Even though, most Staphylococci are present as normal flora in human body, they remain a versatile and potent pathogen since it is one of the most common cause of nosocomial and community acquired infection. They are associated significantly with various self-limiting to severe life threatening infection due to its ability to produce biofilm on inert as well as living tissues [2].
A total of 161 clinically significant Staphylococci were studied. More than half of the isolates were CNS (98, 61%) as compared to S. aureus (63, 39%). Some studies have found high number of S. aureus than CNS [16–18]. The study by Bose et al. shows 111 (62.01%) were S. epidermidis and 68 (37.99%) were S. aureus among 179 Staphylococcal isolates [19]. In a study carried out by Gad et al. [20], out of 292 isolates of urine and catheter, 53 (18.2%) staphylococcal strains were identified (S. aureus represented 6.2% and S. epidermidis represented 12%).
Among the isolates, five different species of coagulase-negative staphylococci were encountered; S. epidermidis (58, 59%), S. saprophyticus (19, 19%), S. haemolyticus (9, 9%), S. hominis (8, 8%) and S. capitis (4, 4%). The present results reveal that S. epidermidis are the most frequently isolated species. The findings of the present study are in agreement with the various studies which shows S. epidermidis as the most common CNS [21–23]. Staphylococci are commensal of skin and commonly gain access to site of skin puncture and deep cuts which most time cause uncomplicated infections but at times may develop into complicated infections leading to systemic failure [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]. Both S. aureus and CNS were found to be resistant to penicillin 60 (95.2%) and 92 (93.9%) followed by erythromycin 59 (93.6%) and 74 (75.5%) respectively. Fortunately, the S. aureus and CNS were found to be susceptible to common antibiotics as tetracycline (100%), 85 (86.7%) and chloramphenicol 62 (98.4%) and 91 (92.9%) respectively.
S. aureus infections are very common and MRSA continues to be a serious and dreadful challenge as their prevalence is reported to be increasing exponentially. Due to the extensive exploitation of therapeutic agents, CNS also have lost susceptibility to many antibiotics and generating a major problem [30]. The present study reported MRSA as 56(34.8%), MSSA as 7(4.3%), MRCNS as 65(40.4%) and MSCNS as 33(20.5%) among 161 Staphylococci. The prevalence of MRSA is 47.05% (48) among 102 S. aureus which is lower than the result reported from south India [31]. In studies carried out in similar settings in Nepal, 75.6%, 69.1% and 54.9% MRSA were reported, fairly higher than present study [32–34]. The difference in prevalence of MRSA may be because of the factors like healthcare facilities available in the particular hospital and rationale antibiotics usage which varies among hospital in different parts of the world. The important reservoirs of MRSA in hospitals/ institutions are infected or colonized patients and transient hand carriage is the predominant mode for patient to patient transmission. But the considerable increase in the prevalence of MRSA has been observed globally [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 Arciola et al. [38} and Mathur et al. [39]. The consistency and color of the colony developed depends not only strains of bacteria, nutrient composition, origin of specimen, physiology of isolates as well as incubation time.
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 sub-inhibitory presence of some antibiotics [17, 38]. Studies have demonstrated biofilm formation via PIA-independent 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.