ARTICLE
Endocervical biofilms in women with endogenous infections in the lower genital tract: study

Biopelículas endocervicales en mujeres con infecciones endógenas del tracto genital inferior: estudio in vitro

Alicia Farinati 1, Daniyil Semeshchenko 1, Martín Santalucía 1, Melina Marqués 1, Adrián Orsini 1

2013
Vol. 25 - Nº.04
Pag.190 – 195

ABSTRACT

Introduction:

The biofilm is one life form of microorganisms (MOs). On mucous membranes of women without and with endogenous infections, they are part of the normal microbiota and cause pathologies. We have demonstrated previously the participation of biofilms in chronic forms of vulvovaginal candidiasis (VVC), the influence of other microorganisms in its formation and evolution, in bacterial vaginosis (BV) and aerobic vaginitis (AV).

Objective:

To analyze the endocervical biofilms in women with or without vaginal infections (VI) comparing them with vaginal biofilms.

Methods:

We studied 22 women, 9 non-pregnant (NP) and 13 pregnant (P). Each patient was gynecologically evaluated, and a vaginal sample (VS) was taken with an aspersorium and an endocervical sample was taken with cytobrush (CB). We performed a fresh examination, pH determination and amine test. Both samples were inoculated in suitable culture medium. After each one, Gram staining and optical microscopy with crystal violet were performed for the study of BF. These were put into Sabouraud broth. All samples were incubated at 35°C for 20-24 hours.

Results:

We have discovered 9 women without pathology and with normal microbiota (NM) and 13 with vaginal infections (VI): bacterial vaginosis (BV) - 6 (4P); vulvovaginal candidiasis (VVC) - 4 (3P); vaginitis and intermediate microbiota (IMB) - 3 (1E). The notable differences were: inflammatory response in the cytobrush compared to the one found in the vaginal samples of women with vaginal infections (10/13), including women with bacterial vaginosis who did not have inflammatory response in the vaginal sample. In the cytobrush of women with normal microbiota, this response occurred only in 1 case (1/9). It was also observed the formation of microfilms of Gram-positive cocci (mostly spp) in the cytobrush of 84.6% (11/13) of the women with vaginal infections and in 66.6% (6/9) of the women with normal microbiota. Among the latter, mixed biofilms were observed in 3 cases with the presence of Gram-positive Bacilli ( (anaerobic) or ).

Conclusion:

Something that called our attention was that the formation of biofilms of and other species of and in the cytobrush of women with vaginal infections in whose vaginal samples these microorganisms were not observed nor recovered significantly. This is a risk since they can initiate an upper genital tract infection (UGTI). In the 4 P with BV, this risk is added to the risk associated with the BV. The question is whether the complications arising from this in pregnancy are not a result of such behavior. In the women with normal microbiota, the biofilms that have Gram-positive cocci can also represent a notable risk in the moment of performing instrumental procedures.

RESUMEN

Introducción:

Las biopelículas constituyen una de las formas de vida de los microorganismos (MOs). En las mucosas de mujeres sin y con infecciones endógenas, integran la microbiota normal y desarrollan patologías. Previamente hemos demostrado la participación de las mismas en las formas crónicas de las candidiasis vulvovaginales (CVV), la influencia de otros microorganismos en su conformación y evolución, en la vaginosis bacteriana (VB) y en las vaginitis aeróbicas (VA).

Objetivo:

Analizar las biopelículas endocervicales en mujeres sin y con infecciones vaginales (IV), comparándolas con las BP vaginales.

Métodos:

Estudiamos 22 mujeres, 9 no embarazadas (NE) y 13 embarazadas (E). Cada paciente fue estudiada ginecológicamente y se tomó muestra vaginal (MV) con hisopo y muestra endocervical con citobrush (EC). Se realizó examen en fresco, determinación del pH y prueba de aminas. Ambas muestras fueron inoculadas en medios de cultivo adecuados. A cada muestra se efectuó la coloración de Gram y se realizó la capa celular sobre el dispositivo de vidrio (DV) para el estudio de las BP. Los DV se colocaron en caldo Sabouraud. Todas las muestras se incubaron a 35°C durante 20-24 horas.

Resultados:

Encontramos 9 mujeres sin patología y con microbiota normal (MN) y 13 con infecciones vaginales (IV): vaginosis bacteriana (VB) - 6 (4E); candidiasis vulvovaginal (CVV) - 4 (3E); vaginitis y microbiota intermedia (VMI) - 3 (1E). Las diferencias notables fueron: hallazgo de respuesta inflamatoria en el EC comparada con la encontrada en la MV en las mujeres con IV (10/13), incluyendo a las mujeres con VB que no presentan respuesta inflamatoria en la MV. En el EC de las mujeres con MN dicha respuesta ocurrió sólo en 1 caso (1/9); y formación de BPs de cocos Gram-positivos (la mayoría spp) en el EC del 84,6% (11/13) de las mujeres con IV y en el 66,6% (6/9) de las mujeres con MN. En estas últimas se observaron BPs mixtas en 3 casos con la presencia de bacilos Gram-positivos, ( (anaerobio) o ).

Conclusión:

Llama la atención la formación de BP de y otras especies de y en el EC de mujeres con IV en cuyas MV no se observan ni se recuperan significativamente estos microorganismos. Esto constituye un riesgo ya que las mismas pueden iniciar una infección del tracto genital superior (ITGS). En las 4E con VB, este riesgo se suma al de la VB y cabe preguntarse si las complicaciones derivadas de la misma en la gestación no son el producto de dicho comportamiento. En las mujeres con MN las BP de cocos Gram-positivos podrían representar un riesgo notable para el desarrollo de ITGS en el momento de efectuar maniobras instrumentales.

Keywords

biofilms
vaginal tract
cervical tract
women.

Palavras-chave

biofilmes
tracto vaginal
tracto cervical
mujeres

INTRODUCTION

In the lower genital tract, the microorganisms (MOs) that compose the normal or usual microbiota can colonize or infect the vaginal mucosa, making biofilms (BF) of single species or mixed ones1. Both the colonizing MOs and those producing vaginal and endocervical infections can determine, in pregnant women during pregnancy and at labor, the contraction of congenital or perinatal infections.

A BF is a very dynamic sessile community of MOs, characterized by cells that are irreversibly joined to a substrate or interface between them, saturated in an extracellular matrix of polymerized substances produced by them and that show a changed phenotype with regard to the growth and gene transcription index2,3,4,5. They can have an important role, both in the infections and for protection. In general, the lower genital tract content is studied taking into consideration the planktonic MOs, but not the BF, since we do not know many physiopathological aspects that happen during the colonization and/or infections.

In the vagina, BFs of lactobacilli are responsible for the wider production of lactic acid that decreases the vaginal pH and prevents, thus, the colonization by pathogenic or potentially pathogenic MOs6.

The usual endocervical microbiota has not been deeply studied and we can assume that it can allow adherence of other MOs that are different from the vagina, due to its more alkaline pH and histological configuration with the cylindrical epithelium tissue. Therefore, in the pathology due to sexually transmitted infections (STIs), like gonorrhea and chlamydia, produced by Neisseria ­gonorrhoeae (NG) and Chlamydia trachomatis (CT), respectively, we know that adherence is produced in the cylindrical epithelial cells instead of in those from the vaginal stratified epithelium7. The association of NG with the receptor is essential for the invasion of the epithelial cell. However, this can vary since members of the CD66 receptors family were identified for several Opacity (Opa) proteins of NG, which mediate the interaction with phagocytes and the passage through epitheliums7,8. CT is linked through bridges of some polysaccharides that are established between the surface of the elementary body and the cellular receptor9.

Is has not been reliably known if the same happens to endogenous infections, since it was always investigated the vaginal tract and not the endocervical one. Probably, the establishment of BF of other MOs in the endocervix is mediated by similar mechanisms, which according to environmental conditions can be changed by the presence of some adhesins, such as those we have seen in these STI agents.

OBJECTIVE

To analyze the behavior of MOs as BF in the endocervix (BFC) in women with and without endogenous vaginal infections (VI) comparing them with the vaginal BF.

METHODS

We have studied 22 women, 9 that non-pregnant (NP) and 13 pregnant (P). Each patient was gynecologically studied. It was taken their vaginal sample (VS) with cotton swabs and endocervical sampling with cytobrush (EC). The following exams were performed: wet mount test, pH determination, and amine testing with HOK at 10%. Both samples were inoculated in suitable culture medians: Sabouraud agar, Cystine Lactose Electrolyte Deficient (CLED) agar, trypticase soy agar (TSA). Gram coloration was performed with each sample and the optical microscopy on the glass equipment (GE) for the BF analysis. The GEs were put in Sabouraud broth. All the samples were incubated at 35oC for 20 to 24 hours.

RESULTS

We found 9 women (5 NP and 4 P) without pathology and NM and 13 with VI: bacterial vaginosis (BV) 6 (4P); vulvovaginal candidiasis (VVC) 4 (3P); vaginitis and intermediate microbiota (VIM) 3 (1P) (Graphics 1 and 2).

Distribution of patients according to the vaginal content findings.

Distribution of patients according to their condition and kind of vaginal content.

The most important differences were:

  • • Inflammatory response in EC comparable to that found in MV in women with VI (10/13), women with BV that did not present inflammatory response in the MV were also included (Graphic 3 and Figure 1). In the EC of women with NM, this response happened only in 1 case (1/9).

  • • Formation of BF of Gram-positive cocci (most of them were Enterococcus spp) in the EC of 84.6% (11/13) women with VI and in 66.6% (6/9) women with NM (Graphic 4). In the latters, mixed BFs were found in 3 cases with presence of Gram-positive bacilli (Actinobaculum (anaerobic) or Actinomyces ) (Figures 2, 3 and 4).

    Inflammatory response in the vagina and endocervix.

    Biofilms of Gram-positive cocci in the EC of women with vaginal infections and normal microbiota.

    Bacterial vaginosis - Endocervix - Positive inflammatory answer - Gram staining, 1000x.

    Actinobaculum (anaerobic) or Actinomyces - Endocervix - Gram staining, 1000x.

    Endocervical biofilm, Gram-positive cocci. Gram staining, 1000x.

    Endocervical biofilm, Gram-positive cocci. Gram staining, 1000x.

    In the cases 12 and 16, one can see the relevant difference on the varied formation of biofilms at vaginal and endocervical levels (Figures 5, 6, 7, 8, 9, 10, 11 and 12). In the Figures 8, 9, 10, 11 and 12, BF presence of negative Staphylococcus coagulase (NSC) (Staphylococcus epidermidis ) is very notable and, nevertheless, these MOs are not seen neither in the vagina nor in their corresponding BF.

    Case 12: direct vaginal exudate. Gram staining, 1000x.

    Case 12: vaginal biofilm. Gram staining, 1000x.

    Case 12: direct endocervical exudate. Gram staining, 1000x.

    Case 12: endocervical biofilm. Staphylococcus epidermidis (SCN). Gram staining, 1000x.

    Case 16: direct vaginal exudate. Gram staining, 1000x.

    Case 16: vaginal biofilm. Gram staining, 1000x.

    Case 16: direct vaginal exudate. Gram staining, 1000x.

    Case 16: endocervical biofilm. Gram staining, 1000x.

    DISCUSSION

    The formation of BF is an important characteristic of NM constitution from the mucosae in our organism. As it is known, the BFs have two kinds of behaviors in our organism, like NM or a pathogenicity and resistance factor in places that are usually sterile or with prosthesis10,11. Their formation participates in the infectious pathology of the lower genital tract together with immunological and allergy factors12.

    We studied the in vitro vaginal BFs both in special equipment and in optical microscopy13,14,15,16. We have demonstrated that in mixed BFs of yeasts and Escherichia coli , the non-albicans Candida species make it easier, due to exuberance of their exopolysaccharide. The bacteria that produce glycocalyx as an adherent material could also have an important role in the pathogenesis of infections by Candida spp, not only in the urogenital level but also in other places. These behaviors would explain the almost constant presence of singe species or mixed BFs in the vaginal tract. In the endocervical tract, the possibilities of BFs formation can be different due to some factors, such as the epithelium characteristics, the number of MOs, the pH and the micro-atmosphere. It is known that in the case of NG colonized over the endocervix cells, the anaerobically induced genes and/or those that codify proteins that participate in the anaerobic breathing are needed to form a BF, whereas the genes responsible for the proteins that take part in the aerobic breathing are less abundant in the BF17. For all these reasons, the BFs in the endocervix may have a different behavior than the vaginal BFs.

    Our findings call the attention to the BF formation of Gram-positive cocci. From the 17 cases of Gram-positive cocci, 11 were of Enterococcus spp, 2 of Streptococcus spp and 4 of SCN, 3 of which were of women with NM. This is a risk since these BFs may progress to the upper genital tract and create an infection therein, which is usually known as pelvic inflammatory illness or infection (PII) and recently as upper genital tract infection (UGTI). In the 4 P women with BV, this risk is an addition to the one of BV and one may ask if the complications from BV in pregnancy are not the product of the sum of such entities. In women with NM, although Gram-positive cocci BFs happen in lower proportion, they can also represent a risk worthy of consideration for the development of a UGTI when performing instrumental maneuvers.

    The inflammatory response in endocervix in BV cases, when absent, could be happening due to the blockage of interleukin (IL)-8 that is in the vagina and not in the endocervix. Although it is increased in the IL-1 β vagina and therefore we expect an inflammatory response, it is not produced through the hydrolytic enzymes of anaerobic bacteria that, together with Gardnerella vaginalis , form an abnormal microbiota in the BV18,19. We do not know if it happens with such ILs in the endocervix, however there is a different microbiota in the BF with a possible distinct activity compared to them. Data found in literature detail the IL concentrations in cervicovaginal washings and in studies about planktonic MOs20.

    Another theme for discussion with regard to BF from the genital tract is the possible influence in the appearance of late sepsis in newborns. Sepsis in such age range is divided into: early sepsis that is manifested in the first 72 hours for 7 days, and the late one, whose incidence peaks are between the second and third weeks21. The Gram-negative bacilli were the most important representations in the 1960s together with emergence of group B Streptococcus in early sepsis22,23. Recently, Gram-positive MOs represent up to 70% of neonatal sepsis in North America, and SCN is the most prevalent in late sepsis, especially in children born with low gestational age and those with low weight (lower than 1,500 g)24.

    In general, it is confirmed that MOs in neonatal late sepsis come from the environment25 or the use of central venous catheters, mechanical ventilation, parenteral nutrition or other invasive procedures26,27,28,29. Few investigators associate the presence of such MOs with a possible colonization from the labor channel.

    Detection of SCN BF in the endocervix of a pregnant woman suggests a risk since the MOs that form them may persist, firstly as colonizers and then as infections, in neonates that normally are treated with anti-microbes with difficulties of approaching the BFs.

    In the common studies regarding lower genital tract infections and during pregnancy, it is more common to give more attention to vaginal infection and forget about investigating what is happening in the endocervix, with the exception of NG or CT. However, endocervix is part of the labor channel and eventually MOs present may affect the fetus before or in labor.

    The described findings allow proposing that further endocervical investigation be performed, independently from the one that is done to investigate NG and CT, especially in women with risks of premature birth or birth of low weight infants.

    CONCLUSION

    Formation of BF of Enterococcus and other species of Streptococcus and Satphylococcus in the EC of women with VI calls the attention, since in their VM such MOs are not significantly seen or recovered. This is a risk, since they may initiate an infection in the UGTI. In the 4 P with BV, this risk is an addition to that of BV and one should ask if the complications from it in pregnancy are not the product of such behavior. In women with NM, the EC BFs of Gram-positive cocci happen in lower proportion.

  • Conflict of interests

    Conflict of interests: The authors declared no conflict of interests.

    REFERENCES

    1. Farinati A, Marqués M, Ocariz MM, Devenutto L. Interacción in vitro de aislamientos urogenitales de spp y Prensa Med Argent. En prensa 2015.
    2. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annu Rev Microbiol. 1995;49:711-45.
    3. Sutherland I. Biofilm exopolysaccharides: a strong and sticky framework. Microbiology. 2001;147(Pt 1):3-9.
    4. Branda SS, Vik S, Friedman L, Kolter R. Biofilms: the matrix revisited. Trends Microbiol. 2005;13(1):20-6.
    5. Davey ME, O'Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev. 2000;64(4):847-67.
    6. Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001;9(1):34-9.
    7. Ison CA, Crabtree HL, Lammel CJ, Brooks GF. Expression of protein II by clinical isolates of In: Achtman M, Kohl P, Marchal C, Morelli G, Seiler A, Thiesen B, editors. Neisseriae 1990. New York: Walter de Gruyter; 1991. p. 597-662.
    8. Mosleh I, Boxberger HJ, Sessler M, Meyer T. Experimental infection of native human ureteral tissue with : adhesion, invasion, intracellular fate, exocytosis, and passage through a stratified epithelium. Infect Immun.1997;65(8):3391-8.
    9. Zaretzky F, Pearce-Pratt R, Philips DM. Sulfated polyanions block Chlamydia infection of cervix-derived human epithelia. Infect Immun.1995,63(9):3520-6.
    10. Wilson M. Bacterial biofilms and human disease. Sci Prog. 2001;84(Pt. 3):235-54.
    11. Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated communities. Annu Rev Microbiol. 2002;56:187-209.
    12. Witkin SS, Linhares JM, Giraldo P. Bacterial flora of the female genital tract: function and immune regulation. Best Pract Res Clin Obstet Gynaecol. 2007;21(3):347-54.
    13. Farinati A, Lopez S, Morgillo P, Rodriguez Estoup V, Vazquez G. Dispositivos médicos, biopelículas y concentración antimicrobiana. Libro de Resúmenes del 9º Congresso de la Sociedad Argentina de Infectología - SADI; 2009 Jun 11-12. Mar del Plata, Argentina.2009.
    14. Farinati A, Miquelarena A, Tajan G, Vazquez G. Biofilms developed in vitro of vaginal microbiota from sexually active women. Abstracts of the 50 Interscience Conference on Antimicrobial Agents and Chemotherapy - ICAAC; 2010 Sep12-15. Boston, United States. 2010.
    15. Farinati A, Marqués M, Orsini A, Arcos M. Vaginal biofilm (VBF) dynamic from normal to the endogenous infection: in vitro study in sexually active women (SAW). Proceedings of the 3 European Congress on Microbial Biofilms - EUROBIOFILMS; 2013 Sep 9-12. Ghent, Belgium. 2013.
    16. Farinati A, Marqués M, Sibert L, Troncoso A, Orsini A. Estradiol hemisuccinate (EH) activity in vitro on vaginal microbiota biofilm. Abstracts of the 51 Interscience Conference on Antimicrobial Agents and Chemotherapy - ICAAC; 2011 Sep 17-20. Chicago, United States.
    17. Falsetta M, McEwan A, Jennings M, Apicella M. Anaerobic metabolism occurs in the substratum of gonococcal biofilms and may be suatained in part by nitric oxide. Infect Immun. 2010;78(5):2320-8.
    18. Cauci S. Vaginal Immunity in Bacterial Vaginosis. Curr Infect Dis Rep. 2004;6(6):450-6.
    19. Cauci S, Guaschino S, De Aloysio D, Driussi S, De Santo D, Penacchioni P, et al. Interrelationships of interleukin-8 with interleukin-1 beta and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women. Mol Hum Reprod. 2003;9(1):53-8.
    20. Kyongo JK, Jespers V, Goovaerts O, Michiels J, Menten J, Fichorova RN, et al. Searching for lower female genital tract soluble and cellular biomarkers: defining levels and predictors in a cohort of healthy Caucasian women. PLoS One. 2012;7(8):e43951.
    21. Qazi SA, Stoll BJ. Neonatal sepsis: a major global public health challenge. Pediatr Infect Dis J. 2009;28(Suppl 1):S1-2.
    22. Donowitz LG. Nosocomial infection in neonatal intensive care units. Am J Infect Control. 1989;17(5):250-7.
    23. Bizzarro MJ, Raskind C, Baltimore RS, Gallagher PG. Seventy-five years of neonatal sepsis at Yale: 1928-2003. Pediatrics. 2005;116(3):595-602.
    24. Stoll BJ, Hansen N, Fanaroff AA, Wright LL, Carlo WA, Ehrenkranz RA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network. Pediatrics. 2002;110(2 Pt 1):285-91.
    25. Brady MT. Health care-associated infections in the neonatal intensive care unit. Am J Infect Control. 2005;33(5):268-75.
    26. Bolat F, Uslu S, Bolat G, Comert S, Can E, Bulbul A, et al. Healthcare-associated infections in a Neonatal Intensive Care Unit in Turkey. Indian Pediatr. 2012;49(12):951-7.
    27. Freeman J, Goldmann DA, Smith NE, Sidebottom DG, Epstein MF, Platt R. Association of intravenous lipid emulsion and coagulase-negative staphylococcal bacteremia in neonatal intensive care units. N Eng J Med. 1990;323(5):301-8.
    28. Távora ACV, Castro AB, Militão MAM, Girão JE, Ribeiro KC, Távora LGF. Risk factors for nosocomial infection in a Brazilian neonatal intensive care unit. Braz J Infect Dis. 2008;12(1):75-9.
    29. Healy CM, Baker CJ, Palazzi DL, Campbell JR, Edwards MS. Distinguishing true coagulase-negative infections from contaminants in the neonatal intensive care unit. J Perinatol. 2013;33(1):52-8.

    Address for correspondence:


    . Calle Monseñor Larumbe 12 - 10 Piso C - Martinez. Buenos Aires, Argentina. Postal code: 1640. Phone: +54 (11) 4792-8531. E-mail: farinati@fibertel.com.ar

    History

    Received: 29/06/2014

    Accepted: 16/08/2014

    Views: 228