Detection of Repetitive DNA Sequence and Outer Membrane Lipoprotein in Local Isolates of Pseudomonas aeruginosa

Hawraa Ahmed,Mohammad Sabri Abd Al-Razzaq,Zainab Hmood
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Keywords : Pseudomonas aeruginosa , Box-element , Lipoprotein I.
Medical Journal of Babylon  13:3 , 2016 doi:1812-156X-13-3
Published :25 December 2016

Abstract

A total of 100 clinical samples were collected during this study which were obtained from patients suffering from different infections such as UTI, skins, burns, wounds and from sputum in patient with respiratory diseases, only (12) isolates of P.aeruginosa isolated. At first molecular detection of P.aeruginosa (OprI) was done by using specific PCR primer. It was found that OprI marker was observed in all bacterial isolates (100%) with molecular size (250bp).The results showed that Box-element at a molecular size (500 bp) gave positive in only 9 ( 75% ) isolates. This will reflect on the distribution of some genes among different isolates of P.aeruginosa isolated from different sources.

Introduction

Paeruginosa is consider one of common gram negative bacteria ,have rod-shaped, P.aeruginos aadapted in numerous environmental conditions and also role in biodegradation of the compounds present in the environment [1]. This organism has many mechanisms of resistant to the antibiotics, so the treatment of Pseudomonas infections is very difficult when compared to other bacterial infections caused by other bacteria [2].Pseudomonas have outside proteins called Lipoproteins “I and L” are forming membrane of P.aeruginosa which is blamable for resistance of Pseudomonas to antibiotics . Due to these proteins are originate just in this bacteria , it can be useful and responsible aspect for fast identification of P.aeruginosa [3]. Both outer membrane proteins OprI and OprL consider very important for P.aeruginosa because these proteins play important roles in interaction of this bacteria with environment also help bacteria to resistance antibiotics because present these proteins (specific outer membrane proteins) play important role in efflux transport systems that effect cell permeability[4]. It was detected OprI and OprL by using molecular method and has seen that OprI and OprL considers dependable factor for P.aeruginosa and we can distinguish the P. aeruginosa from different organisms by detect OprI for the genus and OprL for species of this organism [5]. However, the presence of Box-elements which consist of repetitive DNA sequences may express on Pseudomonas genome length and evolution so, these elements can be used to distinguish Pseudomonas strains isolated from different sources particularly those from soil sources and also to study the phylogenic groups [6]. Indeed, the BOX-PCR protocols were favorable for the rapid molecular characterization of phosphate solubilizing for P.aeruginosa, especially at the strains level . The BOX - PCR technique used to distinguished between isolates,especially the isolates that were not easily distinguished by dependent on other phenotypic and phylogenetic techniques such as 16S rDNA[7]. The aim of this study is to isolate Pseudomonas aeruginosa from different clinical samples and to invasion are some cytotoxic factors in the local isolates.

Materials and methods

1- Specimens Collections:
The specimens are obtained from different sites of infections (burns , wounds and sputum s), Each swabs were taken carefully from the sites of infections and placed in tubes containing ready-made media to maintain the swab wet until taken to laboratory. While urine samples were generally collected from catheter tube in sterilized screw-cap containers and then each samples were inoculated on culture media and incubated aerobically at 37C? for 24 hours.
2-Identification of P.aeruginosa isolates:
A total of 100 clinical samples were  collected  during this  study  being obtained from patients suffering from different infections such as UTI, skins, burns, wounds  and  from sputum in patient with respiratory diseases. The patient ages  ranged from (15-60) years  old who  admitted  to  two main hospitals in Hilla city: Al-Hilla General Teaching Hospital, and Mergan Teaching Hospital during a period of three months lasting from (October 2015 to January 2016). It  was  found that (12) P.aeruginosa isolates were  recovered where  Six isolates (50%) were isolated from burns  patient  and  three  isolates (25%) from  catheters  patient  who have  UTI  or renal failure and  Only  two  isolates (16.6%) from  wound  and  one isolate (8.3%) from  sputum   as  shown in Table (1).




Results

Molecular Detection for Virulence Factors: 4 -1 Detection of Outer Membrane Lipoprotein OprI: Molecular detection of P.aeruginosa (OprI) was done by using specific PCR primer. It was found that OprI marker was observed in all bacterial isolates (100%) with molecule size (250bp) as shown in figure(1). This result was agreement with result obtained by[5]and [8]who detected OprI gene by PCR and they found the ability P.aeruginosa to produce OprI gene for all isolate . Also, wessel et al [9]said that OprI is highly abundant in the outer membrane and can exist in a free and peptidoglycan bound form. 4-2 Detection of Box-element Box-element is considered a repetitive DNA sequences present in P. aeruginosa that is used to distinguish between strains and evolution strains of Pseudomonas, So this technique may make discrimination between pathogenic and environmental isolates come from soil. The results showed that in this technique for Box-element at a locus (500 bp) gave positive in only 9 (75%) isolates, as shown in figure (2), So this result mean that all the nine isolates strains transmitted through the hospital environment and not from the soil environment as mentioned by Nassir [6]who reported that highly similarity in binding patterns of clinical and environment isolate which about 45-99% and 61-100%. The Box primer sequence was pointed used in PCR technique to detected Varity in the number and division of this bacterial repetitive sequence in the clinical sample of P.aeruginosa genomes[10]. As well as Box-PCR technique has been used to classify and distinguished different strain of many bacteria. The ensure of the stability of the typing test of Box-PCR is lead to the fact that high distinguished power with reproducibility, stability fast turnaround times and cost-effective alternatives for typing bacteria [11]. BOX-PCR pattern does not depend on the culture age of the strain to be analyzed[12] and fingerprinting output can be easily analyzed by computer assisted methods[13].In a study carried out by [14] it was indicated that genotyping of P.aeruginosa strains could not be associated to the phenotypic characteristics studies.

Discussions

Molecular Detection for Virulence Factors: 4 -1 Detection of Outer Membrane Lipoprotein OprI: Molecular detection of P.aeruginosa (OprI) was done by using specific PCR primer. It was found that OprI marker was observed in all bacterial isolates (100%) with molecule size (250bp) as shown in figure(1). This result was agreement with result obtained by[5]and [8]who detected OprI gene by PCR and they found the ability P.aeruginosa to produce OprI gene for all isolate . Also, wessel et al [9]said that OprI is highly abundant in the outer membrane and can exist in a free and peptidoglycan bound form. 4-2 Detection of Box-element Box-element is considered a repetitive DNA sequences present in P. aeruginosa that is used to distinguish between strains and evolution strains of Pseudomonas, So this technique may make discrimination between pathogenic and environmental isolates come from soil. The results showed that in this technique for Box-element at a locus (500 bp) gave positive in only 9 (75%) isolates, as shown in figure (2), So this result mean that all the nine isolates strains transmitted through the hospital environment and not from the soil environment as mentioned by Nassir [6]who reported that highly similarity in binding patterns of clinical and environment isolate which about 45-99% and 61-100%. The Box primer sequence was pointed used in PCR technique to detected Varity in the number and division of this bacterial repetitive sequence in the clinical sample of P.aeruginosa genomes[10]. As well as Box-PCR technique has been used to classify and distinguished different strain of many bacteria. The ensure of the stability of the typing test of Box-PCR is lead to the fact that high distinguished power with reproducibility, stability fast turnaround times and cost-effective alternatives for typing bacteria [11]. BOX-PCR pattern does not depend on the culture age of the strain to be analyzed[12] and fingerprinting output can be easily analyzed by computer assisted methods[13].In a study carried out by [14] it was indicated that genotyping of P.aeruginosa strains could not be associated to the phenotypic characteristics studies.

References

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