Resistance profiles and biofilm formation of coagulase negative staphylococci isolated from clinical specimens in a tertiary care hospital in Palestine

Background: Coagulase-negative staphylococci (CoNS) represent one of the major resistant nosocomial pathogens where its biofilmrelated infections often fail to respond to antibiotic chemotherapy. Here, we studied the resistance profiles and biofilm formation in CoNS isolates from clinical specimens at Al Shifa hospital in Gaza, Palestine. Methods: This study was carried out from March to July 2013 and included 81 clinical isolates. Identification and antibiotic susceptibility testing were performed using VITEK-2 system. The presence of nuc and mecA genes was performed using multiplex PCR. Qualitative and quantitative biofilm assays were performed using standard methods. Results: Of the 81 clinical CoNS isolates, S. haemolyticus was the most common species (34, 42%), followed by S. epidermidis (26, 32.1%) and S. saprophyticus (13, 16%). The majority of isolates (83.9%) were from surgery, ICUs, pediatrics and medicine wards and the most common source was pus (28, 34.6%). Antibiotic resistance was highest against aminoglycosides, β-lactams, carbapenems, cephalosporins, fluoroquinolones, fosfomycin and macrolides. Though, no resistance was detected against rifampicin, vancomycin, teicoplanin, nitrofurantoin, linezolid and mupirocin. The antibiotic resistance among MR-CoNS was significantly higher than that among MS-CoNS. Nearly 88.9% of isolates were multidrug resistant with higher percentage among MRCoNS. Most S. epidermidis (76.9%) isolates were biofilm producer, with statistically significant association between methicillin resistance and biofilm production. Resistance profiles and biofilm formation of coagulase negative staphylococci isolated from clinical specimens in a tertiary care hospital in Palestine Nahed A. Al Laham1, Emad Abou Elkhair2, Abdallah Bashir2, Nahed Abdelateef3 1 Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al Azhar University-Gaza, Gaza Strip, Palestine. 2 Department of Biology, Faculty of Science, Al Azhar University-Gaza, Gaza Strip, Palestine. 3 The Central Laboratories, Ministry of Health, Gaza Strip, Palestine. Contact information: Dr. Nahed A. Al Laham. Address: Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al Azhar University-Gaza, P. O. Box 1277, Gaza Strip, Palestine.  nahedallaham@gmail.com The InTernaTIonal arabIc Journal of anTImIcrobIal agenTs ISSN: 2174-9094 2017 Vol. 7 No. 3:4 doi: 10.3823/814 This article is available at: www.iajaa.org / www.medbrary.com 2 Introduction Coagulase-negative staphylococci (CoNS) are now representing one of the major nosocomial pathogens and among the most frequently isolated bacteria in the clinical microbiology laboratories [1-5]. They are responsible for bacteremia, endocarditis, mediastinitis, meningitis and progressive joint destruction mainly in patients with neutropenia, indwelling foreign devices, intravascular catheters or other foreign bodies [1, 3, 4]. The clinical most relevant CoNS are Staphylococcus epidermidis, S. lugdunensis, S. saprophyticus , and S. capitis [6, 7]. Biofilm production by CoNS, specially S. epidermidis , is considered as an important factor in the pathogenesis of implanted medical devices associated infections [1, 2, 8, 9]. Biofilm formation takes place in four successive phases: the attachment of the bacteria to biotic or abiotic surface; the proliferation and accumulation of bacteria in multilayered cell clusters; the growth of biofilm into a thick and structured layer, and finally the detachment and circulation of single cells or cell agglomerates via the bloodstream [1, 2]. Many literature surveys revealed that CoNS showed high resistance against most of the commonly used therapeutic antibacterial agents including methicillin. Irrespective of geographical locations, a worldwide SENTRY study showed that, about 70-75% of CoNS are resistant to methicillin [10]. There is a significant increase in the methicillin-resistant coagulase negative staphylococci (MR-CoNS) infections and these bacteria have recently started to gain resistance to other widely used antibiotics [11-14]. The increased recognition of pathogenic potential of CoNS and emergence of drug resistance among them justify the need to identify various species of CoNS and determine their antibiotic resistance pattern. Epidemiological data about CoNS in Palestine are either scarce or insufficient [15]. To the best of our knowledge, this is the first report describing clinical CoNS infection within the Gaza Strip hospitals. To that end, we conducted this study to determine the frequency of CoNS species isolated from various clinical specimens and to assess their resistance profile to most commonly used antibiotics at Al Shifa hospital in Gaza Strip. Moreover, biofilm forming capacity of S. epidermidis isolates was investigated. Materials and Methods Study design and setting This study was conducted for five months between March and July 2013 at the clinical microbiology laboratory of the largest medical complex hospital

Biofilm production by CoNS, specially S. epidermidis , is considered as an important factor in the pathogenesis of implanted medical devices associated infections [1,2,8,9]. Biofilm formation takes place in four successive phases: the attachment of the bacteria to biotic or abiotic surface; the proliferation and accumulation of bacteria in multilayered cell clusters; the growth of biofilm into a thick and structured layer, and finally the detachment and circulation of single cells or cell agglomerates via the bloodstream [1,2].
Many literature surveys revealed that CoNS showed high resistance against most of the commonly used therapeutic antibacterial agents including methicillin. Irrespective of geographical locations, a worldwide SENTRY study showed that, about 70-75% of CoNS are resistant to methicillin [10]. There is a significant increase in the methicillin-resistant coagulase negative staphylococci (MR-CoNS) infections and these bacteria have recently started to gain resistance to other widely used antibiotics [11][12][13][14].
The increased recognition of pathogenic potential of CoNS and emergence of drug resistance among them justify the need to identify various species of CoNS and determine their antibiotic resistance pattern. Epidemiological data about CoNS in Palestine are either scarce or insufficient [15]. To the best of our knowledge, this is the first report describing clinical CoNS infection within the Gaza Strip hospitals. To that end, we conducted this study to determine the frequency of CoNS species isolated from various clinical specimens and to assess their resistance profile to most commonly used antibiotics at Al Shifa hospital in Gaza Strip. Moreover, biofilm forming capacity of S. epidermidis isolates was investigated.
in Gaza Strip, Al-Shifa hospital with 500 acute care beds. The study was approved by the department of human resources and development in the ministry of health (MOH) and by the Helsinki committee at the MOH in Gaza Strip (Approval no. PHRC/ HC/36/14).

Sampling and bacterial isolates
Eighty one non-duplicate CoNS isolates associated with diverse clinical infections were collected. The isolates represent different sources including pus, urine, blood, sputum, burn and surgical swabs of hospitalized patients in surgery, pediatrics, medicine, burns, gynecology and ICUs departments. These isolates were collected in 2013, out of a total unique 1121 bacterial isolates.

Conventional techniques
CoNS isolates were identified phenotypically based on colonial morphology, Gram stain, and by using the following methods: catalase test, tube coagulase test, Pastorex™ Staph Plus latex agglutination (Bio-Rad, Hercules, California), and the Staph ID 32 API system (bioMérieux, France) according to the manufacturer's instructions. VITEK-2 automated system was used to identify CoNS to the species level using VITEK-2 database, version 4.03 (bioMérieux, Marcy l'Etoile, France). Screening for MR-CoNS was done by testing for oxacillinand cefoxitinresistance using the VITEK-2 Compact Gram Positive Card (bio-Mérieux, Paris, France).

Molecular techniques
DNA isolation: Isolates were overnight subcultured in brain heart infusion broth and pelleted by centrifugation at 5, 000 rpm for 15  minutes. After cooling to room temperature, the suspension was centrifuged at 10, 000 rpm for 5 minutes, and the supernatant was used directly as template for PCR reactions [16].
Detection of nuc and mecA genes:A multiplex PCR assay was used for detection of the nuc gene to exclude any coagulase negative S. aureus isolate and mecA gene for detection of methicillin-resistance among CoNS isolates [17,18].Briefly, primers mecA-1 (5'-GGGATCATAGCGTCATTATTC-3') and mecA-2 (5'-AACGATTGTGACACGATAGCC-3') for the gene mecA and nuc-1 (5'-TCAGCAAATGCAT-CACAAACAG-3') and nuc-2 (5'-CGTAAATGCACTT-GCTTCAGG-3') for the gene nuc were used in a multiplex PCR reaction on a T100™ Thermal Cycler (BioRad, USA) under the following conditions: an initial 5-min denaturation step at 95°C, followed by 30 cycles of 1 min of denaturation at 95°C, 1 min of annealing at 59°C, and 2 min of extension at 72°C; with a final extension step at 72°C for 10 min. The sizes of the amplicons were 530bp and 280bp for mecA and nuc genes, respectively.

Antimicrobial susceptibility testing
Antibiotic susceptibility testing for the CoNS isolates was performed with the automated VITEK-2 Compact automated system, using the VITEK-2 Compact Gram Positive Card (bio-Mérieux, Paris, France); Multidrug-resistant (MDR) phenotype was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories used in this study [19].

Biofilm production
The biofilm forming capacity of only S. epidermidis strains was investigated qualitatively and quantitatively by methods previously described. For qualitative biofilm production, a simple method using growth on Congo red agar (CRG) for detecting biofilm formation was used [20]. With brain heart infusion agar containing Congo red stain at 0.8 g/ liter (BDH Chemicals Ltd., Poole, United Kingdom), biofilm-positive strains yield black colonies with a dry crystalline surface appearance, while biofilmnegative strains mostly give a red colonies.
For quantification assay of biofilm-forming capacity, tests similar to those described previously were applied [9,21,22]. Briefly, S. epidermidis strains were cultivated overnight in 10 ml of tryptic soy broth (TSB) supplemented with 0.25% (wt/vol) glucose. Cultures were diluted in TSB supplemented with 0.25% (wt/vol) glucose to a final OD578 of 0.1 and 200 µl of the cell suspension was used to inoculate sterile, 96-well flat-bottom polystyrene microtiter plates (Greiner bio-one; Cellstar). After cultivation for 24 h at 37°C, the wells were emptied and the contents were gently washed three times with 200 µL of PBS (pH 7.2) to remove free-floating planktonic bacteria. The plates were air dried for 10 min., and the remaining surface-adherent cells were stained with 0.1% safranin (Serva) for 30 s. Absorbance at 490 nm (A490) was measured with a Micro-ELISA Autoreader (SpectraMAXGeminiXS; Molecular Devices). Wells containing only sterile TSB served as a background control; their average A490 value was subtracted from all experimental readings. S. epidermidis RP62A and S. carnosus TM300 were used as positive and negative controls, respectively. Each assay for each strain was performed in quadruplicate.

Statistical analysis
The results were tabulated, encoded and statistically analyzed using Statistical Package for Social Sciences (SPSS ® ) program version 17 (Chicago, IL, USA). Fisher's exact 2-tailed and Pearson's χ 2 tests were used for categorical variables to compare frequencies of CoNS species and MR-CoNS positivity and negativity. Likewise, percentages as of antibiotic resistance profiles of MR-CoNS and MS-CoNS were compared with Pearson's χ 2 or Fisher's exact test as appropriate. P-value were calculated and P< 0.05 was considered statistically significant.

Identification and distribution of CoNS and MR-CoNS species
Out of a total 1121 bacterial isolates, 81 strains of CoNS that belong to six species were identified. S. haemolyticus was the most common species isolated (34, 42%), followed by S. epidermidis (26,32.1%) and S. saprophyticus (13, 16%) ( As stated in Table 2, the highest numbers of S. haemolyticus and S. epidermidis strains were recovered from pus specimens (41.2%, 34.6% respectively), while S. saprophyticus was mainly recovered from urine and swab specimens (38.5% each). According to the type of hospital ward, the highest numbers of S. hominis, S. epidermidis and S. haemolyticus strains were recovered from surgery ward (40%, 30.8%, 29.4% respectively), whereas S. saprophyticus was mainly recovered from pediatric and surgery wards (23.1% each) ( Table 2). As shown in Table 3, most of S. haemolyticus and S. epidermidis isolates were methicillin resistance (94.1%, 88.5% respectively) with a statistical significant difference in comparison to its counterpart     methicillin sensitive (P< 0.0001). Surprisingly, all S. saprophyticus isolates were methicillin sensitive.

Antimicrobial resistance profiles
The antimicrobial resistance profile of CoNS isolates to the 28 antibiotics tested was shown in Table 4 and Figure 1. Resistance to multiple classes of antimicrobials was observed among CoNS isolates, in-cluding aminoglycosides, β-lactams, carbapenems, cephalosporins, fluoroquinolones, fosfomycin and macrolides. However, no resistance was detected to any of the following antibiotics: rifampicin, vancomycin, teicoplanin, nitrofurantoin, linezolid and mupirocin. Yet, a very low to low resistance rate was observed to tigecycline, moxifloxacin and clindamycin ( (Table 4).

Biofilm production
Out of 26 isolates of S. epidermidis examined, a significant number (20,76.9%) produced biofilms as verified by formation of black colonies with a metallic sheen after 24 h of incubation on CRA plates and by polystyrene microtiter plate assay (P < 0.0001). 82.6% of which were in MR-CoNS, and 33.3% in MS-CoNS. Methicillin resistance was found to be significantly higher in biofilm positive strains (19,82.6%) than in biofilm negative strains (4, 17.4%) (P <0.0001) ( Table 5). Also, biofilm producer strains were significantly higher among CoNS isolates recovered from pus, swabs and blood specimens (P = 0.0013, P = 0.01, P = 0.028 respectively).

Discussion
Data regarding CoNS and MR-CoNSin Palestine are insufficient, with only one report from Nablus, Palestine [15]. Previous reports from Gaza Strip and West Bank of Palestine have mostly focused on epidemiology of S. aureus and MRSA both in hospital and community settings [23][24][25][26][27]. Here, we described for the first time the resistance profiles and biofilm formation in CoNS isolated from clinical specimens in Gaza, Palestine. S. haemolyticus was the most common species isolated (34, 42%), followed by S. epidermidis (26, 32.1%). This finding is comparable to the findings in a study conducted in China where the prevalence of S. haemolyticus was the highest (34.1%), followed by S. epidermidis (27.4%) [4]. Also in another recent study in Brazil, S. haemolyticus was the most prevalent species [9]. However, many studies worldwide showed S. epidermidis is the major isolated species followed by S. haemolyticus or other CoNS [10,11,[28][29][30]. These differences in CoNS species rates from the worldwide literature are depending on the country, hospital specialty, study design and setting. In this study, the most common clinical source of CoNS was pus (34.6%), followed by swabs (23.5%), urine (18.5%) and blood (14.8%). Two Indian studies showed that pus specimens were the most common source of CoNS [31,32]. Furthermore, these results are nearly similar to the study done in Pakistan in which percentage of CoNS isolates in pus was 36.7%, and in swab was 17.3% but their findings in regard to the blood (45.9%) was higher [12]. Also, other studies revealed higher CoNS isolates from blood cultures as in Pakistan (53.5%) [33], Jordan (30.9%) [11], Tunisia (29%) [34], and Iran (25.4%) [35]. Yet, in a recent study from Jordan and in accordance with our findings, the ICU and surgery wards accounted for the majority of all CoNS isolates obtained (41%) [11]. A plausible explanation for the differences in the isolation rates of CoNS from clinical source and wards between different countries and even some time in the same country may be due in part to differences in sample size or frequency of sampling and using different conventional and/or molecular methods among others for diagnosis. Screening for MR-CoNS revealed that 74.1% were MR-CoNS which is nearly similar to the studies done at Pakistan (70.3%) [12], China (70%) [4], USA (74%) [36], and India (68.4%) [37]. Likewise, prevalence of MR-CoNS in various parts of Europe is in the range of 60-75% [38]. On the other hand, there were significant higher rates of MR-CoNS in studies conducted  [10,39]. Yet, other studies found less prevalence of MR-CoNS in comparison to our results as from Iran (50%) [30] and India (32.7%) [28]. In this study, the resistance profile of all CoNS isolates showed complete to high resistance to most of the antibiotics tested. This pattern of resistance was shown in previous studies in different countries around the world [4,8,[10][11][12][13]15]. High resistance to the aforementioned antibiotics could be mainly due to excessive use, misuse, and great prescription of these medication in Gaza for both hospital and community acquired infections and also in agriculture and animal feeding. Moreover, the lack of an antibiotic policy and the availability of antibiotics sold without a medical doctor prescription in Palestine worsen the case [27]. In view of the high resistance rates of CoNS to these aforementioned antibiotics, treatment of infections caused by CoNS, particularly S. epidermidis and S. haemolyticus at Al Shifa hospital with these antibiotics may not be effective. In contrast, no resistance of CoNS isolate was detected to rifampicin, vancomycin, teicoplanin, nitrofurantoin, linezolid and mupirocin, and very low resistance was detected to tigecycline and moxifloxacin (3.7%, 8.4% respectively). Our findings are in agreement with many studies that showed full activity of most of these aforementioned antimicrobials against clinical CoNS isolates [8,11,13,15].Conversely, many reports from around the world showed different percentage of resistance to vancomycin, teicoplanin and linezolid ranging from 0.8% to 13% [4,12,39]. We think that this zero resistance in this study to glycopeptides and linezolid may be due to its limited prescription and use in Gaza Strip hospitals. So, the full sensitivity to vancomycin, teicoplanin and linezolid can work perfectly against clinical CoNS isolated in Gaza hospitals as indicated against S. aureus in recent reports from Gaza Strip [24,27]. On the other hand, a very low resistance rate was observed to tigecycline and moxifloxacin (3.7% and 8.4% respectively). MR-CoNS showed low to moderate resis-tance ratios against clindamycin (18.3%), tetracycline (35%), fusidic acid (38.3%) and gentamycin (60%). This picture contradicts a study conducted in Turkey where significantly higher resistance to clindamycin (72%), tetracycline (60%) and gentamycin (90%) was reported. Yet, fusidic acid resistance (25%) was lower than in our findings [40]. The least resistance species found in this study was S. saprophyticus. Same recent report from Jordan found low resistance rate of S. saprophyticus isolated from urine samples [11]. The resistance rate of MR-CoNS isolates to β-lactam inhibitor antibiotics, cephalosporins, tetracycline, gentamicin, tobramycin, levofloxacin, SXT, and fusidic acid were significantly higher than those of MS-CoNS isolates (P value < 0.05) ( Figure  1). This picture is mirrored others elsewhere [4,8,[10][11][12][13]40]. MDR phenotype was detected in 88.9% of CoNS isolates. This high level of MDR in our CoNS collection is similar to many studies conducted in different countries around the world as from China [4], Australia [5], Nigeria [8], Saudi Arabia [10], Jordan [11], Pakistan [12], India [13], West Bank of Palestine [15] and Turkey [40].
Our study has some limitations. First, it is worthwhile to mention that a number of our isolates could be contaminants and not real pathogens. Second, we performed this study for short time in one referral hospital in Gaza Strip. So, this may underestimate or overestimate the real prevalence of CoNS species and MR-CoNS. Third, molecular typing was not performed, such that the clonality of the CoNS strains cannot be assessed and also the biofilm associated genes were not investigated.

Conclusion
Several themes of potential concern are raised by our findings. Most noteworthy is the high prevalence of MR-CoNS and its high resistance profile against most agents tested in this study except for new or not commonly used antibiotics in Gaza Strip. Also, most of S. epidermidis isolates were biofilm producers. Furthermore, high numbers of CoNS isolates were shown to be MDR. These results highlight the critical need for monitoring and managing the usage of antibiotics in our hospitals and community.