|Year : 2016 | Volume
| Issue : 4 | Page : 789-800
Detection of intercellular adhesion icaAD genes in Staphylococcus aureus and coagulase-negative staphylococci and their role in biofilm production
Nahla A Melake, Ghada R Hendawy, Azza Z Labib, Ahmed B Mahmoud, Eman H Salem MD
Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||16-Nov-2014|
|Date of Acceptance||18-Jan-2015|
|Date of Web Publication||21-Mar-2017|
Eman H Salem
Osman EbnAffan, Menouf-Menoufia Governorate, 32511
Source of Support: None, Conflict of Interest: None
This study aimed to investigate the resistance patterns of nosocomial and community-acquired Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and screen methicillin-resistant and vancomycin-resistant isolates by phenotypic and genotypic methods. It also aimed to determine the biofilm-forming capacity and detect icaA and icaD genes responsible for polysaccharide intercellular adhesion synthesis and analyze the association between the biofilm-forming capacity of staphylococcal isolates and their multidrug resistance patterns.
Biofilms have dramatically increased resistance to antibiotics. The genes and products of ica locus [icaR (regulatory) and icaADBC (biosynthetic) genes] have been demonstrated to be necessary for biofilm formation and virulence.
Materials and methods
The study was carried out on specimens collected from Menoufia University Hospitals. Isolation, identification, and antimicrobial susceptibility of staphylococcal isolates were carried out using standardized microbiological methods. Phenotypic biofilm detection was carried out by microtiter plate adherence assay, the Congo red agar method, and the modified Congo red agar method. All clinical isolates (CIs) of S. aureus and CoNS demonstrating reduced susceptibility to methicillin and vancomycin and showing the ability to form biofilm were tested for the presence of methicillin-resistant gene (mecA), vancomycin-resistant genotypes (vanA and vanB), and biofilm-producing genes (icaA and icaD) by means of multiplex PCR.
About 82.4% of S. aureus isolates were methicillin-resistant S. aureus, whereas only 76.5% of them were positive for the mecA gene. VanA-positive gene was detected in 10.3% of S. aureus isolates. Regarding CoNS isolates, 76.9% were negative for the mecA gene and 78.8% were methicillin-resistant coagulase-negative staphylococci. About 10% were positive for the vanA genes in CoNS isolates. Biofilm formation was detected in 45.6 and 41.2% of S. aureus and in 76.9 and 55.8% of CoNS isolates, as detected by microtiter plate and Congo red agar, respectively. Ica genes were detected in 38.2% of S. aureus CIs and in 63.5% of CoNS CIs.
The biofilm-forming ability of staphylococcal isolates correlated with clinical significance and drug resistance. Biofilm-forming ability in the absence of icaA and icaD genes highlights the importance of further genetic investigations of ica- independent biofilm formation mechanisms.
Keywords: biofilm formation, Congo red agar, coagulase-negative staphylococci, icaA gene, icaD gene, microtiter plate adherence assay, Staphylococcus aureus
|How to cite this article:|
Melake NA, Hendawy GR, Labib AZ, Mahmoud AB, Salem EH. Detection of intercellular adhesion icaAD genes in Staphylococcus aureus and coagulase-negative staphylococci and their role in biofilm production. Menoufia Med J 2016;29:789-800
|How to cite this URL:|
Melake NA, Hendawy GR, Labib AZ, Mahmoud AB, Salem EH. Detection of intercellular adhesion icaAD genes in Staphylococcus aureus and coagulase-negative staphylococci and their role in biofilm production. Menoufia Med J [serial online] 2016 [cited 2018 Apr 23];29:789-800. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/789/202525
| Introduction|| |
Staphylococcus aureus is a major cause of hospital-acquired infections, causing high morbidity and mortality throughout the world. The proportion of methicillin-resistant Staphylococcus aureus (MRSA) has risen worldwide during the last two decades. The recommended treatment for multiresistant MRSA is glycopeptides, particularly vancomycin .
Since the emergence of vancomycin resistance in enterococci in 1988 and the in-vitro demonstration that its resistance genes (vanA and vanB) are transmissible to other bacterial species including S. aureus, emergence of vancomycin resistance in clinical staphylococci has become a great concern .
During the last decade, coagulase-negative staphylococci (CoNS) have emerged as a major cause of nosocomial infections. They constitute a major component of the normal skin and mucosal microflora, and are particularly responsible for catheter-related and medical device-related sepsis. They present unique problems in the diagnosis and treatment of infections . Low-level vancomycin-resistant CoNS has been reported .
The biofilm formation capacity is associated with antimicrobial resistance and is considered widely as a virulence factor. Staphylococcus epidermidis was the first species to be described as a biofilm producer; however, the same ability is encountered in S. aureus and other CoNS species . The most clearly characterized component of staphylococcal biofilms is the polysaccharide intercellular adhesin (PIA) encoded by the ica operon present on the bacterial chromosome comprising the icaA, icaD, icaB, and icaC genes and a transposable element, IS256 .
The icaA gene codifies the N-acetylglucosamyl transferase enzyme responsible for synthesizing PIA. This enzyme is not very active in vitro, but coexpression of the icaD gene increases the activity. IcaB is deacetylase responsible for deacetylation of mature PIA and the transmembrane protein IcaC is involved in externalization and elongation of growing of biofilm .
It has been suggested that both biofilm and ica genes are markers of clinical significance. In addition, the presence of indwelling medical devices in patients can induce bacterial biofilm formation, which results in persistent infection .
This study was performed at Menoufia University Hospital to investigate the resistance patterns of nosocomial and community-acquired S. aureus andCoNS and screen methicillin-resistant and vancomycin-resistant isolates by means of phenotypic and genotypic methods. We aimed to determine the biofilm-forming capacity among studied staphylococcal isolates and detect the icaA and icaD genes responsible for PIA synthesis and analyze the association between the biofilm-forming capacity of staphylococcal isolates and their multidrug resistance patterns.
| Materials and Methods|| |
The study was conducted from December 2012 to June 2014. It involved 250 patients (136 male and 114 female patients) with various nosocomial infections who were admitted to Menoufia University Hospitals (group I). The patient personal and clinical histories were taken, including age, sex, associated comorbidities, history of drug administration, admission to ward and/or ICUs, duration of hospitalization and exposure to invasive procedure. Group II involved 90 healthy persons (volunteers) (37 male and 53 female individuals). They were not exposed to antibiotics or to the hospital environment in the 3 months preceding sample collection.
The clinical specimens (n = 250) were collected and processed according to standardized microbiological methods . Swabs were obtained from volunteer noses and processed according to standardized microbiological methods. All staphylococcal isolates were maintained on trypticase soy broth containing 20% glycerol at -80 C for further study .
Antimicrobial susceptibility tests were carried out for staphylococcal isolates by the disk diffusion method against different antimicrobial agents (Oxoid Argentina): ampicillin (10 μg), penicillin (10 μg), amoxicillin/clavulanic acid (20/10 µg), gentamicin (10 µg), tetracycline (30 µg), ciprofloxacin (5 µg), ceftriaxone (30 µg), teicoplanin (30 µ), chloramphenicol (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), erythromycin (15 μg), linezolid (30 µg), and rifampicin (5 µg). Results were interpreted according to Clinical and Laboratory Standard Institute guidelines 2013 .
Detection of methicillin-resistant staphylococci was carried out by means of the cefoxitin disk diffusion method (30 μg) (for S. aureus: sensitive≥18 mm, intermediate 15–17 mm, and resistance≤14 mm; for CoNS: sensitive≥25 mm and resistance≤24 mm). The vancomycin-supplemented agar screening test (4 µg/ml for S. aureus and 8 µg/ml for CoNS) was used for staphylococcal isolates to detect vancomycin-intermediate and vancomycin-resistant isolates  and the results were confirmed by means of the vancomycin minimum inhibitory concentration (MIC) agar dilution method ,. The MIC interpretive criteria for S. aureus were as follows: sensitive≤2 µg/ml, intermediate 4–8 µg/ml, and resistance≥16 µg/ml; and those for CoNS were sensitive£4 µg/ml, intermediate 8–16 µg/ml, and resistance≥32 µg/ml.
Biofim detection was determined by two methods:
- Polystyrene adherence assay [microtiter plate test (MTP)] where overnight bacterial growth was subcultured onto trypticase soy agar (Oxoid) plus 5% glucose and then resuspended in trypticase soy broth plus 5% glucose. Aliquots of 100 μl were inoculated in nine parallel wells of a 96-well polystyrene plate. After incubation for 48 h at 37 C, the plates were softly shaken and rinsed with PBS and then fixed with 150 μl absolute methanol for 10 min. The attached bacterial material was stained by adding 150 μl crystal violet (1% w/v) for 20 min. The plates were rinsed with tap water (to remove excess crystal violet dye) and the amount of attached material was measured by solubilization of the crystal violet dye in 150 μl of 33% glacial acetic acid. The optical density was measured with an ELISA reader at a wavelength of 570 nm. Interpretation of biofilm production was made according to the criteria of Stepanović et al. .
- The second method was the use of Congo red agar (CRA). The medium comprised brain heart infusion broth at 37 g/l, sucrose at 50 g/l, Congo red dye at 0.8 g/l, agar at 10 g/l, and 1000 ml water. Congo red stain was prepared as concentrated aqueous solution and autoclaved separately from other medium constituents and was then added when the agar had cooled to 55 C. Phenotypic characterization of biofilm production was performed by culture of staphylococcal isolates on CRA plates aerobically for 24 h at 37 C. A five-color reference scale was used to accurately determine all color variations shown by the colonies. Isolates presenting bright black and dry opaque black were classified as biofilm producers, whereas red, pink, and Bordeaux colonies were classified as negative . The modifications included changing the concentration of Congo red dye (0.4 g/l) and sucrose, omission of glucose (10 g/l), and replacement of BHI brain heart infusion broth and agar by blood base agar-2 (BAB-2) (40 g/l). Inoculated agar was incubated for 48 h at 37 C and subsequently for 2–4 days at room temperature. A black color was interpreted as biofilm-producing strains, in contrast to red colonies, which were interpreted as non-biofilm-producing strains .
The molecular study was conducted by using multiplex PCR. Detectionof mecA, vanA, and vanB genes was carried out for all methicillin-resistant and vancomycin-intermediate and vancomycin-resistant isolates. The presence of IcaA and icaD genes was investigated to confirm the biofilm-forming ability. DNA was extracted and purified using the Jena Bioscience Etraction Kit Jena Bioscience GmbH (Loebstedterstrasse 80- 07749 JENA)-Germany. The used primers were designed and synthesized by Qiagen (Germany) ([Table 1]). PCR was carried out in a total volume of 50 µl consisting of 25 µl Taq green PCR Master Mix (2×), 1 µl forward primer, 1 µl reverse primer, 1 µl template DNA, and 22 µl nuclease-free water. The PCR conditions for the study of genes included heating at 94 C for 3 min, followed by 30 cycles at 94 C for 1 min, annealing at 54 C for 30 s and at 72 C for 1 min, and a final elongation step of 72 C for 7 min. The study was conducted using a Thermocycler Apparatus (Biometra, Germany). Synthesized DNA fragments were detected on 1.5% agarose gels by ethidium bromide staining. A DNA ladder (100–1000 bp) was used to estimate allele sizes in base pairs (bp) for the gel .
| Results|| |
The present study comprised two groups. Group I included 250 inpatients. Their ages ranged from 3 days to 77 years (mean 30.45±10.32 years). Group II included 90 healthy persons. Their ages ranged from 15 to 60 years (mean 35.43±12.53 years).
In group I, 245 strains were isolated. The most frequently isolated organisms were S. aureus (27.5%), Klebsiella spp. (27.3%), and CoNS (21.2%). Thirty-three specimens of group I (13.2%) showed mixed cultures (two isolates from each specimen), whereas 38 (15.2%) specimens showed no bacterial growth. Regarding group II, 88 isolates were obtained. The most frequently isolated organisms were S. aureus (47.7%) and CoNS (37.5%) [Figure 1].
|Figure 1: Culture results of the studied groups. CoNS, coagulase-negative staphylococci.|
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Staphylococcal isolates were isolated significantly more often from older patients ( > 50 years) (37.5%) compared with other age groups (P< 0.05). Male patients (52.5%) were affected by staphylococcal infection more often than female patients (47.5%). Patients administrated antibiotics were more liable to staphylococcal infection than others. The longer the duration of stay in the hospital, the more the prevalence of staphylococcal infection with no significant difference. Patients who had undergone invasive procedures were more infected by staphylococcal infection compared with others. Patients suffering from associated diseases (e.g. diabetes, autoimmune disease and/or cancer) were more susceptible to staphylococcal infections than were others. Similar results were obtained with phenotypic-resistant clinical staphylococcal strains [S. aureus clinical isolates (CIs): MRSA and vancomycin-resistant Staphylococcus aureus (VRSA); and CoNS CIs: methicillin-resistant coagulase-negative staphylococci (MRCoNS) and vancomycin-resistant coagulase-negative staphylococci (VRCoNS)] and biofilm-forming staphylococci but with no significant difference across all studied parameters, except for VRSA isolates, which showed significant difference among patients exposed to invasive procedures (P< 0.001) and associated comorbidities (P < 0.05) ([Table 2]).
|Table 2 Demographic and clinical data of patients affected by staphylococcal infection, and phenotypic-resistant staphylococcal strains study|
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Regarding the antibiotic susceptibility tests of staphylococcal clinical and commensal isolates measured by the disc diffusion method, the resistance patterns of S. aureus CIs to various antibiotics were as follows: penicillin, ampicillin, and amoxicillin/clavulanate were 92.6% for each, trimethoprim/sulfamethoxazole was 88.2%, cefoxitin was 82.4%, ceftriaxone was 79.4%, ciprofloxacin was 70.6%, teicoplanin and rifampicin were 67.6% for each, tetracycline was 66.2%, and chloramphenicol was 63.2%. The sensitivity against linezolid was 98.5%. CoNS CIs showed resistance against penicillin (92.6%), ampicillin (90.4%), ciprofloxacin (88.5%), amoxicillin/clavulanate (84.6%), cefoxitin (78.8%), ceftriaxone (75%), chloramphenicol (71.2%), tetracycline (67.3%), rifampicin (63.5%), trimethoprim/sulfamethoxazole (55.8%), and teicoplanin (46.2%). The sensitivity against linezolid was 100%. There was statistically significant difference between the resistance patterns of clinical and commensal staphylococcal isolates against most studied antibiotics (P< 0.05).
Methicillin resistance in CIs was detected in 82.4% of S. aureus and 78.8% of CoNS isolates, whereas 21% of S. aureus and 9% of CoNS isolates were methicillin resistant among commensals on the cefoxitin disc diffusion method. About 68% of S. aureus CIs and 46.1% of CoNS CIs were vancomycin-intermediate or vancomycin-resistant on the vancomycin screen agar test, whereas by MIC determination, 26.5% of S. aureus CIs and 19.2% of CoNS CIs were vancomycin resistant with statistically significant difference between the results of the two tests (all vancomycin-resistant strains were isolated from CIs) [Figure 2] and [Figure 3].
|Figure 2: Methicillin and vancomycin-resistant patterns among staphylococcal isolates as detected by phenotypic methods (all vancomycin-resistant strains were isolated from CIs). CI, clinical isolate; CoNS, coagulase-negative staphylococci; MIC, minimum inhibitory concentration.|
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|Figure 3: Congo red agar: (a) Positive test, staphylococci form black colonies; (b) Negative test, staphylococci form red colonies.|
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The sensitivity, specificity, and accuracy of the cefoxitin disc diffusion test and the vancomycin MIC agar dilution method with reference to PCR results in the detection of the resistance pattern of clinical staphylococcal isolates are shown in [Table 3].
|Table 3 Sensitivity and specificity of cefoxitin disc diffusion method and vancomycin minimum inhibitory concentration (agar dilution method) in relation to polymerase chain reaction in the detection of resistance pattern of clinical staphylococcal isolates|
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MecA gene detection was higher (92.9%) in MRSA CIs than in MRSA commensals. The mecA gene was also detected at a higher rate (97.6%) in MRCoNS CIs compared with MRCoNS commensals, with no statistically significant difference (P > 0.05). Regarding symmetrical measurement, there was moderate κ agreement between the cefoxitin disc diffusion method and PCR in the detection of MRSA, whereas there was substantial κ agreement between the cefoxitin disc diffusion method and PCR in the detection of MRCoNS. There was slight κ agreement between the MIC method and PCR in the detection of VRSA, whereas there was fair κ agreement between the MIC method and PCR in the detection of VRCoNS. No vanB genotype was detected among the studied staphylococcal isolates ([Table 4]).
|Table 4 Comparison between methicillin-resistant and vancomycin-resistant genotypes and phenotype results among Staphylococcus aureus and coagulase-negative staphylococci isolates|
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The degree of biofilm production of S. aureus and CoNS strains was determined according to their optical density on the MTP (optical density cutoff value = average optical density of negative control+3 SD of negative control) ([Table 5]).
|Table 5 Analysis of results of Congo red agar, modified Congo red agar, microtiter plate, and multiplex polymerase chain reaction for detection of biofilm formation of staphylococcal isolates|
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About 55% of MRSA CIs and 3.3% of commensal MRSA were biofilm producers. Regarding MRCoNS CIs, 97.6% were biofilm producers, whereas all commensal MRCoNS were biofilm producers. All VRCoNS strains were biofilm producers, whereas 77.8% of VRSA were biofilm producers ([Table 6]).
|Table 6 Relation between methicillin and vancomycin resistance and the ability to form biofilm in staphylococcal species|
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The percentage of S. aureus CIs and commensal isolates showed biofilm production on CRA, modified Congo red agar (MCRA), and MTP tests (shown in [Table 5]). Ica genes were detected among 43% with no statistically significant difference (P > 0.05) with phenotypic test results. The percentage of CoNS CIs and commensal isolates showed biofilm production on CRA, MCRA, and MTP tests, as shown in [Table 5]. Ica genes were detected among 63.5% with no statistically significant difference (P > 0.05) with phenotypic test results.
The IcaA gene alone was not detected in any isolates, whereas icaD alone was detected in 13.2% of S. aureus CIs, in 4.8% of S. aureus commensals, in 21.2% ofCoNS CIs, and in 27.3% of CoNS commensals. Both icaA and icaD genes were detected in 25.1% of S. aureus CIs and in 42.3% ofCoNS CIs. The total number of isolates containing ica genes constituted 38.2% of S. aureus CIs, 4.8% of S. aureus commensals,63.5% of CoNS CIs, and 27.3% of CoNS commensals [Figure 4] and [Figure 5].
|Figure 4: Prevalence of icaA and icaD genes in staphylococcal isolates. CI, clinical isolate; CoNS, coagulase-negative staphylococci.|
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|Figure 5: Gel electrophoresis of multiplex PCR amplification. (a) Staphylococcus aureus. Lane M: DNA ladder; lane 2: positive mecA, icaD, and icaA (160, 371, and 814 bp, respectively); lane 6: positive mecA (160 bp); lane 10: positive mecA, vanA, and icaA (160, 732, and 814 bp, respectively); and lanes 1, 3, 4, 5, 7, 8, 9, and 11: negative genes. (b) Coagulase-negative staphylococci (CoNS). Lane M: DNA ladder; lane 1: positive mecA and icaD (160 and 371 bp, respectively); lane 4: positive mecA (160 bp); lane 7: positive mecA, icaD, and vanA (160, 371, and 732 bp, respectively); lane 10: positive mecA, icaD, and icaA (160, 371, and 814 bp, respectively); and lanes 2, 3, 5, 6, 8, 9, and 11: negative genes.|
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For S. aureus and CoNS, the sensitivity, specificity, and diagnostic accuracy of phenotypic biofilm detection methods compared with PCR results are shown in [Table 7].
|Table 7 Sensitivity and specificity of phenotypic methods in relation to genotypic method for detection of biofilm production among the studied staphylococcal isolates|
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Results of isolation of S. aureus and CoNS and their methicillin, vancomycin, and biofilm producer phenotypes according to different department are shown in [Figure 6].
|Figure 6: Distribution of different staphylococcal phenotypes and biofilm forming among different departments. CI, clinical isolate; CoNS, coagulase-negative staphylococci; MRCoNS, methicillin-resistant coagulase-negative staphylococci; MRSA, methicillin-resistant Staphylococcus aureus; VRCoNS, vancomycin-resistant coagulase-negative staphylococci; VRSA, vancomycin-resistant Staphylococcus aureus.|
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Results of isolation of S. aureus and their MRSA, VRSA, and biofilm producer phenotypes according to type of specimen as well as results of isolation of CoNS and their methicillin, vancomycin, and biofilm producer phenotypes according to type of specimen are shown in [Figure 7].
|Figure 7: Distribution of different staphylococcal phenotypes and biofilm forming according to type of specimen. CI, clinical isolate; CoNS, coagulase-negative staphylococci; MRCoNS, methicillin-resistant coagulase-negative staphylococci; MRSA, methicillin-resistant Staphylococcus aureus; VRCoNS, vancomycin-resistant coagulase-negative staphylococci; VRSA, vancomycin-resistant Staphylococcus aureus.|
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| Discussion|| |
In the present study, the most frequently isolated organisms from group I were S. aureus (27.8%), Klebsiella spp. (27.3%), and CoNS (21.2%). This result was in agreement with that of Ghoneim et al. , who revealed that the most frequent nosocomial isolates were S. aureus (26.1%), followed by Klebsiella spp. (24.1%) and CoNS (11.9%). The result of the study matched that of Fernandes and Dias, who reported S. aureus as the most prevalent isolate followed by Klebsiella spp. Higher rates of nosocomial S. aureus were isolated by Sasirekha et al.  (77%).
With regard to group II, the most common isolated organisms were S. aureus (47.7%), CoNS (37.5%), and viridans streptococci (11.3%). Similar results were reported by Agarwal and Jain. Our results were higher than those of Ghoneim et al. , who found that 8.2% were carriers of S. aureus, followed by 6% carriers of CoNS, 4.4% carriers of viridans streptococci, and 0.7% carriers of pneumococci.
In the present study 82.4% of S. aureus CIs were methicillin resistant (MRSA). This was in agreement with studies by Thati et al.  and Ghoneim et al. , who found that 81.7 and 71.7% of nosocomial S. aureus isolates were MRSA, respectively. Regarding nosocomial CoNS isolated in the present study, 78% were MRCoNS. Sharma et al.  found that 88% of CoNS were resistant to methicillin, whereas lower rates were reported by Shah et al. , who found that 40% were MRCoNS. Regarding group II, 21% of S. aureus commensal isolates were MRSA and 9% of CoNS commensals were MRCoNS.
The presence of the mecA gene was higher (92.9%) in MRSA CIs than in commensal MRSA (88.9%). The mecA gene was also higher (97.6%) among MRCoNS CIs than among commensal MRCoNS (66.7%). Our results were comparable to those ofSharma et al. , who found that the mecA gene was detected in 80% of clinical strains.
In the present study, compared with PCR results, sensitivity of the cefoxitin disc diffusion method was 100% and specificity was 75%. Our results were comparable to those of Sasirekha et al. , who found that the sensitivity and specificity of the cefoxitin disc diffusion method were 100 and 99%, respectively, and to those of Ghoneim et al. , who found that the sensitivity and specificity of the cefoxitin disc diffusion method in the detection of MRSA were 85.4 and 93.3%, respectively.
Regarding CoNS referral to PCR results, sensitivity of the cefoxitin disc diffusion method was 100% and specificity was 92%, comparable to the results of Jain et al. , who found that the sensitivity and specificity of the cefoxitin disc diffusion test were 80 and 100%, respectively.
False positivity of the cefoxitin disc diffusion method could be due to the hyperproduction of β-lactamases, which may lead to phenotypic expression of methicillin resistance; although they appear methicillin resistant, they do not possess the usual genetic mechanism for such resistance . The phenotypic identification of methicillin resistance by the cefoxitin disc diffusion test is sensitive, simple, and rapid for a screening study.
In the present study, screening of S. aureus isolates by vancomycin screen agar plates revealed that 67.6% of S. aureus were vancomycin intermediate Staphlococcus aureus (VISA)/VRSA, whereas by the MIC method 26.5% were confirmed as VRSA. In CoNS, the result of the vancomycin agar screen test demonstrated that 46.1% were VICoNS/VRCoNS, whereas the result of MIC demonstrated that 19.2% were confirmed as VRCoNS. Chakraborty et al.  found that the screening of VRSA strains by vancomycin agar was 26.7% and was matched with MIC (26.7%) results.
Regarding S. aureus CIs, all of the VRSA isolates were MRSA, whereas Taj et al.  found that 22.2% of S. aureus isolates were VRSA. Lower results were reported by Thati et al. , who found that 4.5% were VISA and 2% were VRSA (all VRSA isolates were MRSA).
Regarding CoNS CIs, determination of MIC revealed that 53.8% were VSCoNS, 26.9% were VICoNS, and 19.2% were VRCoNS. All VRCoNS were MRCoNS. Low value was reported by Natoli et al.  (5.4%), whereas Iweriebor et al.  in South Africa reported that 100% of CoNS isolates were vancomycin resistant.
The vanA gene was detected in 38.9% of VRSA CIs and in none of the commensal S. aureus isolates, and in 50% of VRCoNS CIs and in none of the commensal CoNS. Similar results were reported by Ghoneim et al. , who found that 53.3% of vancomycin-resistant staphylococcal isolates were positive for the vanA gene. A higher rate was obtained by Thati et al. , who found that 85.7% of S. aureus were positive for the vanA gene. In the present study, VISA and VICoNS did not contain vancomycin-resistant genes (vanA nor vanB).
Compared with PCR results, the sensitivity of MIC was 100% and specificity was 52% in S. aureus. Sensitivity of the MIC method was 100% and specificity was 85% in CoNS isolates. Comparable results were reported by Daini and Akano , who found that 52.9% of resistant strains were plasmid-less and contained no vancomycin-resistant genes. Tiwari and Sen  found that all VISA, VRSA, VICoNS, and VRCoNS strains were grown on vancomycin screen agar but none of them could demonstrate the presence of vanA and/or vanB gene by PCR. In contrast, Chakraborty et al.  found that 100% of suspected clinically isolated VRSA strains have vanA and vanB genes.
Therefore, the absence of vanA and/or vanB genes in the present isolates did not rule out that these strains were VRSA or VISA. The mechanism of vancomycin resistance has been extensively studied with the first clinical VRSA strain, Mu50 . Electron microscopy examination of the Mu50 cell suggested that it produces increased amounts of murein monomers and more layers of peptidoglycan in the cell wall. As a result, more vancomycin molecules are trapped in the peptidoglycan layers before reaching the cytoplasmic membrane where peptidoglycan synthesis occurs. Besides the vancomycin-trapping mechanism, Hiramatsu  has suggested that dense accumulation of vancomycin molecules within the thickened cell wall significantly delays the timing of complete inhibition of cell wall synthesis by not allowing efficient penetration of vancomycin molecules through the thickened cell wall layers.
There was a statistically significant difference between the resistance patterns of clinical and commensal staphylococcal isolates against most studied antibiotics. S. aureus CIs were highly sensitive to linezolid (98.5%).
Our results demonstrated that the percentage of biofilm-producing staphylococcal isolates was significantly higher among hospital isolates than among commensal isolates and more among coagulase-negative than coagulase-positive staphylococci. Jain et al.  found that 32% from skin and nasal swabs showed biofilm production, whereas 70% of invasive staphylococcal strains exhibited biofilm-forming ability. Despite the low virulence of CoNS, particularly S. epidermidis, they are well adapted to adhere to smooth metal and plastic surfaces of foreign bodies such as vascular catheters, cardiac devices, and ventricular catheters. MTP identified a higher percentage of biofilm producers compared with CRA and MCRA tests (for S. aureus CIs: 45.6, 41.2, and 42.6%; for CoNS CIs: 76.9, 55.8, and 61.5%, respectively). Comparable results were reported by Saisinget al. , who found that 68.8% of S. aureus and 97.7% of CoNS were biofilm producers, as ascertained by the MTP method, and 62.5% of S. aureus and 84.7% of CoNS were biofilm producers, as ascertained by the CRA method. Iorio et al.  found that 15% of S. aureus isolates were positiveon CRA, and after addition of glucose in MCRA 27.5% were positive, including the previous isolates.MTP assay is the most widely used and is considered the standard test for detection of biofilm formation. It is the most sensitive, accurate, quantitative, and reproducible screening method, and is capable of examining a large number of staphylococcal isolates simultaneously. The CRA method is easier and faster to perform but could probably identify only the strong biofilm producers.
The present study showed that methicillin and vancomycin resistance was higher in slime-producing strains than in non-slime-producing strains with statistically high significant difference. About 55.4% of MRSA CIs and 33.3% of commensal MRSA were biofilm producers. Regarding MRCoNS CIs, 97.6% were biofilm producers, whereas all commensal MRCoNS and VRCoNS strains were biofilm producers and 77.8% of VRSA were biofilm producers. The biofilm environment has been reported to promote the dissemination of antimicrobial resistance genes, probably by facilitating cell–cell contact and consequently increasing genetic transfer. Comparable results were found by Sasirekha et al. andEftekhar and Dadaei, who reported that 61.9 and 57.8% of MRSA isolates have shown the potential to produce biofilm, respectively. Sharvari and Chitrafound 80.8% of MRSA and 60% MRCoNS to be biofilm producers. A higher rate was found in the study by Ando et al.  in Japan who showed that methicillin resistance was higher in slime-producing strains (95.4%) than in non-slime-producing strains.
Our results revealed that, although 57.4% of the S. aureus strains were positive by MTP and CRA (MCRA), they correlated in 30.9% of isolates. In CoNS, although 86.5% of the CoNS were positive on the used methods, they correlated in 51.9% of isolates. Therefore, for both phenotypic methods analyzed, we think that a combination of methods would more accurately predict the presence of the ica gene in staphylococcal isolates. The same conclusion was suggested by Iorio et al. , who found that an increased percentage of biofilm-positive isolates (85%) was observed when they combined the results of both phenotypic methods. On the other hand, low correspondence between the two methods was demonstrated by Nasr et al. , who found that the results of CRA with MTP correlated only in 20% of isolates Our results showed that ica genes were present in 38.2% of S. aureus CIs, 4.8% of S. aureus commensals, 63.5% of CoNS CIs, and 27.3% of CoNS commensals. This matched the results of Sharma et al. , who found that the ica locus was detected significantly more in invasive CoNS strains than in saprophytic CoNS strains. The icaA gene alone was not detected in the studied isolates. The icaD gene was the only gene detected among commensal isolates. Both icaA and icaD genes were detected among 25.1% of S. aureus CIs and 42.3% of CoNS CIs. Comparable results were reported by Diemond-Hernández et al. , who found that 36.1% of CoNS and 69.2% of S. aureus isolates harbored the icaD gene only, whereas the icaA gene was present only among 27.8% of CoNS isolates and none of the S. aureus isolates; 10.3% of S. aureus isolates were positive for the icaAD gene, compared with 37.7% of CoNS.
In this study many isolates were able to produce biofilm phenotypically but they did not contain ica genes. This can be explained by other mechanisms of biofilm production that may coexist, other than ica genes (ica-independent mechanism). PIA-independent biofilms were mediated through the accumulation-associated protein (Aap) . In addition, the biofilm-associated protein (Bap) in CoNS and Bap-related proteins of S. aureus can confer biofilm development independent of PIA production through cell-to-cell aggregation. These reports suggested that proteinaceous cell-to-cell adhesion can substitute PIA-mediated biofilm development in ica-independent biofilms. Besides ica, other genes have been associated with biofilm formation/accumulation, including aap, atlE, and hld .
Our study showed that MRSAs were isolated from all departments. VRSA was significantly higher in the ICU followed by general surgery departments compared with other departments. Biofilm-forming strains were significantly higher in the internal medicine department, followed by the urology department and the ICU. Ghoneim et al.  found that isolation of S. aureus was the highest in the general surgery department followed by the transplantation unit, ICUs, and internal medicine, whereas it was the least in the pediatric department.
As regards the type of specimen, MRSA and VRSA were highly isolated from sputum, pus, and blood. Biofilm-forming strains were highly isolated from urine, followed by blood and pus. Similar results were reported by Ghoneim et al.  regarding the distribution of S. aureus in different specimens. Regarding the distribution of MRSAs in different specimens, Taj et al.  found that most of the MRSA isolates were obtained from pus and wound swabs, followed by urine and blood. In contrast, Sasirekhaet al. found that 71.4% of MRSA isolates were from pus samples, followed by ear swabs and blood. Regarding biofilm formation, Gad et al. found that biofilm-forming S. aureus strains were highly isolated from urinary tract-catheterized patients (83.3%),
Regarding CoNS, MRCoNS was significantly higher in all departments. VRCoNS was significantly higher in internal medicine and the ICU followed by general surgery than in other departments. The biofilm-forming strains were significantly higher in all departments. Agarwal and Jain found that CoNS strains were highly isolated from ICU, and then from surgery, internal medicine, and urology.
MRCoNS was highly isolated from burn wound, blood, urine, and pus. VRCoNS was highly isolated from pus and sputum specimens. Biofilm-forming CoNS strains were highly isolated from blood, urine, and pus. Comparable results were reported bySharmaet al. , who found that CoNS strains were highly isolated from urine, blood, and pus, whereas the least rate of isolation was from tracheal swabs. Shah et al. found that MRCoNS strains were highly isolated from pus, urine, and blood. Agarwal and Jain  demonstrated that biofilm-forming CoNS strains were highly isolated from blood and intravascular catheters.
Long period of hospitalization accompanied by invasive procedures, administration of antibiotics, and presence of comorbid conditions were high risk factors for staphylococcal infection. Ghoneim et al.  found that S. aureus infection (especially multidrug-resistant strains) was significantly higher in patients who stayed for more than 7 days in a hospital, took antibiotics, and suffered from associated comorbidities.
| Conclusion|| |
High rates of staphylococcal multiresistance call for strict antibiotic policy and implementation of strict infection control measures. Continuous monitoring of antibiotic susceptibility patterns of clinically isolated staphylococci is required for selection of appropriate therapy. Biofilm formation correlates with antibiotic resistance and may be used as a marker for clinical significance among staphylococci. The biofilm-forming ability of some isolates in the absence of icaA and icaD genes highlights the importance of further genetic investigations of ica- independent biofilm formation mechanisms.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Wootton M, Howe RA, Hillman R, Walsh TR, Bennett PM, MacGowan AP. A modified population analysis (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus
in a UK hospital. J Antimicrob Chemother 2001; 47
Chakraborty S, Mahapatra M, Bal M, Roy S. Isolation and identification of vancomycin resistant Staphylococcus aureus
from post-operative pus sample. Al Amen J Med Sci 2011; 2
Sharma P, Lahiri KK, Kapila K. Conventional and molecular characterization of CoNS in hospital isolates. Indian J Pathol Microbiol 2011; 54
Yazdani R, Oshaghi M, Havayi A, Pishva E, Salehi R, Sadeghizadeh M, Foroohesh H. Detection of icaAD
gene and biofilm formation in Staphylococcus aureus
isolates from wound infections. Iran J Public Health 2006; 2
Diemond-Hernández B, Solórzano-Santos F, Leaños-Miranda B, Peregrino-Bejarano L, Miranda-Novales G. Production of ica
ADBC-encoded polysaccharide intercellular adhesin and therapeutic failure in pediatric patients with staphylococcal device-related infections. BMC Infect Dis 2010; 10
Kozitskaya S, Cho SH, Dietrich K, Marre R, Naber K, Ziebuhr W. The bacterial insertion sequence element IS 256 occurs preferentially in nosocomial S. epidermidis
isolates association with biofilm formation and resistance to aminoglycosides. Infect Immun 2004; 72
Lappin-Scott H, Bass C. Biofilm formation attachment, growth and detachment of microbes from surfaces. Am J Infect Control 2001; 29
Uckay I, Pittet D, Vaudaux P, Sax H, Lew D, Waldvogel F. Foreign body infections due to S. epidermidis
. Ann Med 2009; 41
Cheesbrough M. Microbiological tests in: district laboratory practice in tropical countries, part II
. Great Britain: Cambridge University Press; 2000. 1–266.
Clinical and Laboratory Standard Institute (CLSI). Performance standards for antimicrobial susceptibility testing. Twentieth informational supplement. CLSI document
. Wayne, PA: CLSI; 2014. M100–M124.
European Society of Clinical Microbiology and Infectious Diseases. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution; European committee for antimicrobial susceptibility testing (EUCAST) of the European society of clinical microbiology and infectious diseases (ESCMID). Clin Microbiol Infect 2000; 9
Stepanović S, Vuković D, Hola V. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. Acta Pathol Microbiol Scand 2007; 8
Freeman DJ, Falkner FR, Keane CT. New method for detecting slime production by coagulase-negative staphylococci. J Clin Pathol 1989; 42
Mariana NS, Salman SA, Neela V, Zamberi S. Evaluation of modified Congo red agar for detection of biofilm produced by clinical isolates of methicillin-resistance S. aureus
. Afr J Microbiol Res2009; 6
Depardieu F, Perichon B, Courvalin P. Detection of the van
alphabet and identification of Enterococci and Staphylococci at the species level by multiplex PCR. J Clin Microbiol 2004; 12
Ghoneim EA, Elhendawy GR, Abdel-Motalib T, Hassan H, Elrefahi HB. Characterization of vancomycin resistant S. aureus
(VRSA) in National Liver Institute, Microbiology Department in Menoufia University Hospitals. Menoufia Med J 2014; 124–137.
Fernandes A, Dias MT. The microbiological profiles of infected prosthetic implants with an emphasis on the organisms which form biofilms. J Clin Diagn Res2013; 2
Sasirekha B, Usha MS, Amruta AJ, Ankit S, Brinda N, Divya R. Evaluation and comparison of different phenotypic tests to detect methicillin resistant S. aureus
and their biofilm production. Int J Pharm Tech Res 2012; 2
Agarwal A, Jain A. Glucose and sodium chloride induced biofilm production and ica
operon in clinical isolates of staphylococci. Indian J Med Res 2013; 6
Thati V, Shivannavar CT, Gaddad SM. Vancomycin resistance among MRSA isolates from intensive care units of tertiary care hospitals in Hyderabad. Indian J Med Res 2011; 134
Shah MU, Akram MF, Usman J, Kaleem F. Incidence and susceptibility pattern of methicillin resistant coagulase-negative Staphylococci isolated from a tertiary care hospital of Pakistan. Jundishapur J Microbiol 2014; 1
Jain A, Agarwal A, Verma RK. Cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. J Med Microbiol 2008; 57
Taj Y, Farhan EA, Shahana UK. Current pattern of antibiotic resistance in S. aureus
clinical isolates and the emergence of vancomycin resistance. J Coll Physicians Surg Pak 2010; 11
Natoli S, Fontana C, Favaro M, Bergamini A, Testore GP, Minelli S. Characterization of coagulase-negative staphylococcal isolates from blood with reduced susceptibility to glycopeptides and therapeutic options. BMC Infect Dis 2009; 9
Iweriebor BC, Ramalivhana NJ, Hattori T, Okoh AI, Obi CL. Vancomycin resistant CoNS isolates from HIV positive patients in the Limpopo Province, South Africa. J Microbiol Antimicrob 2013; 2
Daini OA, Akano SA. Plasmid-mediated antibiotic resistance in S. aureus
from patients and non-patients. Acad J SRE 2009; 4
Tiwari HK, Sen MR. Emergence of vancomycin resistant S. aureus
(VRSA) from a tertiary care hospital from northern part of India. BMC Infect Dis 2006; 1
Cui L, Murakami H, Kuwahara-Arai K, Hanaki H, Hiramatsu K. Contribution of a thickened cell wall and its gultamine nonamidated component to the vancomycin resistance expressed by S. aureus
Mu50. J Antimicrob Chemother 2000; 44
Hiramatsu K. Vancomycin-resistant S. aureus
: a new model of antibiotic resistance. Lancet Infect Dis 2001; 3
Saising J, Singdam S, Ongsakul M, Voravuthikunchai SP. Lipase, protease, and biofilm as the major virulence factors in staphylococci isolated from acne lesions. BioScience Trends 2012; 4
Iorio NLP, Lopes AP, Scheck RP, Barcellos AG. A combination of methods to evaluate biofilm production may help to determine the clinical relevance of Staphylococcus
in blood cultures. Microbiol Immunol 2011; 55
Sharvari S, Chitra GP. Evaluation of different detection methods of biofilm formation in clinical isolates of Staphylococci. Int J Pharm Bio Sci 2012; 4
Eftekhar F, Dadaei T. Biofilm formation and detection of IcaAB
genes in clinical isolates of methicillin resistant Staphylococcus
. Iran J Basic Med Sci2011; 2
Ando E, Monden K, Mitsuhata R, Kariyama R, Kumon H. Biofilm formation among MRSA isolates from patients with urinary tract infection. Acta Med Okayama 2004; 4
Nasr R, Abu Shady HM, Hussein S. Biofilm formation and presence of icaAD
gene in clinical isolates of staphylococci. Egypt J Med Hum Genet 2012; 13
Hennig S, Nyunt Wai S, Ziebuhr W. Spontaneous switch to PIA-independent biofilm formation in an ica
-positive S. epidermidis
isolate. IJMM 2007; 297
Otto M. Staphylococcus epidermidis
– the 'accidental' pathogen. Nat Rev Mircrobiol 2009; 7
Gad GF, El-Feky MA, El-Rehewy MS, Hassan MA, Abolella H, El-Baky RM. Detection of icaA
genes and biofilm production by S. aureus
and S. epidermidis
isolated from urinary tract catheterized patients. J Infect Dev Ctries 2009; 3
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]