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29ORIGINAL RESEARCHES
DOI: 10.5281/zenodo.3685641 UDC: 579.861.2+582.282.23+615.015.8
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Antimicrobial susceptibility and biofilm production among Staphylococcus and Candida species
*1,2Greta Balan, 1,2Olga Burduniuc
department of Microbiology and Immunology, Nicolae Testemitsanu State University of Medicine and Pharmacy 2Department of Laboratory Diagnostic in Public Health, National Agency for Public Health Chisinau, the Republic of Moldova
Authors' ORCID iDs, academic degrees and contributions are available at the end of the article
Corresponding author: greta.balan@gmail.com Manuscript received October 01, 2019; revised manuscript February 27, 2020; published online March 10, 2020
Abstract
Background: Biofilms are surface-attached groups of microbial cells that are embedded in an extracellular matrix. One of the main features of biofilms is their resistance to antimicrobial drugs; therefore, the biofilm-based infections are extremely difficult to treat. This study aimed to investigate the biofilm-forming capacity of Staphylococcus spp. and Candida spp. strains isolated from collected clinical samples, as well as to assess their antibiotic susceptibility. Material and methods: The study was conducted on 134 strains of Staphylococcus spp. and 147 strains of Candida spp. isolated from various clinical specimens. Both biofilm formation and antibiotic susceptibility of the isolated strains were studied using contemporary standardized microbiological methods.
Results: The results of the study showed a high biofilm-forming capacity among the clinical strains of Staphylococcus spp. and Candida spp., as well as a higher level of antibiotic resistance in biofilm-producing strains compared to biofilm non-producing ones.
Conclusions: The high rates of antibiotic resistance and biofilm-forming capacity of strains represent a major public health challenge. The study showed a strong correlation between biofilm formation and antimicrobial resistance patterns. Key words: Staphylococcus spp., Candida spp., biofilm formation, antimicrobial resistance.
Cite this article
Balan G, Burduniuc O. Antimicrobial susceptibility and biofilm production among Staphylococcus and Candida species. Mold Med J. 2020;63(1):3-7. doi: 10.5281/zenodo.3685641.
Introduction
The advancement of biomedical science has enabled to study the microorganisms in their natural environment, whereas over 95% of microorganisms existing in nature are in biofilms [1]. Biofilm formation is an important strategy by which microorganisms survive and adapt in natural environments [2, 3].
A biofilm is defined as an aggregate of microorganisms in which the cells adhere to each other on a surface, enclosed in a synthetized extracellular polymeric substance matrix. Biofilms can occur on living or non-living surfaces, being widely spread in nature. The vast majority of bacterial infections may also involve microbial biofilm formation [4].
Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, being protected by a dense biofilm structure, which allows them to cooperate and interact in different manners. The main features of the biofilms are their high resistance to disinfectants and antimicrobial drugs; whereas the thick
extracellular matrix and the outer layer cells protect the interior of the community [5].
Most microorganisms form biofilms as a means of response to a number of factors, including cellular recognition of specific or non-specific attachment sites on a surface nutritional index, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics [6, 7].
It is estimated that microbial biofilms play a major role in over 80% of infections. Sixty percent of healthcare-associated infections are due to biofilm formation on medical implants. Moreover, many chronic diseases are associated with biofilms, such as infectious endocarditis, cystic fibrosis pneumonia, periodontitis, chronic rhinosinusitis, trophic ulcers and otitis media [8].
Staphylococci, predominantly Staphylococcus aureus and Staphylococcus epidermidis, are the disease-causing agents in a series of infections, which are often associated with chro-nicity, difficulty to eradicate and antimicrobial resistance [9]. Staphylococci are ranked first among the etiological fac-
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tors of bacterial infections, along with the annual increase in the number of methicillin-resistant staphylococci (MRS) strains and the occurrence of new antibiotic-resistant bacterial strains, which place this pathology among the emerging infectious diseases [10].
Staphylococcus aureus is an opportunistic pathogen, commonly involved in skin and soft tissue infections. It could be detected in the nasopharynx, skin, eyes, intestine and urogenital tract as part of the normal flora; although in some cases, it might pass through the skin barriers of wounds or surgical incisions, causing infections. In addition, it has the property to adhere and form biofilms on tissues or medical devices. Coagulase-negative staphylococci (CoNS) are considered saprophytic, avirulent or low-virulent microorganisms. However, over the past three decades there has been an increase in human infections caused by CoNS, particularly of S.epidermidis [11].
Levuriform fungi of the genus Candida are found as part of the normal flora in healthy individuals and are involved in the etiology of opportunistic infections, resulting in high mortality rates, particularly in immunocompromised individuals [12]. Candida species are most commonly associated with human diseases due to both virulence factors and bio-film-forming ability. Candida spp. causes systemic diseases and is the fourth most common cause of hospital-acquired blood infections. Candida albicans is the most commonly found species in fungal infections, whereas other species are involved to a lesser extent. However, the increased rate of non-Candida albicans isolation and antimicrobial resistance has become a major challenge for clinicians over the recent years [13].
Most infections caused by Candida spp. are related to biofilm formation on the mucosal surfaces and contaminated medical devices. Some study results revealed that the biofilms, formed by Candida spp. may become resistant to antifungal drugs, including amphotericin B, fluconazole, flucytosine, itraconazole and ketoconazole [14].
Therefore, a current in vitro study of the bio film-forming ability associated with the antimicrobial resistance patterns of Candida spp. and Staphylococcus spp. strains isolated from various clinical biosubstrates is required for the efficient management of these infections.
Material and methods
There have been examined 134 strains of Staphylococcus spp. (88 - S. aureus, 46 - S. epidermidis) and 147 strains of Candida spp. (75 - C. albicans, 24 - C. glabrata, 22 -C. krusei, 14 - C. parapsilosis, 12 - C. tropicalis), isolated from clinical biosubstrates (blood, trophic ulcers, infected wounds, and vaginal secretions) and which have been identified by standard microbiological techniques [15].
Antimicrobial susceptibility testing and the result interpretation were carried out according to EUCAST (The European Committee on Antimicrobial Susceptibility Testing) by using both qualitative methods (Kirby-Bauer disk diffusion assay) and quantitative methods determining
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the minimum inhibitory concentration (E-test, Vitek 2 Compact) [16].
Staphylococcus spp. strains were tested for benzylpeni-cillin, gentamicin, norfloxacin, cefoxitin, chloramphenicol, erythromycin, clindamycin, tetracycline, rifampicin, line-zolid and vancomycin, whereas Candida spp. strains were assessed to fluconazole, itraconazole, amphotericin B, mica-fungin and flucytosine.
Bacteria that showed resistance to at least one preparation out of three or more antimicrobial groups were identified as multidrug resistant strains (MDR) in accordance with the guidelines recommended by the joint initiative of the European Center for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) [17]. The methicillin-resistant or methicillin-sensitive (MSS) patterns of Staphylococcus spp. strains were determined according to the inhibition zone diameters of cefoxitin disk (30mg), based on EUCAST: MSS if the diameter is at least 22 mm; MRS if less than 22 mm. A double disc diffusion test (D test) was used for detecting inducible resistance to clindamycin. The erythromycin (15mg) and clindamycin (2mg) discs are placed at a distance of 12-20 mm measured from the edges of the discs. A flattening of the zone of inhibition around the clindamycin disk (D test positive) is reported as a clindamycin-resistance [18].
Biofilm production by isolated strains was quantitatively determined using the microtiter plate method [19]. For the purpose of study, 150^l of peptonate broth and 15^l of bacterial suspension were added to a 96 well plate and adjusted to the 0.5 McFarland turbidity standard (respectively 1.5x108 CFU/ml), which were previously prepared from 18-24 hour bacterial culture and grown on 5% blood agar. The plates were coated and incubated for 24 hours at 37° C. Subsequently, the level of adhesion of the tested strains to inert substrate was determined by removing the content from each well and then rinsing five times with sterile saline and fixing with cold methanol for 5 minutes. After removing of the methanol, the dried plates were stained with 0.1% violet crystal solution for 30 minutes. The excess stain was removed by washing and the stained biofilm was re-suspended in a 33% glacial acetic acid solution. Thus the obtained suspensions were used to determine the optical density (OD), based on the spectrophotometric absorbance readings at 570 nm colored suspension (A570). The tests were performed in duplicate.
The optical density cut-off value (ODc) is defined as the average OD of negative control + 3x the standard deviation (SD) of negative control. Biofilm formation by the tested strains was assayed and classified according to the adsorption of the violet crystal dye. The isolates were classified into four categories: non-adherent (OD < ODc), poor adherent (ODc <OD < 2xODc), moderately adherent (2xODc <OD <4xODc) and strongly adherent (4xODc <OD).
The reference strains Staphylococcus aureus (ATCC 25923), Candida albicans (ATCC 10231) and Candida tropi-calis (ATCC 750) were used for quality control. Epilnfo 2000 was used in statistical data analysis.
Results
The antimicrobial susceptibility testing results of 134 strains of Staphylococcus spp., revealed that 92 (68.6%) were polyresistant to antibiotics, 69 (51.5%) were methicillin-re-sistant, and 32 (23.9%) were D-test positive.
Staphylococcus spp. strains showed the highest sensitivity levels to vancomycin (100%), followed by tetracycline (88.8%), linezolid (83.6%) and chloramphenicol (82.8%) (fig. 1).
Invasive candidiasis is usually treated with five main groups of antifungal drugs, including azoles, polyenes, al-lylamines, echinocandins and pyrimidine analogues [20]. A study, conducting a susceptibility testing for Candida species to fluconazole, voriconazole, itraconazole, ketoconazole and flucytosine, showed that most Candida spp. strains were sensitive to fluconazole and flucytosine [21].
The studied Candida spp. strains showed different levels of susceptibility to the tested antimycotics. The data analysis showed the highest level of resistance to itraconazole (87.7%) and fluconazole (87.1%), followed by amphotericin B (10.9%) and micafungin (2.7%). All tested strains were found to be sensitive to flucytosine (fig. 2).
Fig. 1. Antibiotic resistance of Staphylococcus spp. (%).
Fig. 2. Antibiotic resistance of Candida spp. (%).
The next stage of the study determined the biofilm formation ability of Staphylococcus spp. and Candida spp. Of the 134 tested staphylococcus strains, 77 (57.5%) produced detectable biofilms. The biofilm status referred to 27 (35.1%) of isolates, which produced strong biofilms, 32 (41.6%) -moderate biofilms and 18 (23.4%) - weak biofilms (fig. 3).
Candida spp. strains produced detectable biofilms in 59.2%. The highest level of biofilm formation ability was recorded in C.glabrata strains (95.8%), followed by C. parap-silosis (57.1%), C. krusei (54.5%), C. albicans (52.0%) and
Fig. 3. The biofilm formation capacity of Staphylococcus spp. (%).
C. tropicalis (41.7%). 44 (50.6%) of Candida spp. strains produced strong biofilms, 29 (33.3%) - moderate biofilms and 14 (16.1%) - weak biofilms (fig. 4).
Fig. 4. Biofilm formation capacity of Candida spp. (%).
It is a well-known fact that bacterial populations in biofilms are considerably more resistant to antibiotics than planktonic cells [22]. Thus, biofilm-producing Staphylococ-cus spp. strains showed a higher antibiotic resistance compared to non-producing strains: benzylpenicillin (100% vs. 94.7%), gentamicin (61.0% vs. 0%), erythromycin (94.8 % vs 45.6%), tetracycline (19.5% vs 0%), cefoxitin (68.8% vs 42.1%), clindamycin (38.9 vs 3.5%), norfloxacin (90.9% vs 47.4%), chloramphenicol (27.3% vs 0%), rifampicin (81.7% vs 29.8%) and linezolid (25.9% vs 3.5%). All strains were found sensitive to vancomycin (tab. 1).
Comparison of biofilm formation ability between methi-cillin-resistant (MRS) and methicillin-sensitive (MSS) isolates of Staphylococcus spp. was carried out. The quantitative and qualitative results showed higher biofilm formation ability in MRS strains for both S.aureus and S. epidermi-dis strains compared to MSS bacteria. Biofilm-producing strains revealed a higher antibiotic resistance, which may lead to treatment failures in MRS infections (tab. 2).
The studies on Candida spp. strain resistance to antifun-gal drugs, as well as biofilm formation capacity, showed a statistical correlation between biofilm formation capacity and antifungal susceptibility (p <0.05) (tab. 3).
Flucytosine is known to inhibit both ribonucleic acid and deoxyribonucleic acid synthesis [23] and was the most effective antifungal agent against biofilm-producing Candida strains, tested within the present study.
Table 1
Antibiotic resistance of biofilm-producing and non-producing Staphylococcus spp.
Antimicrobials Biofilm-producing strains (N=77) Biofilm-nonproducing strains (N=57) p-value
n (%) n (%)
Penicillins
Benzylpenicillin 77 (100) 54 (94.7) p>0.05
Aminoglycosides
Gentamicin 47 (61.0) 0 (0) p<0.0001*
Macrolides
Erythromycin 73 (94.8) 26 (45.6) p<0.0001*
Tetracyclines
Tetracycline 15 (19.5) 0 (0) p>0.05
Cephalosporins
Cefoxitin 53 (68.8) 24 (42.1) p<0.05*
Lincosamides
Clindamycin 30 (38.9) 2(3.5) p>0.05
Fluoroquinolones
Norfloxacin 70 (90.9) 27 (47.4) p<0.0001*
Miscellaneous agents
Chloramphenicol 21 (27.3) 0 (0) p<0.05*
Rifampicin 76 (81.7) 17 (29.8) p<0.0001*
Oxazolidinones
Linezolid 20 (25.9) 2(3.5) p>0.05
Glycopeptides
Vancomycin 0 (0) 0 (0) NA
Note: ^Statistically significant (p<0.05); NA - not applicable.
Table 2
Biofilm formation capacity of MRS and MSS Staphylococcus spp.
S.aureus S.epidermidis
Biofilm production MRSA MSSA Total MRSE MSSE Total
n (%) n (%) n (%) n (%) n (%) n (%)
Strong 16 (26.2) 7 (11.5) 23 (37.7) 4 (25.0) 0 (0) 4 (25.0)
Moderate 17 (27.9) 8 (13.1) 25 (41.0) 6 (37.5) 1 (6.3) 7 (43.7)
Weak 7 (11.5) 6 (9.8) 13 (21.3) 3 (18.7) 2 (12.5) 5(31.3)
Note: MRSA - methicillin resistant S. aureus; MSSA - methicillin sensitive S. aureus; MRSE - methicillin resistant S. epidermidis; MSSE - methicillin sensitive S. epidermidis.
Table 3
Antimicrobial resistance of biofilm- producing and non-producing Candida spp. strains
Antimicrobials n (%) Biofilm-producing strains (N=87) Biofilm-nonproducing strains (N=60) p-value
n (%)
Azoles Fluconazole Intraconazole 87 (100) 87 (100) 41 (68.3) 42 (70.0) p<0.0001* p<0.0001*
Polyenes Amphotericin B 15 (17.2) 1 (1.7) NA
Echinocandins Micafungin 4 (4.6) 0 (0) p>0.05
Pyrimidine analogue Flucytosine 0 (0) 0 (0) NA
Note: ^Statistically significant (p<0.05); NA - not applicable.
Conclusions
The study results revealed a higher biofilm formation capacity in the clinical strains of Staphylococcus spp. and Candida spp. as well as higher rates of antimicrobial resistance in biofilm-producing strains compared to non-producing ones. The obtained data proves a strong correlation between biofilm formation capacity and antimicrobial resistance patterns. The implementation of the relevant antimicrobial susceptibility testing of biofilm-producing strains will improve the management of infections caused by these microorganisms, as well as provide feasible strategies to prevent their spread.
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Authors' ORCID iDs and academic degrees
Greta Balan - https://orcid.org/0000-0003-3704-3584, MD, MPH, PhD. Olga Burduniuc - https://orcid.org/0000-0002-6944-0800, MD, MPH, PhD.
Authors' contributions
GB designed the trial and interpreted the data. OB revised the manuscript critically. Both authors revised and approved the final version of the manuscript.
Funding
This study was supported the Nicolae Testemitsanu State University of Medicine and Pharmacy and National Agency for Public Health. The trial was authors' initiative.
The authors are independent and take responsibility for the integrity of the data and accuracy of the data analysis. Ethics approval and consent to participate
The strains used in this study were obtained from the routine analysis of clinical specimens. Sample collection did not involve direct contact with the patient, thus, no consent was required. The study was approved by the ethics committee of Nicolae Testemitsanu State University of Medicine and Pharmacy from the Republic of Moldova, proceedings No 65/12.04.2017 and No 67/12.05.2017.
Conflict of Interests
No competing interests were disclosed.
