In vitro activity of sanitizers against mono- and polymicrobial biofilms of C. parapsilosis and S. aureus

ABSTRACT

The emergence of disinfectant-resistant microorganisms poses a significant threat to public health. These resilient pathogens can survive and thrive in hospital settings despite routine disinfection practices, leading to persistent infections and the potential for outbreaks. In this study, we investigated the impact of 11 different commercial sanitizers at various concentrations and exposure times on biofilms consisting of clinical and nosocomial environmental isolates of Candida parapsilosis and Staphylococcus aureus. Among the sanitizers tested, 0.5% and 2.0% chlorhexidine (CLX), 10% polyvinyl pyrrolidone (PVP-I), a disinfectant based on quaternary ammonium compound (QAC), 2% glutaraldehyde, and 0.55% orthophthalaldehyde (OPA) demonstrated efficacy against both C. parapsilosis and S. aureus in monospecies and mixed biofilms. Analysis showed that 0.5% CLX and 10% PVP-I had fungicidal and bactericidal activity against all biofilms. However, the sanitizer based on QAC and 0.55% OPA proved to be bacteriostatic and fungicidal against both monospecies and mixed biofilms. In mixed biofilms, despite the last four sanitizers exerting fungicidal action, the reduction of fungal cells was approximately 4 log₁₀ CFU/mL compared to monospecies biofilms, showing that the interaction provided more resistance of the yeast to the sanitizer. Formation of mixed biofilms in hospital settings can create an ecological niche that enhances the survival of pathogens against routine sanitization procedures. Therefore, effective sanitization practices, including regular cleaning with effective sanitizers, should be implemented to prevent C. parapsilosis/S. aureus biofilm formation in healthcare settings.

INTRODUCTION

Yeasts belonging to the Candida genus are a group of single-celled fungi that have the ability to colonize various sites in the human body. However, under certain circumstances, Candida can cause opportunistic infections (1). Candida albicans is the most commonly isolated species in human infections (2, 3). However, the members of the Candida parapsilosis complex, especially Candida parapsilosis sensu stricto, have become increasingly responsible for invasive infections in the nosocomial setting, particularly in patients who are immunocompromised, have indwelling medical devices, or receive broad-spectrum antibiotics (3). In recent years, outbreaks of C. parapsilosis infections have been reported in various countries around the world (4 – 9). Studies also show the unusual persistence of this agent in the nosocomial setting, probably linked to the hability of C. parapsilosis thriving in a range of environmental settings, including hospital surfaces, medical equipment, water sources, and the hands of healthcare workers (10, 11).

Candida parapsilosis has been shown to exhibit resistance to various classes of antifungal agents, including azoles and echinocandins, which are commonly used in the treatment of candidiasis. This resistance is often attributed to the overexpression of efflux pumps and alterations in the target enzymes (7, 11). In addition, C. parapsilosis can produce biofilms, which can protect the fungus from the effects of antimicrobials including the sanitizers (11). In fact, it has been suggested that the increased use of sanitizers in healthcare settings is contributing to the emergence of multidrug-resistant strains (12). Furthermore, we have previously highlighted evidence indicating that the ability of C. parapsilosis to form biofilms in intravenous catheters and medical devices, as well as colonize healthcare workers’ hands, is a significant risk factor that contributes to invasive infections and clonal outbreaks in intensive care unit (ICU) settings (8, 13, 14).

Due to the heterogeneous nature of microorganisms in hospital environments, generated biofilms are usually polymicrobial, involving species from different kingdoms such as bacteria and fungi, or species of the same genus (15). The interspecies interactions displayed by such biofilms are relevant to colonization, host response, drug resistance, and disease progression (15, 16). Staphylococcus aureus is frequently recovered from hospitalized patients and is a common opportunistic agent in skin and soft tissue infection. The bacteria can penetrate skin barriers through wounds or surgical incisions, causing infections (17). Moreover, S. aureus has the ability to adhere and form biofilms on tissues or medical devices, leading to significant mortality and morbidity in patients with wounds (18). Multiple mechanisms for attachment by S. aureus have been identified, and associations have been found between biofilm formation and the degree of pathogenicity (19).

Strategies to prevent microorganism transfer include antisepsis/disinfection provided by (i) chlorine and chlorine-based derivatives used for disinfecting tonometer heads, spot-disinfection of countertops and floors, blood spills, training manikins, laundry, hydrotherapy tanks, and the water distribution system in hemodialysis centers and hemodialysis machines; (ii) hydrogen peroxide used as disinfectant on inanimate surfaces; (iii) peracetic acid and aldehydes [glutaraldehyde (GLA), orthophthalaldehyde (OPA)] for disinfection of medical instruments; (iv) iodine-releasing agents (iodophores) and biguanides [chlorhexidine (CLX)] used as antiseptics; and (v) quaternary ammonium compounds (QACs) used as disinfectants (20 – 25). Asepsis prevents microorganism entry, antisepsis inhibits microorganisms in a specific environment, and disinfection eliminates microorganisms from surfaces using disinfectants (24). Antiseptics and disinfectants are generically called sanitizers according to Brazilian legislation (21).

The prevention of biofilm formation in hospital environments is crucial for preventing the spread of infections (12), and disinfection plays a vital role in this prevention process. Therefore, this study aims to evaluate the effectiveness of sanitizers against both C. parapsilosis sensu stricto and S. aureus ATCC 25923 in monospecies and mixed biofilms. All C. parapsilosis sensu stricto strains are clinical and environmental hospital strains that are resistant to fluconazole and biofilm formers.

RESULTS

Table 1 displays the results of the efficacy testing of all commercial sanitizers evaluated in this study. Among them, six sanitizers, CLX (0.5%, 3 min), CLX (2.0%, 1 min), polyvinyl pyrrolidone (PVP-I; 10%, 3 min), alkyl dimethyl benzyl ammonium chloride (ADBAC) 0.08% plus alkyl benzyl ammonium chloride (ABAC) 0.02% (3 min), GLA (2%, 30 min), and OPA (0.55%, 10 min), demonstrated effectiveness against both C. parapsilosis and S. aureus in both monospecies and mixed biofilms.

TABLE 1.

Effectiveness of all sanitizers against eight strains of C. parapsilosis and S. aureus in monospecies and mixed biofilms ^a

Recommended use	Active constituent/use concentration	Exposure time	C. parapsilosis biofilms	S. aureus biofilm	Mixed biofilms
Topical use	CLX 2.0%	1 min	E	E	E
	CLX 0.5%	3 min	E	E	E
	PVP-I 10%	3 min	E	E	E
Environment surfaces	NaClO 0.02%	60 min	I	I	I
	NaClO 1%	30 min	I	I	I
	ADBAC 0.32%	10 min	I	I	I
	H2O2 (4.25%) + DDAC (5.6%) pure	10 min	I	b	b
	H2O2 (4.25%) + DDAC (5.6%) 1:25	10 min	I	b	b
	PMS 1%	10 min	I	I	I
	ADBAC (0.08%) + ABAC (0.02%) pure	3 min	E	E	E
	ADBAC (0.08%) + ABAC (0.02%) 1:20	6 min	E	E	E
Sterilization of non-autoclavable medical devices	PAA (3.5%) + H2O2 (2.6%) 0.1%	3 min	E	b	b
	PAA (3.5%) + H2O2 (2.6%) 0.2%	10 min	E	b	b
	GLA 2%	30 min	E	E	E
	OPA 0.55%	10 min	E	E	E

^a ADBAC, alkyl dimethyl benzyl ammonium chloride; ABAC, alkyl benzyl ammonium chloride; CLX, chlorhexidine gluconate; GLA, glutaraldehyde; DDAC, didecyl dimethyl ammonium chloride; H₂O₂, hydrogen peroxide; PAA, peracetic acid; NaClO, sodium hypochlorite; OPA, D-orthophthalaldehyde; PMS, potassium monopersulfate; PVP-I, polyvinyl pyrrolidone; I, ineffective; E, effective; min, minutes.

^b Impaired result due to high catalase activity of the S. aureus.

Both topical chlorhexidine solutions (2.0% and 0.5%), used for hands disinfection, and the tested iodophore, used for skin disinfection during surgeries, were effective (Table 1). The hypochlorite-based disinfectants, commonly used for surface disinfection, were found to be ineffective, as were two of the three QAC-based sanitizers. Only one QAC-based sanitizer, specifically ADBAC 0.08% plus ABAC 0.02%, showed effectiveness against all biofilms (Table 1). Regarding the sanitizers for use in non-autoclavable medical devices, both GLA and OPA were effective against all biofilms (Table 1). The high activity of S. aureus catalase hindered the accurate assessment of the effectiveness of the combination of peracetic acid and hydrogen peroxide against monospecies S. aureus biofilms and mixed biofilms, using the methodology employed in the assays, despite its effectiveness against C. parapsilosis biofilms. In Brazil, hospitals exclusively utilize peracetic acid combined with hydrogen peroxide formulations.

The average number of cells in the C. parapsilosis biofilm after incubation for 24 h or 48 h at 37°C showed no statistically significant difference (P > 0.05). Therefore, we will consider the 24-h biofilms for exposure to sanitizers. The monospecies biofilms of C. parapsilosis and S. aureus had an average of 9.49 ± 0.45 log₁₀ CFU/mL and 9.17 ± 0.575 log₁₀ CFU/mL, respectively. In contrast, for mixed biofilms, C. parapsilosis and S. aureus had an average of 6.60 ± 0.13 log₁₀ CFU/mL and 7.72 ± 0.06 log₁₀ CFU/mL, respectively. Student’s t-test indicated that both C. parapsilosis and S. aureus had significantly fewer cells in the mixed biofilm compared to the monospecies biofilm (P < 0.0001).

Figure 1 illustrates different biofilms: the monospecies biofilm of S. aureus (Fig. 1A), the mixed biofilm of C. parapsilosis and S. aureus (Fig. 1B), and the monospecies biofilm of C. parapsilosis (Fig. 1C). In Fig. 1A, the presence of multiple cell layers is evident, emphasizing the characteristic “grape-like” arrangement of staphylococci. Figure 1B depicts yeast blastoconidia surrounded by a significant number of staphylococci. In Fig. 1C, several layers of blastoconidia are observed, highlighting the elongated nature of blastoconidia in the overlapping regions.

Fig 1.

Representative electron micrographs of biofilms. (A) Monospecies biofilm of S. aureus; (B) mixed species biofilm of C. parapsilosis and S. aureus; (C) monospecies biofilm of C. parapsilosis. Overlapping figures in A and C represent higher magnifications of biofilm cells.

Figure 2 illustrates the CFU/mL values obtained for 38 monospecies C. parapsilosis biofilms, while Fig. 3 displays the results for mixed biofilms formed by 38 C. parapsilosis and S. aureus using effective sanitizers, namely 0.5% CLX, 10% PVP-I, ADBAC 0.08% combined with ABAC 0.02%, and 0.55% OPA. Additionally, it can be observed from the figures that some isolates (7, 12, 23, 29, 31, and 36) exhibited higher log₁₀ CFU/mL values after exposure to the selected sanitizers (Fig. 2 and Fig. 3). Notably, isolates 7 (18E), 12 (26E), and 36 (P5/10S) were isolated from the hands of hospital workers, suggesting potential nosocomial transmission and tolerance of these isolates to the selected sanitizers.

Fig 2.

Determination of colony forming units (CFU) per milliliter (mL) after exposure of C. parapsilosis stricto sensu biofilm cells to selected sanitizers. (A) 0.5% CLX, 3 min; (B) 10% PVP-I, 3 min; (C) ADBAC (0.08%) and ABAC (0.02%) diluted at 1:20 and exposure time of 6 min; (D) 0.55% OPA, 10 min.

Fig 3.

Determination of CFU per milliliter after exposure of C. parapsilosis stricto sensu and S. aureus mixed biofilm cells to selected sanitizers. (A) 0.5% CLX, 3 min; (B) 10% PVP-I, 3 min; (C) ADBAC (0.08%) and ABAC (0.02%) diluted at 1:20 and exposure time of 6 min; (D) 0.55% OPA, 10 min.

Table 2 displays the log₁₀ reduction results obtained after exposing all biofilms to the selected sanitizers. Variations in efficacy were observed among some agents against monospecies and mixed biofilms. The QAC-based (ADBAC 0.08% plus ABAC 0.02%) sanitizer and OPA exhibited bacteriostatic activity against the S. aureus monospecies biofilm. In contrast, all selected sanitizers showed fungicidal activity against C. parapsilosis monospecies biofilms (Table 2). Interestingly, when C. parapsilosis was associated with S. aureus in mixed biofilms, the yeast showed increased resistance to sanitizers, while S. aureus showed no significant change in behavior toward sanitizers (Table 2). When employing two-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparisons, to evaluate untreated monospecies biofilms (control) versus biofilms treated with the chosen disinfectants, all treatments exhibited a statistically significant decrease in cell number (P < 0.0001). The comparisons between the disinfectants themselves indicated that there was no significant difference between PVP-I and OPA (P = 0.995) in the case of S. aureus. However, when performing the same comparisons for mixed biofilms, all exhibited statistically significant differences (P < 0.0001).

TABLE 2.

Logarithmic decreases and resulting antimicrobial activity in monospecies and mixed biofilms of C. parapsilosis and S. aureus strains after using the four selected sanitizers.

Treatment	Log10 CFU/mL ± SD a obtained after exposure		Reduction factor		Antimicrobial activity
	S. aureus	C. parapsilosis	S. aureus	C. parapsilosis	S. aureus	C. parapsilosis
Monospecies biofilms
CLX b (0.5%)	1.14 ± 0.23	0.67 ± 0.08	8.03	9.1	Bactericidal g	Fungicidal g
PVP-I c (10%)	4.75 ± 0.04	1.59 ± 0.37	4.42	8.18	Bactericidal	Fungicidal
ADBAC d (0.08%) with ABAC e (0.02%)	6.48 ± 0.15	1.69 ± 0.03	2.69	8.08	Bacteriostatic	Fungicidal
OPA f (0.55%)	5.90 ± 0.59	0.53 ± 0.13	3.27	8.94	Bacteriostatic	Fungicidal
Mixed biofilms
CLX a (0.5%)	0.60 ± 0.11	1.25 ± 0.02	7.12	5.35	Bactericidal	Fungicidal
PVP-I b (10%)	3.55 ± 0.02	2.56 ± 0.01	4.17	4.04	Bactericidal	Fungicidal
ADBAC c (0.08%) plus ABAC e (0.02%)	5.52 ± 0.02	2.21 ± 0.03	2.20	4.39	Bacteriostatic	Fungicidal
OPA f (0.55%)	4.48 ± 0.11	2.55 ± 0.09	3.24	4.05	Bacteriostatic	Fungicidal

^a SD, standard deviation.

^b CLX, chlorhexidine gluconate.

^c PVP-I, polyvinyl pyrrolidone.

^d ADBAC, alkyl dimethyl benzyl ammonium chloride.

^e ABAC, alkyl benzyl ammonium chloride.

^f OPA, D-orthophthalaldehyde.

^g Adopted criterion BS EN 13697:2015 + A1:2019.

DISCUSSION

Our study demonstrates the effective inactivation of C. parapsilosis and S. aureus cells in monospecies and mixed biofilms by topical and non-autoclavable medical device sanitizers. However, most sanitizers for use on fixed surfaces were ineffective against these biofilms. This is concerning as chlorine-based sanitizers, replaced by QAC or ammonium persulfate-based sanitizers, may not effectively combat the biofilm formation.

Previously, our research group demonstrated the effectiveness of hydrogen peroxide with acetic acid against C. parapsilosis sensu stricto and Candida orthopsilosis biofilms generated by isolates obtained from a hydraulic circuit in a hemodialysis unit (26). Furthermore, we assessed the susceptibility of environmental isolates of Aspergillus spp. and Fusarium spp. to disinfectants at various concentrations and time exposures, as well as the in vivo toxicity of residual concentrations of peracetic acid in mice. The exposure of fungal biofilms to disinfectants revealed sensitivity to acetic, citric, and peracetic acids, although the concentrations and exposure times varied depending on the fungal genus. Mice exposed to residual doses of peracetic acid for 60 weeks exhibited anatomopathological, hematological, and biochemical changes (27). Additionally, our research group has shown through previous studies (8, 13) that C. parapsilosis strains isolated from the hospital environment share the same genetic profile as strains causing infections, particularly candidemia in hospitalized patients. Similar findings have been reported in previous studies (28 – 32), reinforcing that environmental surfaces may act as reservoirs for persistent colonization by C. parapsilosis. Another important point is that all strains of C. parapsilosis studied were biofilm formers that provide increased protection against drugs, disinfectants, and adverse environmental conditions (15, 33). Furthermore, all C. parapsilosis strains tested exhibited in vitro resistance to fluconazole by point mutations in the ERG11 gene (13). This mutation inhibits fungal growth by interfering with the synthesis of ergosterol, a necessary component of fungal cell membranes (15). Although studies on the association between drug and sanitizer resistance in bacteria are well established in the literature, research on fungi in this area is still limited (34). However, it is known that fungal species can develop resistance to sanitizers through mechanisms such as altering target proteins/enzymes, efflux pumps, membrane permeability/drug uptake, and biofilm formation, which are regulated by resistance genes (35). Understanding the relationship between sanitizer resistance and antimicrobial resistance in fungi is crucial for effective management, considering their mobility and potential for environmental spread (35).

In the case of S. aureus, particularly the nasal carriage increases the risk of infections and invasive disease (36). Decolonization efforts have been implemented through the use of topical nasal agents and antiseptic body washes, particularly in patients undergoing cardiac and orthopedic procedures (36, 37). Furthermore, the formation of mixed biofilms on hospital setting can potentially create an ecological niche that enables pathogens to more effectively colonize and enhance their survival against routine sanitization procedures. Consequently, this may lead to an increased prevalence of pathogens and higher risk of infections (15). Therefore, effective sanitization practices should be implemented, including regular cleaning of environmental surfaces with effective sanitizers to prevent C. parapsilosis/S. aureus biofilm formation.

CLX has demonstrated efficacy against all the biofilms studied. Previous researches by La Fleur et al. (38), Alvendal et al. (39), and Baldino et al. (40) have shown that this antiseptic exhibits antifungal activity against C. albicans biofilms, while Fathilah et al. (41) reported that CLX is effective against non-albicans Candida species such as C. tropicalis and C. krusei in free-growing or planktonic form. Furthermore, Hayden et al. (42) found CLX to be effective against biofilms and planktonic cells of Candida auris. Regarding S. aureus, Park et al. (43) propose that prolonged CLX utilization could potentially exert selective pressure on specific methicillin-resistant Staphylococcus aureus (MRSA) clones within the hospital setting, leading to the induction of biofilm formation.

Furthermore, PVP-I demonstrated bactericidal and fungicidal properties against all C. parapsilosis and S. aureus biofilms within 3 min of exposure. Similar findings were reported by Hoekstra et al. (44), who tested the effectiveness of PVP-I ointment on mixed biofilms consisting of C. albicans and MRSA. Additionally, PVP-I has demonstrated effectiveness against bacterial biofilms, including those formed by S. aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, even at concentrations as low as 0.25%. Regarding fungal species like C. albicans, Aspergillus fumigatus, and C. auris, PVP-I has shown efficacy at concentrations ranging from 6.25% to 3.125% (45).

Although it is known that the biofilm matrix can interfere with the diffusion of antimicrobials (46), the two antiseptics evaluated in the study effectively penetrated and disabled both bacteria and fungi within monospecies and mixed biofilms. CLX has been reported to promote leakage of cytoplasmic constituents, induce cytoplasmic coagulation, and disrupt lipid bilayer membranes in bacteria (47), while iodine rapidly penetrates microorganisms and oxidizes critical proteins, nucleotides, and fatty acids, resulting in cell death (20).

The 1% sodium hypochlorite solution, also known as Dakin’s solution, has been reintroduced for healing diabetic foot wounds and septic surgical wounds to address concerns over antiseptic toxicity and antibiotic resistance (48). Our study found that it was ineffective against all biofilms tested. Usually, sanitizer efficacy testing is conducted using planktonic cells in suspension or dried onto a surface, and does not take into account the complexity of biofilm communities (21, 25). As a result, sanitizer oncentrations that are effective against planktonic cells often fail to inactivate biofilm-associated cells of the same strain (12). Compared to the planktonic state, cells in the biofilm state display specific characteristics, including modified physiological status, production of extracellular matrix, presence of drug efflux pumps, and altered gene regulation (49). These traits contribute to the inherent resistance of biofilms to sanitizers. Our electron micrographs confirmed the formation of robust biofilms by either S. aureus or C. parapsilosis, as well as by both microorganisms together. Notably, mixed biofilms demonstrated a higher prevalence of S. aureus over C. parapsilosis, with the former appearing to bind to the matrix that covers the blastoconidia.

QAC-based sanitizers are preferable to sodium hypochlorite for disinfecting fixed surfaces in healthcare settings due to their stability under various conditions. QACs act by disrupting intermolecular bonds between phospholipids, leading to microbial inactivation by damaging membrane integrity and releasing cytoplasmic contents (50). QACs are biodegradable and can be found in varying concentrations in indoor and outdoor environments, usually below the minimum inhibitory concentration values, ranging from 400 ppm to 500 ppm, and often below 1,000 ppm as in Lysol (0.1% m/vol) (51).

In this study, it was observed that the use of a QAC-based sanitizer led to a significant reduction of C. parapsilosis cells in single-strain biofilms compared to mixed biofilms, with reductions of approximately 8 log CFU/mL and 4 log CFU/mL, respectively. However, the efficacy of the sanitizer against S. aureus biofilm cells, whether in monospecies or mixed biofilms, was limited, with only a logarithmic reduction of 2.7 log CFU/mL and 2.20 log CFU/mL, respectively. Studies have shown that microorganisms in mixed biofilms exhibit greater resistance to sanitizers, particularly those based on QAC, compared to monospecies biofilms (52, 53). This phenomenon is commonly observed in polybacterial biofilms and is believed to be caused by increased production of extracellular polymeric substances (EPS) or enhanced interspecies communication (54). In the case of mixed bacterial-fungal biofilms, the α-glucan component of the EPS matrix is known to play a crucial role in promoting cell-cell cohesion between the bacterial and fungal cells (15). Nevertheless, further comprehensive research is required to elucidate the mechanisms of interactions, whether synergistic or antagonistic, between the two microorganisms, as well as the mechanism of action of QAC-based sanitizers on these biofilms.

Potassium monopersulfate (PMS) is a sanitizer commonly recommended for surface and medical device disinfection according to Brazilian legislation (23). However, our research findings showed that PMS may not be effective against C. parapsilosis and S. aureus monospecies or mixed biofilms. Recent studies have highlighted a concerning connection between outbreaks in healthcare facilities and the use of recommended sanitizers (55 – 57). These outbreaks have been attributed to errors in the proper use of sanitizing solutions or incorrect disinfection of medical devices. Unfortunately, current investigations into these outbreaks often focus solely on tracing the sources and routes of pathogen transmission, without sufficient consideration of the possible involvement of disinfectant resistance (12). In our previous study conducted at a Brazilian public hospital hemodialysis center, we identified 53 isolates of C. parapsilosis sensu stricto and 47 isolates of Candida orthopsilosis from the hydraulic circuit (58). When evaluating the sensitivity of these isolates to commonly used sanitizers in this environment, we found that all of them exhibited resistance to the standardized sanitizer used for hemodialysis machine disinfection, which contained sodium hypochlorite at a concentration of 500 ppm or 0.05% (26). Altogether, this information highlights the urgent need for developing effective disinfection strategies to eradicate mixed biofilms of non-Candida albicans Candida and bacteria in various settings, particularly in hospitals.

Experiments with sanitizers used in reusable medical devices showed that exposure to 2% GLA for 30 min or 0.55% OPA for 10 min can significantly reduce the number of viable yeasts in biofilms. However, it should be noted that GLA has been withdrawn from use in some countries, including Brazil (21). Although OPA has been confirmed as a fungicide for C. parapsilosis cells in both monospecies and mixed biofilms, it only exhibits bacteriostatic properties for S. aureus in both biofilms (Table 2). This is a significant concern because S. aureus has been shown to form polymicrobial biofilms with Candida species, both in vitro and in vivo experimental conditions (33, 59). Previous study has demonstrated that the pathogenicity of S. aureus is heightened when it coexists with C. albicans. This phenomenon is thought to be a result of both physical interactions and the differential regulation of particular virulence factors that are induced during polymicrobial growth (60).

Conclusion

We demonstrated in this study that the most sanitizers for use on fixed surfaces were ineffective against C. parapsilosis, S. aureus and C. parapsilosis/S. aureus biofilms, while effective inactivation was achieved by topical and non-autoclavable medical device sanitizers. Therefore, for effective sanitization practices, we reinforce the recommendation of regular testing of sanitizers against the local pathogens. Lack of compliance with this practice may help to explain the persistence of pathogens in the nosocomial setting, especially ICU.

MATERIALS AND METHODS

Sanitizers

The sanitizers were purchased commercially in their original packaging and diluted in distilled water at the time of use. The diluted solutions were sterilized through the use of 0.22 µm membrane filters (Sartorius Minisart, Gottingen, Germany). The exposure time of biofilms to the sanitizers was determined based on legislation, literature, or recommendations provided by the product manufacturer, as an indication of use (Table 3).

TABLE 3.

List of sanitizers used in this study according to indication, trade name, active constituent, use concentration, and reference

Recommended use	Trade name/manufacturer	Active constituent/abbreviation/concentration	Use concentration	Exposure time	Reference
Topical use (antiseptic)	Riohex, Rioquímica, São José do Rio Preto, SP, Brazil	CLX	2.0%	1 min b	(21, 22)
			0.5%	3 min b
	Riodeine, Rioquímica, São José do Rio Preto, SP, Brazil	10% PVP-I	1% active iodine	3 min b	Manufacturer (22, 24)
Environment surfaces disinfection	Solynt, Guarulhos, SP, Brazil	NaClO 12%	0.02% (200 ppm) a	60 min b	Manufacturer (21 – 25)
			1% (10.000 ppm) a	30 min b
	Lavanda, Johnson & Johnson Consumer Health (Brazil) Ltda. SP, Brazil	QAC constituted by ADBAC	0.32%	10 min b	Manufacturer
	Peroxy 4D, Spartan, Sumaré, SP, Brazil	QAC constituted by H2O2 (4.25%) plus cocobenzyl alkyl dimethyl ammonium chloride, DDAC (5.6%)	1:25	10 min b	(21 – 25)
			1:100	10 min b
	Virkon, B. Braun, São Gonçalo, RJ, Brazil	PMS, 50 g	1%	10 min b	Manufacturer (22)
	Lysol, Reckitt Benckiser (Brazil) Industrial Ltda. SP, Brazil	QAC constituted by ADBAC (0.08%) and ABAC (0.02%)	Pure	3 min b	Manufacturer (21 – 25)
			1:20	6 min b
Sterilization of non-autoclavable medical devices	Puristeril, Fresenius Medical Care, Jaguariuna, SP, Brazil	PAA (3.5%) plus H2O2 (2.6%)	0.1%	30 min b	Manufacturer (21 – 25)
			0.2%	10 min b
	Glutaron, Rioquímica, São José do Rio Preto, SP, Brazil	GLA	2%	30 min b
	Cidex, Johnson & Johnson, SP, Brazil	OPA	0.55%	10 min b (room temperature)

^a ppm, parts per million.

^b min, minutes.

Microorganisms used and preparation of inocula

Thirty-eight Candida parapsilosis sensu stricto isolates and the Staphylococcus aureus ATCC 25923 strain were employed in the experiments. The 38 isolates (Table 4) were recovered from both clinical samples and nosocomial environment during candidemia outbreaks in adult ICUs from Brazilian medical centers previously described (8, 13, 14). All C. parapsilosis isolates were identified using VITEK 2 (BioMérieux, Marcy l’Etoile, France), as well as by restriction fragment length polymorphism (RFLP) using the SADH gene and the Ban I enzyme, and genotyped using the Multilocus Sequence Typing technique (13, 14). Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for isolates identification (13). All isolates were found to be resistant to fluconazole and demonstrated positive biofilm formation (13, 14). S. aureus cells were obtained from an overnight culture in yeast-peptone-dextrose (YPD) broth at 37°C, agitated at 100 rpm, and washed with sterile phosphate-buffered saline (PBS) through centrifugation (61). The inoculum was standardized to a concentration of 1.5 × 10⁸ CFU/mL in YPD broth using a nephelometer (Densimat, BioMérieux, Italy). Candida parapsilosis cells were cultured for 18–24 h in YPD broth at 30°C with shaking, washed twice with PBS, and adjusted to a concentration of 1.5–3 × 10⁶ CFU/mL using a nephelometer (Densimat) (61).

TABLE 4.

Origin and nomenclature of C. parapsilosis sensu stricto isolates employed in the study

Collection location
Infusion bomb	BI 1031
Bedside cart	CBL 914; CBL 936; CBL 1003; CBL 1017; CBL 1022; CBL 1028; CBL 1031
Bedrail	C 935; C 936
Healthcare workers	4D; 13D; 14D; 17D; 22D; 29D; 2E; 4E; 7E; 8E;11E; 12E; 17E; 18E; 22E; 23E; 26E; 27E; 29E; 30E; P2/9N; P5/10S; P8/10N
Cardiac monitor	M 935

Evaluating the efficacy of sanitizers against biofilms

For these experiments, all sanitizers (Table 3), S. aureus, and eight isolates of C. parapsilosis randomly selected were used. Aliquots of 200 µL of either S. aureus or C. parapsilosis cell suspension were added to each well of a 96-well microtiter plate to form monospecies biofilms, or a mix of both (100 µL of S. aureus and 100 µL of C. parapsilosis) to form mixed biofilms. After incubation for 24 h at 37°C, the wells were treated with sanitizers at specific concentrations and exposure times as displayed in Table 3 (62 – 64). Subsequently, the biofilms were neutralized with a solution composed of 10 g Tween 80, 1 g lecithin, 0.5 g L-histidine, 2.5 g Na₂S₂O₃, and 3.5 g C₃H₃NaO₃ (65) and scraped off. Control wells without sanitizers were included in all experiments. To assess the viability of the treated and untreated cells, 10 µL aliquots were seeded on brain heart infusion (BHI) agar and incubated for 48–72 h at 37°C (66). These assays enabled the determination of sanitizer efficacy against monospecies and mixed biofilms.

Evaluation of the effectiveness of selected sanitizers against the biofilms

Based on the findings of the study on effective sanitizers, four were selected for their utilization in hand hygiene, skin disinfection, environmental use, and medical device disinfection, to measure the logarithmic reduction of biofilm cells. The sanitizers were tested on both monospecies and mixed biofilms from 38 C. parapsilosis isolates (Table 2) and the S. aureus strain. After the exposure time to the sanitizers, the biofilms were washed with sterile PBS and scraped off. They were then transferred to 0.9 mL of neutralizer solution in microcentrifuge tubes and sonicated for 5 min. Following a 10-fold dilution in PBS, 100 µL of the biofilm solution was seeded on Mannitol agar (S. aureus) and CHROMagar Candida (C. parapsilosis) plates. The CFU/mL was calculated from the colonies counted on the plates and multiplied by the dilution. The log reduction factor (LRF) was used to quantify the mean log reduction in viable cells. This was calculated as the log₁₀ mean density of untreated cells subtracted from the log₁₀ mean density of treated cells (67). Sanitizers that reduced the viable cell count by >4 log₁₀ for bacteria and >3 log₁₀ for fungi were considered bactericidal or fungicidal (21, 68). Sanitizers with lower values were considered bacteriostatic and fungistatic, respectively. All experiments were carried out in triplicate at three different occasions.

Scanning electron microscopy assay

To analyze the morphology and architecture of monospecies and mixed biofilms, we followed a previously published protocol (69). In summary, circular glass coverlips were used as substrates within 12-well cell culture plates (Corning). Cell suspensions containing 5.0 × 10⁶ Candida cells either 1,5 x 10⁸ S. aureus cells per milliliter in YPD were dispensed onto the appropriate wells and incubated at 37°C to allow biofilm formation. To fix the biofilms, we placed them in a solution of 2% formaldehyde (vol/vol) and 3% glutaraldehyde (vol/vol) in 0.1 M potassium phosphate buffer (pH 7.2–7.4) for 48 h. After three washes with 0.1 M phosphate buffer, the cells were postfixed with 1% (wt/vol) OsO4 and dehydrated using a series of ethanol washes (30%–100%). Next, the biofilms were critically dried in CO₂ (MS 850, Electron Microscopy Sciences). To enhance conductivity, the specimens were coated with gold using a Denton Vacuum Desk II coater. Finally, the processed samples were observed using a scanning electron microscope (JSM 5410: JEOL, Tokyo, Japan). This experimental procedure was repeated with three replicates.

Statistical analysis

The data analysis was performed using GraphPad Prism 9.00 software for Windows (GraphPad Software, Inc.). To compare the growth of C. parapsilosis biofilms incubated for 24 h and 48 h, as well as the number of cells in monospecies and mixed biofilms, Student’s t-test was utilized. For comparisons between non-exposed and exposed biofilms to sanitizers, two-way ANOVA and Tukey’s test were employed for multiple comparisons. The significance level was set at P < 0.05 to determine statistical significance.