and those with close contact sports as well as those who live in close proximity, are also exposed to Staphylococcal infections [5].
A notable major obstacle in the treatment of S. aureus infections is their ability to develop resistance to antimicrobials. Few decades ago and up to this date, the emergence of methicillin resistance S. aureus (MRSA) has spread worldwide and is almost endemic in most healthcare facilities and is increasingly becoming one of the prominent causes of death in the United States [6][7].
Biofilms form when bacteria aggregate in communities where cells are embedded in an extracellular polymeric compounds matrix attached to a surface [8]. This extracellular matrix is produced by the bacteria itself and composed of exopolysaccharides (EPSs), proteins and other macromolecules such as DNA [9]. Biofilm contribute in protecting the microorganism from host defenses and prevent the effective penetration of antimicrobial agents [10].
A wide range of bacterial pathogens produce and persist in biofilms. S. aureus is one of the most effective biofilm-forming organisms [11]. The ability of biofilm formation is considered as an important contributing factor for the initiation and establishment of chronic infection by S. aureus [12]. Biofilm formation by S. aureus may lead to the delay of re-epithelialization of the infected tissues, leading to increased healing time [13].
It is not fully known whether S. aureus biofilm forming ability qualify it to colonize/infect certain (90.5%) when compared with the biofilm non-producer MRSA (9.5%). Importantly, 89.2% of biofilm-producing S. aureus were multidrug resistant. sites or tissues. In addition, correlating biofilm forming ability to antimicrobial resistance is necessary in the battle of combating S. aureus and its drug resistance.

Sample Collection and Processing
A total of 196 isolates were collected from three major hospitals (Al-Shifa, Al-Aqsa and Al-Nasser Pediatrics hospitals) from different departments and different sites of infection. Isolates were obtained from the microbiology department along with patient's data.
The sampling period started from April 2018 and continued for eight months. All isolates were transferred to Islamic University of Gaza (IUG) Microbiology Research laboratory. Each isolate was then streaked onto the surface of Blood Agar and Mannitol Salt Agar plates and incubated for 24 hours at 37°C for purity check and identification purposes. Glycerol with Brain Heart Infusion Broth (BHIB) were used for long-term storage of isolates.

S. aureus identification
After the incubation period, colonies were identified based on colony color and morphology in addition to Gram stain and the final identification was performed using conventional biochemical tests (e.g, catalase, coagulase, and DNase test). Only confirmed S. aureus isolates werefurther tested.

Antimicrobial susceptibility testing
The susceptibilities of the isolates to ceftaroline, cefoxitin, chloramphenicol, ciprofloxacin, clarithromycin, clindamycin, gentamicin, linezolid, oxacillin, penicillin, rifampicin, tetracycline and trimethoprim-sulfamethoxazole, were determined using the disk diffusion method according to the quidelines and interpretive criteria of the Clinical and Laboratory Standards Institute (CLSI) [14]. A 0.5 McFarland standardized inoculum was spread using cotton swab onto the surface of two Muller Hinton (MH) agar plates. The appropriate antimicrobial disks were applied and plates were transferred to a refrigerator for 30 minutes and then was incubated for 24 hours at 37 o C. The zone of inhibition was measured and interpreted as sensitive, resistant or intermediate according to the CLSI.
Tissue culture plate method A single colony of S. aureus was picked from an overnight-incubated Blood agar plate and inoculated into 2 mL of BHIB supplem ented with 0.25% (wt/vol) glucose (15). The broth was incubated overnight at 37 °C. The culture was then diluted 1:100 with fresh medium. A sterile individual well in 96 flat-bottom polystyrene wells was filled with 200 μL of the diluted culture. Negative control contained broth only and a Positive control (A control strain of biofilm forming S. aureus (ATCC: 6538) was used. The control strain was also processed in a similar manner. The plate was incubated at 37 °C for 72 hours. After incubation, the content of each well was removed. Each well was washed three times with 250 μl of DW. After 15 min, plates were stained for 5 min with 0.2 ml of 2% crystal violet per well. Excess stain was removed and rinsed off by placing the plates under running tap water. The plates were air-dried.
The adherent cells were re-solubilized with 160 μl pure methanol per well [16]. The optical density of each was measured at 570 nm. Results interpreted according to the followings criteria; OD <0.5 (negative), OD ≥ 0.5 (positive) (17). Wells containing only sterile BHIB served as a background control; their average absorbance value was subtracted from all experimental readings [15].

Tube adherence method
An aliquot of 0.1 mL of bacterial culture (obtained by adjusting turbidity to 0.5 McFarland standards) was transferred to glass test tube containing 10 mL BHIB which was incubated at 37°C for 72 hours.
The medium was then removed and the tubes were washed with distilled water, air dried and biofilm formation were assayed by 2% crystal violet [16]. Visible lining of the wall and bottom of the tube by presence of a film were considered as positive.

Data analysis
Collected data were summarized, tabulated and analyzed using Statistical package for Social Sciences (SPSS) software. Chi square test was used to detect significant differences among hospitals and or samples. The results are presented as tables and figures.
All the isolates were obtained from clinical samples, collected from different sources The pus group includes pus from different sources, wound and ear charge samples (N=171), while the body fluids group contains urine, blood, plural and cerebrospinal fluied (N=10). The group labelled as "Others" contain sputum, bone, skin and soft tissue samples (N=15).

Antimicrobial susceptibility test
As shown in Table 1, the highest resistance rate was against penicillin G (92.9%), followed by cefoxitin (80.6%) and oxacillin (67.9%); both of which are used to detect methicillin resistance, while the lowest resistance rate was against linezolid and ceftaroline (1%) Out of 13 antimicrobials used in this study, pinicillin G had the lowestt activity in all three hospitals in the study as showen in Table 2. Chi sqaure statistical analysis showed significanct differences among hospitals with regard to resistance against several antibiotics (P value ≤ 0.05) with Al-shifa hospital showing the highest resistance rate for most of the tested antimicrobials.

Multiple Antibiotc Resistance index (MARI) and multidrug resistance
MAR index was calculated for each isolates by dividing the number of antibiotics that the isolate was resistsnce to by the number of all antibiotics which the isolates was tested for. Any isolate with an index of more than 0.2 is considered as MDR. Among the 196 isolates, 71.4% (N=140) were classified as MDR with a MAR index (≥0.2).

MRSA and MSSA
To identify MRSA, cefoxiten antibiotic disks were used, 80.6% of the isolates were resistant to cefoxitin. The highest resistance to cefoxitin was found at Al-Shiffa hospital 90.4% followed by Al-Nasser pediatrics hospital (79.4%) and Al-Aqsa hospital (70.9%)

Biofilm results
Among 196 isolates, 88.8 % (N=174) were biofilm producers, while only 11.2% (N=22) did not show biofilm formation by the Tissue Culture Plate method. The biofilm-producing isolates percentage in Al-Shiffa hospital was 94.0%, while the percentage was 82.3% and 91.2% in Al-Aqsa and Al-Nasser Pediatics hospitals respectively. Biofilm formation was 100% positve in all specimen type except pus, the percent was (87.1%). About 74.0% (N=145) of the isolates were biofilm producers by the Tube Method distributed among hospitals (79.5%),(67.1%), (76.5%) for Al-Shiffa, Al-Nasser pediatrics and Al-Aqsa hospitals respectively. The biofilm formation was 100% in the specimens that comes from body fluids while it was (71.9%) in the pus group and (80.0%) in the other specimen types as presented in Table 3. Table 4 showes the biofilm forming ability versus antimicrobial resistance. For all the antimicrobialsu-sed, the majority of the resistant strains was biofilm producers but the results don't reach statistically significant level. A total of 125 (89.2%) MDR isolates were biofilm producers while the remaining 15 isolates (10.7%) were non-biofilm producers as presented in Table  5. (No statistical significant differnce was found).

Disscusion
All S. aureus isolates examined in this study were at least resistance to one of the 13 antimicrobials tested. The lowest antimicrobial resistanc was 1.0% for both ceftaroline and linezolid, however, these two antimicrobials are not used in treatment protocols    in Gaza strip hospitals and are not available in the local drug market. Linezolid is a worldwide effective and well tolerated antimicrobial in patients with S. aureus infections. In a study done in Nepal, linezolid showed the highest rate of susceptibility (100%) [18]. In other study done in Cleveland showed that there is an 10.4% emergence of linezolid-resistant S. aureus after prolonged treatment of cystic fibrosis [19]. Thus, linezolid should be considered in treatining MRSA infections.
The highest resistance rate was for penicillin (92.9%), this rate is higher than the percents conducted in two studies in 2016 and 2017 that was 65.9% and 62% respectively, in Gaza Strip [20][21], and was lower than Elbayoumi study that done in 2011, the resistance percent for penicillin was 100% [22] and Hujir study in 2006 (94.0%) [23].
Also oxacillin resistance percent in this study was higher than the result obtained in tow previous studies done in 2006 and one study in 2017 [21][22][23]. These variations could be explained by the variation of sample size, types of samples tested and of course by the specific area covered by these studies.
The rate of resistance for trimethoprim-sulfamethoxazole, ciprofloxacin and chloramphenicol was higher than the percent in Elmanama study in 2016 [20].On the other hand, the resistance to rifampicin and clindamycin was lower than that in 2006 and 2016 (20)(21)(22)(23). This increase in the susceptibility for these antimicrobial may be due to the limited prescription and use in the treatment procedure during the recent years.
The increase in the antibitics resistance rates in this study could be due to the excessive use, misuse and the absence of restriction on the prescription of these antibiotics in Gaza strip [24].
For most antimicrobials tested in the study, Al-Shiffa hospital demonstrated the highest rate of resistance, the differences between hospitals may be due to the differences in the treatment protocols between them, the varied level of commitment of infection control measures and procedures and the high number of patients.
Eight of the 13 antibiotics tested in the study showed the highest rate of resistance in the surgury department, with a staistically significant level for ciprofloxacin (P=0.01), gentamycin (P=0.030) and tetracyclin (P= 0.048). This may be due to the excessive use of these antimicrobial agents in the surgury departments in Gaza Strip hospitals with the absence of clear guidelines. Therefore, it's very important to focus on the choice and duration of antimicrobials agents that used either as pre or post operative prophylaxis or post infection [6].
For detecting MRSA,cefoxitin disk was used rather than methicillin and oxacillin because cefoxitin would overcome the failure of routine oxacillin disks to detect heterogeneous MRSA, it is a good indicator of pinicillin binding protein 2a in S. aureus isolates that carry mecA gene [21]. Therefore, testing of cefoxitin give more reproducible and accurate results than testing with methicillin or oxacillin. This fact was eveident by the results of this study where oxacillin resistance rate was 67.9% while cefoxitin was higher (80.6%).
The overall MRSA percent in this study was 80.6% and this is higher than the results of study conducted in 2006 in Gaza strip which was only 22% [23], and higher than another study done in 2015 (56.3%) [25]. In 2017, MRSA percent in southern "Israel" was 40% [26], while in other neighboring countries like Egypt, Jordan and Lebanon MRSA percent was 52%, 56% and 30% respectively [27].
This increase in MRSA percent in Gaza strip in this study over the last decade maybe due to the presence of high percent of health care worker carrying MRSA according to study done in 2017 [14], the weak infection control procedur and the absence of protocols for diagnosis, detection and quarantine for MRSA patients in Gaza Strip hospitals.
High percent of MRSA was found in pediatric departments (93.8%) and surgery departments (85.1%). In a study done in Gaza Strip hospitals in 2017 for detecting nasal carrige MRSA among health care worker the highest percent of MRSA was in surgery department (35%) [21]. This high resistance rate in these departments may be due the crowdeness, high work load, weak infection control procedure and shortage of stuff.
The prevalence of biofilm formation varied between the two testing methods (88.8% and 74.0% (TCP and TM). Also, there was a little variation betwen the two methods used by a study in Nepal, where biofilm formation was (69.8%) and (65.1%) for TCP and TM, respectivily [28].
Tube method is an easy and low cost test, but it's less accurate than TCP method because it depends on the personal observation (visual inspection) as a qualitaive methode, it can be used as screning test for biofilm formation. While TCP method is a qantitative method that can detect even the weak biofilm producer strains [28].
The present study, shows that the biofilm-producing isolates were associated with higher incidence of antimicrobial resistance when compared with non-producer isolates. In addition, the major rate of the MDR isolates were biofilm producer (89.2%), particularly among the isolates that were identified as MRSA (90.5%). However, theses differences did not reach a statistically significant level.
These results is concordant with a study done in Nepal (2018) concluded that higher rate of antimicrobial resistance is demonstrated among biofilm producers than in biofilm nonproducers. The biofilm-positive strains have a higher tendency to exhibit multidrug resistance and methicillin resistance compared to biofilm-negative strains [28]. Another study showed that 97.5% of biofilm producers from burn wound samples were MRSA [29]. A study done in Spain, found that all the staphylococcal isolates that produced biofilm except one harbor horizontal transfer of antibiotic resistance genes [30].
In addition, our results is concordant with Belbase study in Nepal that showed higher rates of multidrug resistance and methicillin resistance were found among biofilm-producing isolates in comparison to biofilm non-producing isolates [18]. Also, a recent study from Jordan, has demonstrated that most MRSA strains can produce biofilm, and one methicillin-sensitive S. aureus (MSSA) isolate typed as spa t955 showed biofilm formation potential through protein deposition [31]. Therefore, we suggest that biofilm forming capacity of MRSA strains are important factor in developing infection.

Conclusion
The findings in this study demonstrated that higher rates of antimicrobialresistance areexhibited by biofilm producers than by nonproducers biofilm isolates. The biofilm-positive strains have a higher tendency to exhibit multidrug resistance than by nonproducers biofilm isolates. In addition, among MRSA isolates, the majority were biofilm producer. The Journal is an open access peer-reviewed journal that publishes scientific papers about all aspects of antimicrobials. The journal will publish original research articles, reviews, brief reports and case reports dealing with basic and clinical antibacterial agents, antiviral, antiprotozoals, antituberculuous, antifungal and antihelminthes agents. All manuscripts must be prepared in English, and are subject to a rigorous and fair peer-review process. Accepted papers will immediately appear online. The journal aims to advance the knowledge, attitude and the research of chemotherapy in the Arabic world in cooperation with international, national scientific and public societies as well as research centers with similar aims and objectives.