Isolation and identification Streptococcus pneumonia from contact lenses of conjunctivitis and keratitis patients with bacteria resistance to antibiotics.

: There are different methods for identification bacteria,we used culture methods,biochemical tests and molecular method,the aim of this study to decide the measure of efficient select gene for the recognition of the S. pneumonia by molecular tests -specific polymerase chain reaction(PCR),the assay targeting lytA gene from keratitis and conjunctivitis in patients and clients wearing contact lenses in several centers in Erbil city, the results showed that out of 12 investigated S.pneumoniae isolates by culture methods and biochemical testes , lytA gene is found in 6(50%).all 6 of the positive lytA isolates encapsulated ,Norfloxacin and Seftazidime were the best antibiotics (100% ,83.3%) respectively, most infections was keratitis(cornal infections),in addition the majority of patients and clients were among females 78(60%),32(24.6)of patients and clients in the average age ranging from(15-20)years ,80(61.5%) of infections recoded of those who are putting cosmetic lens.


Introduction
Acontact lens is athin lens placed directly on the surface of the eye.there are various uses for it, such as therapeutic reasons for correcting vision otherwise cosmetics or for both reasons ,Many reasons leads people to wear contact lenses ,for instance some people use contact lenses to keep away from wearing glasses or to gain a more aesthetic appearance of their eyes,while others wear contact lenses for the purposes of optical reasons (Zhivov etal.,2007)etal.,2007).Streptococcus pneumoniae is an important cause of keratitis (Pepose and Wilhelmus,1992) S. pneumoniae keratitis commonly follows surgery or trauma to the eye and is more common in patients with coexisting ocular disease Ten thousands of bacterial keratitis cases are reported in USA each year (Arnaud and Tristan,2011) ,keratitis due to S. pneumonia symptoms are the clear dome-shaped tissue on the front of your eye that covers the pupil and iris.This ulcerations will expand quickly, for two days.It become harder especially among immune depressed patients (Mah etal.,2014;Bhave and Chamie,2008) ,many signs appears among keratitis patients such urgently eye( pain, redness) excess tears,feeling that something in eye,photophobia and weak vision (Ng etal.,2016)Many virolence genes support S. pneumonia invasive for instance capsule which promoting attachment to epithelial surfaces and prevent bacteria from phagocytosis by immune system of host , S. pneumonia has many virolence genes such as lytA that encodes surface protein autolysin, represent potential targets for the specific detection of S. pneumoniae corresponds to a right pneumococcus (Nagai etal.,2001;Canvin etal.,1995;Berry etal.,1989),autolysin are endogenous enzymes that specifically degrade the covalent bonds of the cell walls(degrading peptidoglycan) (Tahereh etal.,2015) and eventually can induce bacterial lysis (Berry etal.,1989),autolysinhave been postulated to play a variety of physiological roles on wall growth, wall turnover, cell separation, lysis induced by antibiotics, and pathogenicity, one of the bestcharacterized autolysins, the major pneumococcal lytA amidase (Feldman etal.,1990) the pneumococcal amidase has a modular organization, the Nterminal domain provides the catalytic function, whereas the C-terminal domain, which consists on six repeated sequences, is responsible for binding specificity to the cell wall.
There are many studies conducted the virulence of S. pneumonia, Sanz and his company in 1992 found out that parent strains of S. pneumoniae are more virulent than mutated LytA strains, (Berry etal., 1989)and (Feldman etal.,1990)respectively but two main hypotheses about S. pneumonia virulence,the First explained that autolysis promotes the release of the intracellular toxin pneumolysin (Ply) , Ply is an important determinant of virulence and second that Ply interferes with several defense systems, including inhibition of ciliary beating.

Material and Methods
All 130 swabs samples were collected from eye infections from contact lens wearer in several eye centres in Erbil Governorate during 2016 and 2017 for ages ranging between(14-64)years old.all samples used were collected under aseptic condition and safety precautions, It were taken from patients and clients suffering from eye infections due to complications caused by wearing contact lense for both purposes (cosmetics,therapeutic),samples were taken from keratitis ,conjuctivitis and from(routine tests) healthy clients .1) and 3µl of template DNA (bacterial cell suspension), deionized Sterile distilled water was added to make a final volume of 25 µl.followed by PCR product was electrophoretically separated on agarose gel (1%, containing 0.5 µl/ml ethidium bromide.PCR programe amplifications were carried out in thermocycler using the program as previously described by (Tahereh etal.,2015),The oligonucleotide sequences of primer used in this study are scheduled in Table 1.

Result
Out of 130 samples obtained from keratitis and conjunctivitis ,three types of pathogens were isolated, The overall result shows Pseudomonas species has the highest isolation rate 14(10.8%),followed by S.pneumoniae 12(9.2%) and Staph species has 3(2.3%) as showen in Table (2), this study focused on streptococcus pathogen, 12(9.2%) of isolates were identified S. pneumoniae through a characteristic spherical or ovoid form and appeared achain of two(diplococci) or more bacteria cocci which grow mostly in pairs and in chains of cells, αhemolytic colonies, Catalase negative, Optochin susceptible, Bile soluble.Confirmation was also provided by PCR targeting the S. pneumoniae specific lytA gene (S.pneumoniae species specific), out of 12 investigated S. pneumoniae isolates, lytA gene were found in 6(50%) as shown in figures (1,2) , The incidence of S.pneumoniae respect to the gender and age groups of the patients and clients was found to be more in females Table(3), in the age group (male and female) between (ten twenty) years with contact lens for cosmetic purposes as shown in figure (3) and Table (3).The percentage of S. pneumoniae in order with site of infection; keratitis 5(42%) > both infection(keratitis,conjunctivitis)3 (25%) < (16.5%)for each of periodic checks and conjuctivitis as given in    S.pneumoniae is a member of human microbial flora, set up on the mouth, pharynx, it causes a variety of diseases including keratitis and conjuctivitis, S.pneumoniae is considered the top causes of bacterial keratitis (Ng etal.,2016 ;Bhave and Chamie, 2008).S.pneumoniae usually don't cause any hurt.But the combination of a lot of them on our contact lenses and any small scrape on our eyes can be very dangerous.S.pneumoniae easily spread to eyes by hands.

Identification
the responsible microorganism that cause conjunctivitis and keratitis an important steps for the ophthalmologist and the treatment will be in a correct way, phenotypic characterization such as metabolic enzymes ,optochin test as well as bile solubility of the S.pneumoniae strains were present in some pneumoniae-mitis, pseudopneumoniae strains, traditional tests doesn't give a precise diagnosis .
Our study classified S.pneumoniae in second rank with little difference (two isolates) further than Pseudomonas that not in agreement with (Green etal.,2008) which have concluded Pseudomonas are the most microbes were isolate in eye infections among lenses wearers followed by Staphylococcus.Streptococcus in third world countries is the most common pathogen isolated from keratitis , While largely infection types in developed countries are lachrymal sac or of conjunctival blistering and Pseudomonas as well as staphylococcus is the mainly microbes isolated.
This study showed that an encapsulated isolates were exist in half cases50%, which is the most important virulence factors in S. pneumoniae,in same time 50% of cases caused by noncapsolated S. pneumoniae which means in addition to the capsule it is other pathogenic factors are required by S.
pneumoniae for virulence (Arnaud and Tristan,2011;Kelly etal.,1994) , some previous studies have different conclusions, In a study conducted by (Erin etal., 2002;Reed etal.,2005;Norcross etal.,2010)(Brayn etal.,1992;Blue etal.,2003),whereas (Thomas etal.,2012)showed 100% of their isolates positive result(295-bp).theyshowed also that lytA gene is specific to S. pneumoniae with the exception of bile-insoluble pneumococci.,alsothe method published by Sheppard et al. (2004) targeting the lytA gene constitutes a sensitive and specific assay for distinguishing S. pneumoniae from its close relatives in the mitis group.This is due to differences in the lytA gene sequence of S. pneumoniae and the other mitis group streptococci.This study suggests that the bileinsoluble pneumococcal strains test negative in the lytA gene PCR and (Daniel etal.,2006)studyproved that S.pneumoniae harbored typical lytA alleles (927-bp-long) they result that detection lytA gene by polymerase chain reaction (PCR) assay permits fast and reliable recognition of accurate S. pneumoniae strains as well as characterizes an improved diagnostic tool for the study of pneumococcal.italso save time and effort greater than the classical culture method especially when the sample is blood, cerebrospinal fluid, or pleural fluid (Berry etal.,1989;Versalovic etal.,2011;Thomas etal.,2012;Tahereh etal.,2015).
virulence factors are different among S.pneumoniae strains that cuses different diseases,due to differences in the genetic material that took place in many ways for example S.pneumoniae strains that eye infections has gentetic profile different from that adapted for lung infections or tonsils of the same host,may be the reasons returned to different in adaption period and changes occur in that period such transformation between normal flora and S.pneumoniae which leads to exchange genetic information with other bacteria,another reasons that S.pneumoniae genom is containing BoxB elements which spreded in multiple copies which gives varying gene expression as well as plasticity (Aguiar etal.,2008), (Mogens etal.,2008)Infectious keratitis and conjuctivitis affects both males and females.Afemale preponderance in this study(60%) that was agreed with (Keay etal.,2006;parmar etal.,2007).
In addition(61.5%) of patients were wearing cosmetics lens has been noted,may be due overload attention by women in external appearance,most infection recorded in the teenager group, that concluded age has important role to influence the aetiological agent, may that returns to ignorance hygiene matters and neglect by wearers such as lack of affirmation to visiting the eye doctor at least once a year, sleeping or napping with contacts in, and swimming while wearing contacts.not replacing lenses as often as prescribed, not regularly replacing storage cases and less likely to be instructed on appropriate lenses use and basic hygiene rules (Richard etal.,1991;Arnaud and Tristan,2011).

Fig
Fig.(2): number and percentages of the positive S.pneumonia ,lytA gene and capsule samples.

Table ( 3): Isolation rate of S.pneumoniae from different samples of patients and clients.
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