Antibiotic susceptibility pattern of biofilm producing Staphylococci isolated from different clinical samples

Background: Biofilm mediated infection by Staphylococci have a significant negative impact on patient health and necessitate reliable method for detecting biofilm producers. The ability of isolates to produce biofilm make them resistant to host immune response as well as available antibiotics. This study aims to detect biofilm producing ability among clinical staphylococci by phenotypic methods and presence of icaAD genes as well as their antibiotic profile. Methods: A total of 4063 different clinical specimen received in the tertiary care hospital of Nepal were examined and Staphylococci were identified following standard microbiological procedure. The antibiotic resistivity pattern was detected by Kirby Bauer disc diffusion method whereas biofilm formation was detected by three phenotypic methods viz. congo red agar (CRA), tube method (TM) and tissue culture plate (TCP) method. Furthermore, icaAD genes were detected by PCR method. Results: A total of 161 Staphylococci were isolated comprising S. aureus (63, 39.1%) and CNS (98, 60.9%). The isolates were found to be resistant to penicillin and erythromycin. Strong biofilm formation was detected among 6 (3.7%), 22 (13.7%) and 35 (21.7%) by CRA, TM and TCP method respectively. Similarly, among 24 (14.9%) isolates icaAD genes were detected. Biofilm formation was found to be correlated with methicillin resistance. Conclusion: The study showed significant association between phenotypic production of biofilm and presence of ica genes. The biofilm producing isolates were found to be resistant to antibiotics than biofilm non producers.


Background
Staphylococci are Gram positive bacteria often residing as normal flora in human being but now emerging as one of the most common agent for hospital acquired infection [1]. Various diseases associated with this organism ranges from minor skin infection to life threatening endocarditis and septicemia. Staphylococci are most often associated with chronic infections of implanted medical devices [2,3,4]. Biofilm is a major virulent factor along with other agents that attribute to its pathogenesis. Biofilm infections characteristically are refractory to antibiotic treatments leading to treatment failures and relapse of infections. In addition, biofilms are also the source of metastatic infections because of their dispersal mechanism once they get matured [5]. According to the CDC, 65% of infections are associated with biofilms formed by the most notorious pathogens such as S. aureus, S. epidermidis and P. aeruginosa [6]. Biofilms can be defined as multicellular communities of bacteria, immobilized by an extracellular polymeric matrix produced by the bacteria, which can be attached to various biotic and abiotic surfaces [7]. The biofilm formation is mediated by Polysaccharide Intercellular Adhesion (PIA) which is 1, 6 linked 3 N-acetylglucosamine polymer responsible for cell-cell attachment and is the gene product of icaADBC [8]. Once grown as a biofilm, the embedded bacteria are protected from various physical, chemical and biological stresses. They develop high resistance to mechanical interference, mechanisms of innate and acquired host defenses, and antibiotic treatment. In fact, biofilms can resist antibiotic concentration 10-10,000 folds higher than those required to inhibit the growth of free floating bacteria [9,10].
The knowledge on correlation of biofilm formation and antibiotic resistance as well as biofilm producing genes among clinical staphylococci will be helpful for preventive and therapeutic management of staphylococci infection and developing new strategies in their treatment.

Methods
The hospital based cross sectional descriptive study was conducted at KIST Medical College and hospital, Imadol, Lalitpur, Nepal. A total of 4063 different clinical samples like blood, urine, wound swab, different types of tips received in clinical microbiology lab of KIST Hospital were subjected to microbial analysis. All the clinical specimens were processed by standard microbiological technique as described by Cheesebrough [11]. The isolates were identified as staphylococci following Gram staining and different biochemical tests. Coagulase enzyme production by slide and tube method and DNase production were used to confirm the isolates as S. aureus. The species of CNS were identified based on simplified scheme proposed by Cunha et al. [12]. The antibiotic susceptibility test were performed towards various antibiotics by the modified Kirby Bauer disk diffusion method within the guidelines of Clinical and Laboratory Standard Institute (CLSI) [13].

Detection Of Biofilm Formation
Three phenotypic methods i.e. Congo Red Agar method (CRA), Tube method (TM) and Tissue Culture Plate Method (TCP) and polymerase chain reaction (PCR) for the detection of ica genes were used for detection of biofilm formation ability of isolates. All tests were performed using Staphylococcus epidermidis ATCC 35984 as positive control and repeated three times.

Congo red Agar method (CRA)
CRA medium was prepared with brain heart infusion broth 37 g/L, sucrose 50 g/L, agar (10 g/l) and Congo Red indicator 8 g/L. First Congo Red stain was prepared as a concentrated aqueous solution and autoclaved (121ºC for 15 minutes) separately from the other medium constituents. Then it was added to the autoclaved brain heart infusion agar with sucrose at 55ºC. CRA plates were inoculated with test organisms and incubated at 37ºC for 24 h aerobically. Black colonies with a dry crystalline consistency indicated biofilm production [14].

Tube Adherence method
A loopful of test organisms was inoculated in 10 mL of trypticase soy broth with 1% glucose in test tubes. The tubes were incubated at 37ºC for 24 h. After incubation, tubes were decanted and washed with phosphate buffer saline (pH 7.3) and dried. Tubes were then stained with crystal violet (0.1%).
Excess stain was washed with deionized water. Tubes were dried in inverted position and observed for biofilm formation. Biofilm formation was considered positive when a visible film lined the wall and the bottom of the tube. The amount of biofilm formed was scored as 1 (weak/none), 2 (moderate) and 3 (high/strong) [15].

Tissue Culture Plate Method
Organisms isolated from fresh agar plates were inoculated in 10 mL of Brain Heart Infusion (BHI) broth supplemented with 2% sucrose. Broths were incubated at 37ºC for 24 h. The cultures were then four times. This removed free floating bacteria. Biofilm formed by bacteria adherent to the wells were fixed by 2% sodium acetate and stained by crystal violet (0.1%). Excess stain was removed by using deionized water and plates were kept for drying. Optical density (OD) of stained adherent biofilm was obtained by using micro ELISA auto reader at wavelength 570 nm [15]. The genomic DNA was extracted as previously described using the DNA extraction Kit following the manufacturer instructions (Thermo Fischer).
The sequences of icaA and icaD (accession number U43366) were taken from the GenBank sequence of the National Center for Biotechnology Information (NCBI) database. Primers specific for icaA and icaD were designed by the Primer3 program and were purchased from Solis Biodyne (Denmark). The primer used for the detection of icaA was forward 5'-TCTCTTGCAGGAGCAATCAA and reverse 5'-TCAGGCACTAACATCCAGCA primer generating a product size of 188-bp. Similarly, for detection of icaD, 5'-ATGGTCAAGCCCAGACAGAG was used as a forward primer and 5'-CGTGTTTTCAACATTTAATGCAA was used as a reverse primer with the product size of 198 bp. The PCR product was analyzed in 2% agarose gel stained with SYBR safe (Invitrogen) dye.

Data analysis
The statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, United States) software.
Chi-square test was used to compare between groups of clinical isolates and P-values < 0.05 were considered statistically significant.   (Table 3).

Detection Of Biofilm Formation
Among all the Staphylococci isolates, black colonies were produced by 6 (3.7%) isolates in CRA while 16 (10%) isolates were moderate biofilm producers that showed Bordeaux colored colonies.
Remaining 139 (86.3%) isolates were found to be biofilm non-producers whose colony color was pink to red. Strong biofilm production was observed only among CNS.
While TCP method detected 5(3.1%) biofilm producers among S. aureus and 14 (8.7%) among CNS. In total of 161 isolates, 24 (14.9%) isolates were found to possess both icaA and icaD genes comprising 6 (3.7%) S. aureus and 18 (11.2%) CNS isolates. None of the genes were identified in 137 (85.1%) isolates. (Table 4). Methicillin resistivity among ica positive isolates In total of 161 isolates, 24 (14.9%) isolates were found to possess both icaA and icaD genes comprising 6 (3.7%) S. aureus and 18 (11.2%) CNS isolates. None of the genes were identified in 137 (85.1%) isolates. The ica genes were harbored by methicillin resistant than methicillin sensitive isolates of both S. aureus and CNS (Table 5). Evaluation of different methods for the detection of biofilm production When different methods for the detection of biofilm formation was analyzed, it was found that TM method is statistically significant when compared with presence of ica genes whereas other two phenotypic methods were statistically insignificant (Table 6).   [24]. It has been noticed in several studies that the S. epidermidis is the most frequently isolated species in nosocomial infections.
Staphylococci are commensals as well as pathogens of human beings and because of their versatile nature they were isolated from different clinical samples. Out of 161 Staphylococci, the highest number of CNS were isolated from blood 54 (33.5%) and S. aureus from W/P 47(29.2%). Increased antibiotic resistance, in addition to the increased frequency of invasive surgery, increased use of intra vascular devices, and increased number of patients with immune compromised status because of HIV infection or immunosuppression after transplantation or cancer treatment, has led to sharp increases in the incidence of S. aureus bacteremia and S. aureus infective endocarditis [25,26] and is associated with significant mortality and morbidity. Bloodstream infection with the S. aureus is associated with mortality rate of about 30% and the incidence is increasing [27].
In order to fight bacterial infections successfully, the rapid recognition of proper treatment modalities are critical. The determination of antibiotic susceptibility and resistance are keys to this process [28].
Resistance has been observed to every class of antibiotic, regardless of whether it was derived from natural or synthetic origins. The emergence of antimicrobial resistance among Staphylococci isolates is one of the important factor in nosocomial infection. About 90% of the S. aureus strains found in hospitals are now resistance with penicillin G. With the extensive exploitation of therapeutic agents, CNS also have lost its susceptibility to most of the available antibiotics and become resistance to most active antimicrobials that is β lactams and other antimicrobial classes [29].  [31]. Likewise prevalence of MRCNS is (12) 25% among 48 CNS isolates which was in accordance with other studies [27,35,36] but opposed with the findings of others [26]. Similarly, prevalence of MRCNS ranging from 48.2-60% has been reported in India [6] which was comparatively higher than our study. Overall, data indicated by this study shows slightly lower rate of MRSA and MRCNS than that reported by other studies.
Pathogenesis of Staphylococci is attributed to a number of virulence factor and biofilm formation is thought to be the most important one. There are number of methods available for biofilm detection.
Both phenotypic and genotypic methods were used to analyze the ability of biofilm production in all isolates. Growth of organism on the surface of CRA media is simple, easy and inexpensive method for detection of slime production. Investigation of biofilm by CRA showed 22 (13.7%) staphylococcal isolates positive for the slime production. Among CRA positive, only 6 isolates formed black colonies representing the strong biofilm production. Variable result was obtained from various researches [19,21,23,37]. Slime formation is not always indicative of biofilm formation in vivo as highlighted by Investigation of biofilm production by the tube method showed 24 (14.9%) isolates as strong biofilm producers, 16 (9.9%) moderate and 121 (75.2%) weak/non-biofilm producers. This result is comparable with Mathur et al. [39] (11.8%) but the data is less than that observed by other researchers [40,41]. The result of tube method is based on visual observation of adherent on the wall of tube. So, it is difficult to discriminate between weak and biofilm negative isolates due to the variability in observed result by different observers.
The TCP method detected 35 (21.7%) strong and 84 (52.2%) weak biofilm producers. The TCP method is a convenient and quantitative technique that directly detects the polysaccharide production by measuring the adherent biofilm by spectrophotometer. TCP is the most widely used and was considered as standard test for the detection of biofilm formation [20,39]. This method has been reported to be the most sensitive, accurate and reproducible screening method for the determination of biofilm production by clinical isolates of Staphylococci and has the advantage of being a quantitative tool for comparing the adherence of different strains [15,39].
Previous studies have shown the presence of ica locus in clinical isolates emphasizing their increased virulence as compared to the saprophytic strains [44,42]. Besides, plethora of studies has demonstrated the causal link between staphylococcal biofilm and the presence of ica operon (icaADBC genes) [38,43,44], which in turn are involved in the PIA production; the most extensively characterized staphylococcal biofilm component. In ica operon, mainly co-expression of icaA and icaD has been demonstrated to be necessary for phenotypic expression of biofilm production in clinical staphylococcal isolates [23,43]. Besides, being reliable yet efficient, PCR of ica genes has been extensively used for the detection of biofilm formation [20,23,43]. In the present study, concomitant presence of icaA and icaD genes was detected in 24 (14.9%) staphylococcal isolates comprising of 6 (3.7%) S. aureus and 18 (11.2%) CNS isolates. Previous studies have also demonstrated the presence of ica genes in clinical staphylococcal isolates. Los et al. [45] showed the prevalence of ica operon in 27.4% nasopharyngeal S. epidermidis isolates from hospitalized patients. Oliveira & Cunha [23] detected ica genes in 40% CNS isolated from clinical specimen and nares of healthy individuals.
Likewise, Cafiso et al. [46] found 35% of the isolates positive for icaA and icaD genes, Silva et al. [47] showed 40% staphylococcal isolates positive for ica genes respectively. Altogether, these results indicate importance of ica genes in biofilm production in device associated infections.
This low rate of ica detection as compared to the previous studies [21,23,38,45,46] may be due to difference in in-vivo and in-vitro conditions possibly contributing to the physiological changes of the pathogen modulating biofilm formation capabilities. For instance, ica genes are expressed in the stressful environment such as high osmolarity, anaerobic condition, high temperature, and subinhibitory presence of some antibiotics [17,38]. Studies have demonstrated biofilm formation via PIAindependent mechanisms in S. aureus [48]. A number of transcriptional regulators have been reported in ica-independent biofilm production. These include araC-type transcriptional regulator or regulator of biofilm (rbf), which controls the biofilm production by novel regulatory mechanism [49].
Likewise, biofilm-associated protein (Bap); the first gene known to form biofilm via icaADBC independent in S. aureus from bovine mastitis isolates. Although initially, it appeared to be absent in human clinical S. aureus isolates, Bap protein has now emerged associated with more than 100 surface proteins that are involved in biofilm formation [50]. In the clinical S. aureus isolates of UAMS-1 strain, mutation of ica locus showed little effect on biofilm formation, thus, suggesting the presence of additional loci relevant to biofilm formation [24]. Also, studies suggest the regulation of biofilm by global regulator SarA in ica-independent mechanisms [43]. However, given the undeniable role of icaADBC in biofilm matrix formation and that PCR enables rapid diagnosis of slime producing virulent strains assays; implementation of genotypic measure is strongly suggested in routine diagnostic laboratory. We reason many factors as environment, nutrition, sub inhibitory concentration of certain antibiotics, and stress (temperature, osmolarity) might play a significant role in biofilm formation resulting in varied frequency of biofilm producers among clinical isolates [17, 38,48].
From a clinical perspective, the discrepancy between genotype and phenotypic resistance expression suggest that a susceptible strain harboring, but not expressing, an antibiotic resistance gene should be regarded as potentially resistant to that antibiotic. Overall, we did not detect a significant presence of antibiotic resistance genes, compared to the great biofilm resistance of the isolates [45].
In consistence with previous studies, CRA and TCP method correlated well in positive results [23,39,46]. However, evidences of false negative results in CRA method while comparing with TCP method suggest that CRA method alone cannot be solely depended upon for the precise detection of biofilm formation. Taken together, in this study, the modified TM method showed the best correlated result with genotypic assay suggesting its importance in routine diagnostic laboratories. Oliveira & Cunha [23] also reported good sensitivity and specificity for the tube test and PCR when analyzing isolates obtained from infection. According to Cunha et al. [12], the test provides reliable results for biofilm detection in CNS and is adequate for routine use.

Conclusion
The study showed a significant association in between phenotypic production of biofilm and presence of ica genes. Taken together, this study demonstrates the high prevalence of methicillin resistant isolates producing biofilms in clinical staphylococcal samples. Since staphylococcal infections have a significant impact on morbidity and mortality, prevention and management of these infections remain a priority. This study, while bringing additional information about the status of biofilm producing clinical strains and their association with multiple antibiotic resistances, highlights the importance of early detection strategies in routine diagnostics. Implementation of those will help to identify biofilm producing cases to prevent occurrence of treatment failures of staphylococcal infections in Nepal.

Limitation Of The Study
The study of all genes responsible for biofilm production other than icaA and icaD genes could not be carried out. The presence of ica genes were not tested with antibiotic resistant genes to confirm the resistivity.