Detection of Phospholipase Enzyme in Bacterial Associated with Vaginitis

Douaa Hamza Khair-Allah,Mohammad S. Abdul-Razzaq,Asmaa K. Gata?a
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Keywords : Phospholipase Enzyme,Bacterial Associated,Teaching Hospital of Hilla,candida albicans
Medical Journal of Babylon  10:4 , 2014 doi:1812-156X-10-4
Published :05 June 2014

Abstract

In this study, (90) vaginal swabs were taken from women suffering from vaginitis, who were admitted to Babylon hospital for maternal and pediatrics, and Teaching Hospital of Hilla, during the period from November 2012 to march 2013 in Babylon province . The patient’s age ranged from (18 - 54 years ). 76 vaginal swabs were taken from non pregnant women , and 14 from pregnant women, 88 samples gave positive results of bacterial growth and only two samples gave no growth bacterial. The most common species of opportunistic bacterial types were, Streptococcus agalactiae (28.4%), S. aureus (21.5%) , Streptococcus mutans (15.9 %) , Coagulase – negative staphylococcus (12.5%), Escherichia coli( 7.9%), Acinetobacter (5.9%), Enterococcus (5.9%), Neisseria.gonorrhoea(2.2%). Production of exteracellular phospholipase D in the presence of arachidonic acid was also detected. It was found that all isolates have ability to produce this enzyme . Also , phospholipase extract was added to the culture media of candida albicans , and it was found that there was an increase in number of candida( which was isolated in this study) after the addition of enzyme extract.

Introduction

The genital tract is the portal of entry for numerous sexually and non-sexually transmitted diseases. A number of bacterial and non-bacterial infections exist that affect the female reproductive tract and cause vaginal discharge. Vaginal discharge is a common symptom in primary health care and is often the second most common gynecological problem after menstrual disorders [1]. Bacterial vaginitis is an infection of the lower female genital tract that occurs predominantly in women after marriage . It is caused by an alteration in the normal vaginal flora in which the normally predominant Lactobacilli are replaced by pathogenic bacteria [2]. The most common organisms present in healthy vagina are Lactobacilli, and candidia Albicans in the most common, However many bacteria may associate with vaginitis such as staphylococcus aureus, Escherichia coli, Group B streptococcus, Listeria monocytogenes, coagulase_negative staphylococcis, Acinetobacter spp., Neisseria. gonorrhoea, Enterococcus spp. Streptococcus mutans [3]. The most important virulence factor are the phospholipases produced by bacteria . There are many ways by which bacterial phospholipases contribute to the development of disease ,direct effects of phospholipases resulting from the hydrolysis of phospholipids leading to loss of membrane integrity and cytotoxicity [4,5]. Certain microbial phospholipases are known to possess haemolytic activity. Phospholipase C from certain microbial source was shown to be devoid of the amino acid sequence associated with haemolytic activity [6]. This study mainly aims to detect phospholipase D activity in microorganisms associated with vaginitis.

Materials and methods

collection of samples : The samples were generally collected from women with vaginitis by using a vaginal speculum Specimens were immediately inoculated on blood agar plates, nutrients agar, Columbia- bloob agar plates and MacConkey s, plates. All plates were incubated at 37°C for 24 – 48 hrs.
Phospholipase D Production
Nutrient agar media was used for the detection of phospholipase D enzyme. After sterilization in autoclave and cooling to 50°C , 1% of arachidonic acid (sterilized by filtration) was added. After the inoculation of this media with bacterial isolates and incubation at 37°C for 24 hours; 3ml of glacial acetic acid was added to precipitate the lipid. The formation of transparent area around the colony of bacteria indicated a positive result for phospholipase enzyme (personal communication by Dr. Mohammad Sabri).
Growth of candida albicans in presence of bacterial phosphlipases extract (personal communication by Dr. Mohammad Sabri).
1- Bacteria supernatant which was confirmed to possess phospholipases is subdetacted for this experiments
2- Candida was previously cultivated on brain heart infusion ( BHI) agar for 24 hr. at 37°C
3-The yeast was then transferred to BHI broth and then dilutions were done to calculate the colony forming unit (CFU).
4- Then,250 ?l of yeast growth was mixed with 100 ?l of bacterial laysate.
5- After incubation for 24 hr. at 37 °C on BHI agar with 1% arachidonic acid ,dilutions were also done and the CFU was calculated for each


Results

Isolation and Characterization: Ninty (90) vaginal swabs were taken from women with abnormal vaginal discharge , who were admitted to Babylon hospital for maternal and pediatrics, Hilla teaching hospital, during the period from November 2012 to March 2013 in Babylon province . The patient’s age ranged from ( 18 - 54 years ).( 76 ) vaginal swabs were taken from non pregnant women, and (14) taken from pregnant women, of these 90 swabs,28swabs were from Opportunistic Bacterial Isolates in Women with Vaginitis Some cases of vaginitis were caused by single bacterial type ,candida albicans was also isolated from cases. The followed types of bacteria were detected, group B streptococci GBS (25), Staphylococcus aureus (19) ,strepcoccus mutans (14), coagulase – negative staphyloccocis (11), E coli (7) , acinetobacter(5) and enteroccoci (5) and N. gonorrhoeae(2) 3.2. Phospholipase D production It was found that most bacterial isolates have the ability to produce phospholipase D enzyme extracellular when arachidonic acid as a substrate is used. It was noticed that phospholipase D is mostly produced by Gram positive bacteria and in less degree by Gram negative bacteria. .shown in table 3. Growth of candida albicans in prsenc of bacterial phosphlipases extract In this study candida albicans was grown in the presence of PLD extract after incubation for 48 hrs. it was found that the number of candida was increase.

Discussions

The outcome of our study revealed that prevalence of isolated among nonpregnant women is high when compared with pregnant women, also it was observed that gram positive bacteria is more prevalence than gram negative bacteria where the rate of isolation of gram positive is 67 % among nonpregnant women. This result is identical to that results obtained from local study in Hilla province where gram positive bacteria was more predominant than gram negative (Taisser 2009 ; ZainabAdil 2011). Also, This study revealed that S. agalactia constitutes one of the predominant organisms incriminated as the major causes of viginitis , because out of the 88 positive isolates, S. agalagtia constituted 25 (28.4%) out of the organisms isolated from the pregnant and non-pregnant women , this was followed by Staphylococcus aureus 19(21.5 %), S. mutans 14(15.9%), Coagulase – negative staphylococcis 11(12.5%), E. coli 7(7.9%) Acinetobacter 5(5.9%), Enterococci 5(5.9%) and N. gonorrhoeae . From these results , S. agalagtiae is predominant among Gram positive whereas E. coli predominant among Gram negative. Phospholipase D enzyme was screened to show the ability of bacterial isolates to produce this enzyme extracellularly .It was found that most bacterial isolates have the ability to produce phospholipase D enzyme extracellular when arachidonic acid as a substrate is used. It was noticed that phospholipase D is mostly produced by Gram positive bacteria and in less degree by Gram negative bacteria. The results of this study revealed that most isolates of S. aureus , C.N.S and S.agalatiae were able to produce this enzyme (89.4%, 72.7%, 60 % respectively ). It was clear that staphylococci isolates are highly predominant of this enzyme followed by streptococci which are presented by St. agalactiae and S. mutans where their isolates produce this enzyme at rate above 50 %. In contrast to enterococcus which failed to produce this enzyme , this may be due to absence of genes involved for production of enzyme , or the time of enzyme induction is not enough or there is a problem in secretion of such enzymes. Also ,Escherichia coli isolates were able to produce this enzyme extracellarly as in Gram positive bacteria although vagina is not the natural habitat of this enzyme .Moreover Acinetobacter isolates have the ability to produce this enzyme at a rate 60%. This is a real pathogen in vaginitis and also it resembles N. gonorrhea . It s diplococci but the later failed to produce this enzyme . The results of this study resemble the study done by walev et al. [7 ] who found that S. aureus have the ability to produce PLD but previous studies done by Colee and Proulx [8] have found that S. aureus have no ability to produce this enzyme. The results shown that 71.9% E.coli produced phospholipase D, Lee et al , [9] reported that E. coli phospholipase D. Songer et al [10] , also found that E. coli was expression of the phospholipase D gene . 60 % of Acinetobacter isolates also had been product phospholipase D Jacobs et al [11], reported that phspholipase D major virulence factor of Acinetobacter. Also 50% of S. mutans produced PLD. In this study, the results showed that all Enterococcus and N. gonorrhea isolates were negative for PLD production. Bacterial PLD are important to adhere to and to invade primary cervical cells [12] . In this study candida albicans were grown in the presence of PLD extract after incubation for 48 hrs. It was found that the number of candida was increase. When PLD extract of S. arueus , S. mutans and E. coli it was used. It was ,known that PLD increase the rate of candida cell division , this will explain why candida albican were overcome in some of vaginitis although it is not a real pathogen , this will return to the ability of bacterial pathogen to produce extracellular PLD which in turn will increase the division of candida and hence increase the number of it. Mclain and Dolan [12] showed that PLD required for dimorphic transition in candida albican. However, some studies indicated that PLD is very important in adhesion and also in stimulation of sporulation in some fungi.

Conclusions

N/A

References

1- Isibor, J. O.1, Samuel, S. O., Nwaham, C. I., Amanre I. N., Igbinovia, Oand Akhile, A. O. 2011. Prevalence of bacterial and Candida albicans infection amongst women attending Irrua Specialist TeachingHospital, Irrua, Nigeria African Journal of Microbiology Research Vol. 5(20) : 3126-3130

2- Tagg, J. R., Dajani, A.S. and Wannamaker ,L.W. 2007. Bacteriocins of gram positive bacteria, Bacteriol. Rev., 40: 722-756.

3- Donders, G.G.G.; Vereecken, A.; Bosmans, E.; Dekeersmaecker, A.; Salembier, G.; and Spitz, B. (2005). Aerobic vaginitis: A bnormal vaginal flora entity that is distinct from bacterial vaginisis. Gynecol. Obstet. Rep. Med. In Daily Practice, 1279: 118-29.

4- Sitkiewicz, I., Stockbauer, K.E., and Musser, J.M., 2006. Secreted bacterial phospholipase A2 enzymes: better living through phospholipolysis. Trends in Microbiol. 15(2), 63-69.

5- Hurley, B.P., and McCormick, B.A., 2008. Multiple Roles of Phospholipase A2 during Lung Infection and Inflammation. Infect. Immun. 76(6), 2259-2272.

6- Zofia Olempska-B. 2008. Phospholipase C Expressed in Pichia pastoris. Chemical and Technical Assessment (CTA)1-7.

7- Iwan Walev , Ulrich Weller , Suanne Strauch, Timothy Foster, and Sucharit Bhakdi 1996 . Selective Killing of Human Monocytes and Cytokine ReleaseProvoked by Sphingomyelinase (Beta-Toxin) of Staphylococcus aureus Infection and Immunity,. 64( 8) 2974–2979.

8- Coleeand R. and proulx. 1975. Phospholipase D Activity of Gram-
Negative Bacteria. J. of Bacteriolgy.124( 3): 1148-1152.

9- Lee JS, Bat-Ochir M, Demirev AV, Nam DH2009 .Molecular cloning of the phospholipase D gene from Streptomyces sp. YU100 and its expression in Escherichia coli.J Microbiol. 47(1):116-22.

10- Songer JG, Libby SJ, Iandolo JJ, Cuevas WA1990 . Cloning and expression of the phospholipase D gene from Corynebacteriumpseu-dotuberculosis in Escherichia coli.J .of Infect Immun. ;58(1):131-6.

11- Anna C. Jacobs, Indriati Hood, Kelli L. Boyd, Patrick D. Olson, John M. Morrison , Steven Carson, Khalid Sayood, Peter C. Iwen, Eric P. Skaar, and Paul M. Dunman 2010. Inactivation of phospholipase D diminishes Acinetobacter baumannii pathogenesis. Infect Immun. 78(5):1952-1962.

12- Edwards JL, Entz DD, Apicella MA: Gonococcal phospholipase D modulates the expression and function of complement receptor 3 in primary cervical epithelial cells. Infect Immun 2003, 71:6381-6391.

13- Nealoo McLain and Joseph W. Dolan 1997. Phospholipase D activity is required for dimorphic transition in Candida albicas. J. of Microbiology 143: 3521-3526.


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