ARTÍCULOS ORIGINALES

Nasal carriage of Staphylococcus aureus and Candida species in immunocompetent individuals

 

María L Nastri¹, Pedro Hecht⁴, Natalia Nastri¹, Virginia Jewtuchowicz^1,2, Alicia Cuesta¹, Juliana Estrugo³, Ariel Gualtieri⁴, Daniel O. Sordelli², Alcira Rosa¹

¹ Department of Microbiology, School of Dentistry, University of Buenos Aires, Argentina
² Department of Microbiology, School of Medicine, University of Buenos Aires, Argentina
³ Department of Periodontology, School of Dentistry, University of Buenos Aires, Argentina
⁴ Department of Biophysics, School of Dentistry, University of Buenos Aires, Argentina

CORRESPONDENCE Dra. Alcira Cristina Rosa Catedra de Microbiologia y Parasitologia Facultad de Odontologia - Universidad de Buenos Aires M.T. de Alvear 2142 - (C1122AAH) Buenos Aires, Argentina E-mail: flianastri@yahoo.com

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ABSTRACT

The aim of this study was to analyze the prevalence of Staphylococcus aureus and Candida species in samples of nasal mucosa from 100 immunocompetent subjects of both sexes, aged 18-70 years, during stomatological clinical examination. Samples were taken from the mucosa of both nasal fossae using sterile swabs. Samples were observed fresh, stained with Gram and Giemsa, and cultured on selective differential media at 37^oC to isolate and identify the selected microorganisms; conventional biochemical tests and commercial equipment and molecular studies using PCR were performed. A digital thermometer-hygrometer was used to measure room temperature at the time of sampling, which was on average 25±2^o C, with relative ambient humidity 66±11%. S. aureus was isolated from 38% of the samples; 4% of the samples were methicillin-resistant (MRSA) strains, with 2% identified genetically as community-acquired (CA-MRSA) and 2% as hospital-acquired (HA-MRSA). Candida was identified in 23% of the samples, with prevalence of C. albicans (19%) followed by C. dubliniensis (3%) and C. krusei (1%). There was significant association between Candida and S. aureus (Chi-squared = 27.75; df = 1; (p<0.001). The nasal cavity is a reservoir and the identification of genus and species contributes to adequate epidemiological surveillance.

Key words: Nasal cavity; Staphylococcus aureus; Candida.

RESUMEN

Portación nasal de Staphylococcus aureus y especies de Candida en individuos inmunocompetentes

El proposito de este trabajo fue analizar la prevalencia de Staphylococcus aureus y especies de Candida en muestras de mucosa nasal de 100 individuos inmunocompetentes de ambos sexos con edades entre 18-70 anos, durante el examen clínico estomatológico. Las muestras fueron obtenidas con hisopos estériles sobre la mucosa de ambas fosas nasales. Se realizo observacion en fresco, tinción de Gram, Giemsa y cultivos en medios selectivos y diferenciales a 37oC para el aislamiento e identificación de los microorganismos seleccionados, pruebas bioquímicas convencionales y equipos comerciales y estudios moleculares mediante la prueba de PCR. Con un termo higrómetro digital se midió la temperatura ambiente cuyo promedio en el momento de la toma en el consultorio fue de 25±2 ^oC y la humedad relativa ambiente fue del 66±11 %. S aureus se aisló en el 38% de las muestras y dentro del mismo, 4% fueron meticilino resistentes (MRSA) siendo genéticamente 2 % de la Comunidad (MRSA-CA) y 2 % Hospitalarios (MRSA-HA). En el 23% de las muestras fue identificada Candida siendo la especie prevalente C. albicans: 19% y en menor proporcion C. dubliniensis: 3% , C. krusei: 1%. Se registro una asociacion significativa entre Candida y S. aureus (Chi-cuadrado=27,75; gl=1; (p<0,001). La cavidad nasal constituye un reservorio y la identificación de género y especie contribuye a la adecuada vigilancia epidemiológica.

Palabras clave: Cavidad nasal; Staphylococcus aureus; Candida.

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INTRODUCTION

Microorganisms in their reservoirs can act as a source of infection and spread. The nasal fossae are an ecological niche that can be colonized by S. aureus, and asymptomatic S. aureus colonization is much more common than infection. Transmission occurs directly by contact with a carrier. Depending on the population studied, the carrier rate ranges from 25% to 50% in the general population and is higher in injection drug users, insulin-dependent patients, hemodialysis patients, patients with skin disorders, intravascular catheters or HIV/AIDS and healthcare providers^{1- 2}. Carriage plays a relevant role in the epidemiology and pathogenesis of infection. In healthy subjects, 20% are persistent carriers, 60% are intermittent carriers and 20% were never carriers³. The increasing multi-resistance of S. aureus to antibiotics is a major issue¹. Staphylococcus aureus (Sa) is a bacterial species often isolated from patients both with hospitalacquired infections (HA-MRSA) and communityacquired infections (CA-MRSA). It has become one of the hospital and community pathogens of great epidemiological interest, and causes mild to lifethreatening infections such endocarditis or septicemias. S. aureus in nasal carriers can also colonize other locations such as intact skin⁴. Our group carried out an analysis of MSRA carriage in a healthy population of patients with gingival- periodontal disease, unrelated to infected patients⁵.
Infections of the paranasal sinuses (maxillary, sphenoid, ethmoid and frontal) and sinusitis due to Candida spp. may be due to translocation by which the fungus has propagated from the nasal cavity. They may also occur as a result of treatment with cytotoxic drugs or processes associated to leukemia, multiple myeloma and others⁶. Studies by Ponikau et al.⁷ and Braun et al.⁸ show high incidence of fungal colonization (91 to 96%) in subjects with chronic nasal sinus pathology, compared to previous studies which had established little relationship between these conditions. The aim of this study was to determine the prevalence of S. aureus (Sa) and identify methicillin resistant species (MSRA) and Candida species (Ca) in samples of nasal mucosa from immunocompetent patients.

MATERIALS AND METHODS

Study population
The study was performed on samples of nasal mucosa from 100 immunocompetent adults of both sexes, aged 18 to 70 years, mean age 43.3±15.4 (54.9% female and 45.1% male) who received care at the School of Dentistry of Buenos Aires University during 2005-2009. Their evaluation included medical-dental history and clinical examination. Patient participation was voluntary and all participants signed informed consent after the purpose of the study had been explained to them.

Exclusion criteria
Pregnant women, subjects with systemic diseases who had taken antibiotics, non-steroidal antiinflammatory drugs and/or corticoids or antifungal medication within six months prior to the sampling were excluded from the study.

Temperature and humidity measurement
A digital thermometer-hygrometer was used to record the temperature and humidity in the room where the nasal mucosa was sampled.

Sample collection
To prevent contamination, the outer area on the nostrils was rubbed with sterile gauze soaked in sterile distilled water, which was then discarded. Nasal mucosa was sampled by rotating a sterile swab on the mucosa in both nasal fossae. The material collected was placed in a sterile tube with a stopper and sent to the laboratory within 2 hours.

Sample processing: Microbiological studies.
Microscopic studies were performed by: a) observation of fresh samples, b) smears stained using Gram and Giemsa techniques. The clinical samples were cultured on selective differential media to isolate the selected microorganisms, and incubated under specific atmospheric conditions. Quality control was performed previously on the culture mediums by using ATCC reference strains, following Cuesta⁹. Isolate phenotypes were identified by using conventional biochemical assays and commercial equipment to verify the presence of morphological and stain characteristics for the genus. Staphylococcus species were identified by using mannitol salt agar and CHROMagarTM Staph aureus (Sa) (CHROMagarR Company, Paris, France) following Cuesta⁹ Platzer ¹⁰ and Fowler¹¹. Sensitivity to antimicrobials was tested according to NCCLS and CLSI (Clinical Laboratory Standards Institute).¹².

Molecular studies
The MRSA strains isolated were subjected to PCR (Polymerase Chain Reaction) in a thermal cycler (Mini CyclerTM, MJ Research Inc.) using Taq polymerase from Invitrogen¹³. We investigated the mecA gene and LukF-PV and LukS PV genes, two contiguous genes co-transcribing and coding for Panton Valentine leukocidin (PVL), which represent one of the genetic markers associated to CA-MRSA⁹. The PCR products were analyzed by gel electrophoresis in 2% agarose with 0.5ug/ml ethidium bromide, following Jewtuchowicz¹⁴ and Von Eiff¹⁵ ATCC positive controls were used for the PCR of methicillin-resistant and PVL carrying strains, following Cuesta ⁹. Candida was identified by using CHROMagar Candida R medium (CHROMagar Company, Paris, France). Yeast species were identified according to the color of colonies, following Odds.¹⁶, observation of micromorphology in milk-Tween 80 1% agar and carbohydrate assimilation profile using commercial system Api ID 32D (a BioMerieux, France)^{17 -18}.

Statistical analysis
Frequency distribution and arithmetic mean were performed for the microorganisms analyzed. Standard deviation and 95% confidence interval for temperature and humidity, and Chi-squared (c2) test for comparison of frequencies in contingency tables, with Yates correction for continuity (significance level p< 0.05).

RESULTS

Average room temperature during sampling was 25 ± 2oC and relative humidity was 66 ± 11% (Table 1). S. aureus was isolated from 38% of the samples; and 4% were methicillin-resistant (MRSA). Genetically, we identified 2 % community-acquired (CAMRSA) and 2% hospital-acquired (HA-MRSA) (Fig.1).

Table 1: Descriptive statistical parameters for temperature and relative humidity

Fig. 1: Distribution of Staphylococcus aureus and methicillinresistant Staphylococcus aureus (MRSA) and its genotypic detection as CA-MRSA (community acquired) and HA-MRSA (hospital-acquired), isolated from nasal fossae.

The four MRSA in the total sample were found in males. Candida was identified in 23% of the samples. The prevalent species were C. albicans (19%), C. dubliniensis (3%) and C. krusei (1%) (Fig 2). Significant association was found between Candida and S. aureus (Chi-squared = 27.75; df = 1; (p<0.001) (Table 2).

Fig. 2: Distribution of Candida species isolated from nasal fossae.

Table 2: Contingency of Staphylococcus aureus and Candida spp

DISCUSSION

The appearance of new species related to human health calls for the development of simple, quick, efficient methods for their identification at clinical microbiology laboratories. The relationship between colonization and infection has not been fully elucidated, although it has been associated to intrinsic factors of the host as well as factors of the microorganism. There is controversy regarding whether S. aureus is considered normal microbiota, as some authors have studied the correlation between S. aureus nasal carriage and the presence of S. aureus bacteremia, having found clonality between the two strains (nasal and blood) in 80% of the cases, whereby nasal carriage of S. aureus might be considered as a factor associated to bacteremia¹⁵.
Moreover, the nasal colonization rate by S. aureus varies according to the population studied. In healthy adults, three types of nasal carriage can be distinguished, associated to host and microorganism factors. These are: a) intermittent carriers (60% of individuals), b) persistent carriers (20%) and c) non-carriers (20%). Longitudinal studies have shown that colonization is transient rather than permanent in a given individual, and may change over time^19,20. Archer¹ reports that there is nasal colonization by Staphylococcus aureus in 20%-30% of the general population and it does not imply the presence of true infection. Kuehnert²¹ analyzed S. aureus nasal colonization and MRSA in 9622 persons one year of age or older as part of a National Health and Nutrition Examination Survey, 2001-2002 in USA, and found that estimated prevalence and colonization of S. aureus and MRSA were 32.4% and 0.8%, respectively, and may vary according to demographics and the organism. The same group²² analyzed nasal colonization by S. aureus from 2001 to 2004 and found that the prevalence of S. aureus colonization decreased from 32.4% in 2001-2002 to 28.6% in 2003-2004 (P < .01), while the prevalence of colonization by MRSA increased from 0.8% to 1.5% (p <.05), concluding that nasal colonization by MRSA has increased in the United States, despite an overall reduction in nasal colonization by S. aureus. Our study found higher Staphylococcus aureus and MRSA prevalence than Kuehnert²¹ and Gorwitz ²². Platzer ¹⁰ detected lower proportions of Staphylococcus aureus nasal carriage (22.7 %) in a healthy population in the city Santiago de Chile. Further studies should be undertaken in larger numbers of patients, and there should be longitudinal studies to analyze nasal colonization by S. aureus and MRSA, to ascertain whether they are persistent or intermittent carriers, as suggested by Kluytman3 and epidemiological surveillance of community transmission should be performed. Candida infection is considered endogenous, caused by commensal species previously present in the subject, with C. albicans being the species with highest incidence. These species should be defined as opportunistic within the general concept of hostparasite relations^23-25. Colonization by the genus Candida in humans usually remains as such due to the different defense mechanisms of the human body against any agent. The intervention of non-specific natural barriers, such as external barriers (skin and mucosa) is considered important¹⁶.
We agree with Torres-Rodriguez²⁶ regarding the use of cultures in chromogenic media for the primary isolation of Candida, because it facilitates species identification. Ponikau et al.⁷ and Marple²⁷ both report that nasal sinus fungal colonization is often sub-diagnosed, and they have created consensuses based on applying diagnostic methods with the aim of detecting fungi and determining their role in the etiopathogenesis of nasal sinus diseases. A range of precise diagnostic methods is currently available for identifying the fungi. However, culture in specific media is the standard of choice for the purpose^7,27. Our study used these media for identifying Candida species. The oral cavity has been proved to be a reservoir and source of dissemination to other parts of the body and a source of transmission to other persons, foods and objects²⁶. Nasal fossae can also be an ecological niche for colonization by the genus Candida. It is necessary to identify not only the genus but also the species in the nasal fossae for proper epidemiological surveillance, as they are reservoirs of opportunistic microorganisms²⁷. Our study found Candida albicans 19%, C. dubliniensis 3%, and C.krusei 1% in nasal mucosa, and did not identify C. parapsilosis. However, according to reports published by certain European currents, it is not possible to specify whether it is only colonization by co-habitation or whether the presence of these microorganisms is a determining factor of chronic disease^{8, 28}.
We agree with Rodero²⁹ that it is not surprising to isolate C. albicans, since it is the yeast most often associated to colonization of skin and gastrointestinal tract. Nevertheless, in recent decades, there has been a change in the spectrum of infections by yeasts and an increase in the appearance of other non-C. albicans yeasts. The results of the study by Martin³⁰ on microorganisms causing sepsis in the United States during the past twenty years show that the incidence of Grampositive bacteria has increased more than that of Gram-negative bacteria, and fungal infections have increased exponentially. Our study detected a significant association between the presence of Candida and Staphylococcus aureus in nasal mucosa. Noverr4 and Peters ³¹ suggest that Candida albicans acts as a scaffolding for Staphylococcus aureus, forming microcolonies and polymicrobial biofilm. Further studies are needed to interpret this association. Temperature and moisture were recorded at the room where nasal mucosa was sampled in order to detect possible exogenous variables in the transportation and isolation of the microorganisms that were found. Further studies on larger numbers of patients will shed light the matter. It would be interesting to perform epidemiological studies in different geographical areas across Argentina in order to carry out surveillance of nasal carriage of these pathogens and the way they relate to other human pathologies.

CONCLUSION

The following were recorded for nasal mucosa samples from immunocompetent patients:
• 38% nasal carriage of Staphylococcus aureus, which included 4% genetically identified as community- acquired methicillin-resistant strains (CA-MRSA) and hospital-acquired methicillinresistant strains (HA-MRSA);
• the most frequently identified Candida species was C. albicans, followed by C. dubliniensis and C. krusei;
• a significant association between Staphylococcus aureus and Candida.

ACKNOWLEDGEMENTS

This work was supported by Grant UBACYT O 016, from Universiy of Buenos Aires.

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