Evaluation of methods to detect in vitro biofilm formation by staphylococcal clinical isolates

Objective Staphylococcus genus comprising both Staphylococcus aureus and coagulase negative staphylococci (CoNS) are widely distributed in nature and can infect diversity of hosts. Indeed, staphylococci are the major pathogens causing biofilm associated infections caused by contaminated hospital indwelling devices. These infections are persistent in nature being highly refractory to various stresses including antibiotics. Implementation of efficient diagnostic techniques for the biofilm production would help minimize the disease burden. Thus, early detection of pathogenic strains producing biofilms warrant the utmost importance in diagnostic laboratories especially in resource limited settings. Result Among 375 isolates collected from different clinical specimens, 214 (57%) were identified as coagulase negative staphylococci and 161 (43%) S. aureus. Detection of In-vitro biofilm formation in these isolates were carried out by three commonly used phenotypic assays and a genotypic assay. While evaluating the results, tissue-culture method with supplemented glucose and sucrose showed the best correlation with the results of genotypic assay. Electronic supplementary material The online version of this article (10.1186/s13104-018-3820-9) contains supplementary material, which is available to authorized users.


Introduction
Staphylococcus spp., widely distributed in nature, colonize the skin and anterior nares of humans. However, upon achieving the favorable environment, they can infect the diversified hosts [1,2] due to the presence of numerous virulence factors including exotoxins, enzymes, surface proteins, ability of biofilm production and acquisition of resistance to multiple drugs [3][4][5].
Biofilm is a structured community of bacterial cells enclosed in self-produced polymeric matrix adherent to an inert or living surface [6][7][8]. As implant devices are increasingly used in medical practice, staphylococcal infections are now considered one of the major nosocomial infections [9,10]. Biofilm associated infections are characteristically refractory to different stresses including host immune defense and antibiotics, leading to persistent infections [2,[11][12][13].
The polysaccharide intercellular adhesin (PIA) is the main biomolecule responsible for cell aggregation and biofilm formation. PIA biosynthesis is carried out by the proteins encoded by the ica operon (icaADBC) [14][15][16]. Given that staphylococcal infections associated with medical devices have significant impact on morbidity, mortality and socio-economic burden, prevention and management of such infections remains a priority. Thus, detection and differentiation of staphylococci in their ability to form biofilm in routine laboratory practice bear great importance to initiate effective treatment measures and minimize unsuccessful antibiotic therapies [7,17].
Various phenotypic methods like Congo-red agar method (CRA), tube method (TM), tissue culture plate method (TCP), electron microscopy, confocal scanning microscopy and bioluminescent assay are available for the detection of biofilm formation in staphylococcal infections [7,18]. Detection of biofilm related genes using PCR techniques have been increasingly used, but Open Access BMC Research Notes *Correspondence: nsrivastava1@amity.edu; neersar@gmail.com 2 Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Noida, UP 201303, India Full list of author information is available at the end of the article this may be infeasible as routine diagnostic in a resourcelimited country like Nepal. Therefore, in the present study, we sought to compare and evaluate the sensitivity and specificity of three most commonly used phenotypic assays with the genotypic assay to detect biofilm production.

Materials and methods
A total of 375 clinical staphylococcal isolates were collected from two tertiary care hospitals from 2015 to 2017. Staphylococci were isolated and identified from various clinical samples by standard microbiological techniques [19]. High biofilm producer strain Staphylococcus epidermidis ATCC 35984 was used as reference strain in all the tests performed. All experiments were performed in triplicate and repeated thrice.

Phenotypic assay
The in vitro biofilm production was measured using phenotypic assays CRA, TM and TCP methods. In CRA method, biofilm production was measured qualitatively described [20]. The black colonies with dark consistency were regarded as strong biofilm producers while the pink colonies as biofilm non-producers.
TM, a qualitative method for the detection of biofilm formation was performed as described [21]. Briefly, biofilm formation was considered positive when a visible film was observed along the inner wall and bottom of tube. Depending on this, isolates were scored as 0, 1, 2 and 3 for absence, weak, moderate and strong biofilm formation respectively. TCP, a quantitative method was used as described by Christensen et al. with slight modification [21], using trypticase soy broth (TSB), TSB with 1% glucose and Brain Heart Infusion (BHI) broth with 2% sucrose. Optical densities (OD) of both the dry plates and eluted stain was measured using micro ELISA auto reader at OD 630 nm. Mean OD value < 0.120, 0.120-0.240 and > 0.240 were classified as non/weak, moderate and strong biofilm adherence respectively [18].

Genotypic assay
Polymerase chain reaction (PCR) was used to detect icaA and icaD genes. The genomic DNA was extracted using a DNA extraction kit following the manufacturer instructions (Thermo Fischer). The forward and reverse primers (Solis Biodyne, Denmark) for icaA used were 5′-TCT CTT GCA GGA GCA ATC AA and 5′-TCA GGC ACT AAC ATC CAG CA respectively. For icaD, 5′-ATG GTC AAG CCC AGA CAG AG as forward and 5′-CGT GTT TTC AAC ATT TAA TGCAA as reverse primer. The PCR product was analyzed in 2% agarose gel stained with SYBR safe (Invitrogen) dye [22].

Statistical analysis
Sensitivity and specificity were evaluated by comparing the result of phenotypic methods with genotypic methods as standard. Different phenotypic methods were also compared with TCP as standard for phenotypic assays. Chi square test was used to evaluate the apparent differences for significance at 95% confidence level using IBM SPSS v 21.0.
Among all isolates, 20 (5.3%) isolates were positive in CRA while 329 (87.7%) isolates were biofilm non-producers with red colonies. It was found that detection of biofilm production by TM method was statistically significant when compared with presence of ica genes whereas CRA and TCP methods were statistically insignificant (Table 1) (Additional file 2: Table S2). We observed 14% sensitivity and 88% specificity while comparing CRA method with the genotypic assay. This shows no good correlation of CRA method with genotypic assay (Table 3).
The biofilm production is accurately confirmed by detecting the genes involved in biofilm formation. But PCR technique as routine diagnosis is impractical in resource-limited countries like Nepal. In this scenario, implementation of easier and reliable phenotypic method would be more appropriate. Therefore, we sought to evaluate CRA, TM, and modified TCP method with standard TCP method. The results revealed the CRA method with the highest specificity (86%) but the lowest sensitivity (8%). With that, the modified TCP method using BHI with 2% sucrose was 80% sensitive with 57% accuracy rate for differentiating biofilm producers and nonproducers. Addition of glucose in TSB corresponded to sensitivity and specificity of 59% when compared with the TCP method. These results suggested that modified TCP method using BHI with 2% sucrose and/or TSB supplemented with glucose, to be more reliable than those without supplements for detecting staphylococcal biofilm production (Additional file 4: Table S4).

Discussion
For high disease burden of biofilm associated staphylococcal infections, a reliable and prompt diagnostic method is essential in health care facilities [2,23]. Therefore, in this study, we evaluated three phenotypic, and a genotypic method of in vitro biofilm detection. To the best of our knowledge, this is first study using genotypic assay to detect in vitro biofilm production in clinical samples in Nepal.
In this study, 375 clinical staphylococcal isolates retained from various specimens were identified as S. aureus and CoNS in 161 (43%) and 214 (57%) isolates respectively. Consistent with previous studies, [24], S. epidermidis was the predominant CoNS species corresponding to 123 (57.5%) isolates. Because of its adaptive ability and highest dominance on human skin and mucosa [25], S. epidermidis has been reported the most prevalent in multiple studies [26,27].
A plethora of studies demonstrate the causal link between staphylococcal biofilm and the presence of ica operon [3,[28][29][30][31][32][33], which in turn are involved in the PIA production; the most extensively characterized staphylococcal biofilm component [7,29,[34][35][36]. In the present study, concomitant presence of icaA and icaD genes was detected in 86 (22.9%) staphylococcal isolates. Among CoNS, 29 (34%) S. epidermidis isolates   [31,34,38]. CRA method showed slime production in 46 (12.2%) staphylococcal isolates. The sensitivity and specificity of CRA method was only 14% and 88% respectively as compared to genotypic assay. Arciola and colleagues also identified eight and six CRA negative isolates possessing ica genes in two consecutive studies [16,39]. Similarly, Cafiso et al. and Fitzpatrick et al. also showed the reduced accuracy of this method to biofilm production [34,40]. All these evidences suggest that, despite being easier and faster, CRA method cannot be relied upon for precise detection of biofilm producers in routine diagnostic laboratory.
TM showed 63 (16.8%) isolates as strong, 66 (17.6%) moderate and 246 (65.6%) weak/non-biofilm producers. The TM results showed 64% sensitivity and 74% specificity as compared to the genotypic assay. Consistence with the previous study [7], TM among phenotypic assays in our study demonstrated the best correlation with genotype assay.
The expression of ica genes in vitro studies have been reported to be highly variable depending on the composition of media as their expression is induced by the stresses with additional sugars [18,41]. In only TSB, 112 (30%) isolates produced biofilm, while adding 1% glucose, the number of biofilm positive isolates increased to 174 (46.4%). This is consistent with the previous studies showing less positive results in TSB only medium [18,42]. Furthermore, the biofilm formation in BHI agar with 2% sucrose drastically increased number of biofilm producers to 229 (61%). When the presence of icaAD genes was compared with TCP method, sensitivity increased on adding 1% glucose and 2% sucrose as compared to TSB only. These evidences suggest that biofilm formation by staphylococci depends on growth conditions. Indeed, the use of sugar as supplement in the media was found to be essential for biofilm formation [7,18]. The use of additional sugar amount in a medium produces a stress condition that stimulates the fermentation reaction, resulting anaerobic condition that favors the production of PIA and consequently increasing biofilm production [8,43]. Taken together, these results indicate that the expression of ica gene is highly variable and induced by many factors including incorporation of sugar, salt, ethanol in the culture media [6-8, 18, 40, 44]. Detection of ica genes by PCR method has been demonstrated to be highly reliable to detect biofilm formation [3,7,29]. However, previous studies have shown evidences that presence of ica gene doesn't always correlate with biofilm production. For example; the study by deSilva demonstrated that only 59% of ica positive S. epidermidis isolates were found to be positive in CRA method [38]. In a study of Cafiso et al., 83.3% of CRA and TCP positive isolates were ica positive [34]. We also observed the presence of icaAD genes in many biofilm-negative strains in phenotypic assays, indicating the importance of genotypic assay in in vitro biofilm detection. However, evidences showing ica independent biofilm production suggest that ica negative results may not always reveal the absence of biofilm production. For instance; the presence of accumulation associated protein (aap) or Bap homolog protein (bhp) have been demonstrated to be responsible for biofilm production, suggesting the presence of PIA independent mechanisms in biofilm formation [37,[45][46][47][48].

Conclusion
The present study demonstrated the causal link between the presence of icaAD genes and biofilm production in the clinical staphylococcal isolates. Although TCP method was found to be superior to other phenotypic assays in terms of specificity and sensitivity, it was not well correlated with the genotypic assay. Taken together, these results suggest the use of genotypic assay along with the TM method in routine diagnostics to detect biofilm producers in clinical samples.

Limitations
Evaluation of biofilm production based merely on different nutrient supplements in vitro phenotypic assay may jeopardize the detections of biofilm production which depend on various factors. In addition, we examined presence of ica genes that are associated with PIA dependent biofilm production. This likely limits the detection of ica independent biofilm production.

Additional files
Additional file 1: