Genetic study of biofilm forming bacteria, isolated from denture and orthodontic devices

Other Title(s)

دراسة وراثية للبكتيريا للغشاء الحيوي و المعزولة من أطقم و أدوات تقويم الأسنان

Dissertant

Husayn, Khulud Abd al-Karim

Thesis advisor

Abd al-Abbas, Munaf Jawdat

University

University of Basrah

Faculty

Science College

Department

Department of Biology

University Country

Iraq

Degree

Master

Degree Date

2013

English Abstract

The bacterial species were identified by molecular diagnosis using 16SrDNA sequencing for only 94 bacterial samples from denture (n=47) and orthodontic (n=47) revealing the following species in denture: Klebsiella pneumoniae [8(40%)], Proteus mirabilis [7(35%)] , Proteus penneri [5(25%)] , Enterobacter cloacae [3(15%)], morganella morganii [2(10%)], Hafnia alvei [2(10%)], Enterobacter aerogenes [2(10%)],Enterococcus faecalis [1(5%)] , Enterobacter faecium [1(5%)], Bacillus cereus[1(5%)], Enterobacter mori [1(5%)] and Citrobacter freundii [1(5%)].Some of these bacterial species were isolated at first time in the world from denture as : Proteus houseri [1(5%)], Klebsiella variicala [1(5%)], Lactococcus lactis [1(5%)], Streptococcus equinus [1(5%)], Acinetobacter baumannii [1(5%1)], Chryseobacterium vietnamense [1(5%)], Klebsiella oxytoca [1(5%)], and Staphylococcus hominis [1(5%)].

But the following species from orthodontic: Klebsiella pneumoniae [8(40%)] , Staphylocpccus aureus [7 (35%)], Bacillus cereus[4(20%)], Enterobacter cloacae [3(15%)], Enterococcus faecalis [3(15%)], Staphylococcus epidremidis [2(10%)], Proteus penneri [2(10%)], Enterobacter faecium [2(10%)], Enterobacter mori [1(5%)], Citrobacter freundii [1(5%)], Staphylococcus worneri [1(5%)] and Serratia marcescens [1(5%)].Some of these bacterial species were isolated at first time in the world from orthodontic as: Staphylococcus pasteuri [1(5% )], Enerobacter ludwigii [1(5% )], Lactobacillus plantarum [2(10%)], Streptococcus anginosus [2(10%)], Pediococcus acidilactici [1(5%)], Bacillus subtilis [2(10%)], Escherichia fergusonii [1(5%)], and Proteus mirabilis [1(5%)].

Furthermore, Proteus penneri, Enterobacter mori and Citrobacter freundii in both denture and orthodontic were isolated at the first time.

The rooted neighbour joining (NJ) phylogenetic tree of isolates from present study (n=28) with 28 type strains from GeneBank showed that Enterobacter cloacae is the out group (root).Moreover, four bacterial strains were recorded in European Nucleotide Archive (ENA),National Centre for Biotechnology Information (NCBI) and Gene Bank as new strains in the world, these are: 71-Chryseobacterium vietnamense "IRQBAS3" (HG003648) and 74-Morganella morganii "IRQBAS4" (HG003649) isolated from dentures, and 7-Enterobacter ludwigii "IRQBAS1" (HG003646), and 34-Enterobacter cloacae "IRQBAS2" (HG003647) isolated from orthodontic.

All these strains showed 99% sequence identity with their reference due to occur of point or frame shift mutation.

Biofilm formation of bacterial isolates from denture (n=47) and orthodontic (n=47) were screened by Congo red agar (CRA) ,tissue culture plate (TCP) and icaAD gene method.

In denture, CRA method appeared the frequency of negative isolates 37(78.7%) of 47 isolates was higher than positive isolates 8(17.02%) of 47 isolates with high significant ( p ≤ 0.01) , and higher than intermediate 2(4.3%) of 47 isolates with high significant ( p≤ 0.01), while frequency of positive isolates was higher than intermediate isolates with high significant ( p ≤0.01).the frequency in orthodontic recovered very different results 4(8.5%) of 47 isolates appeared positive result , 2(4.3%) of 47 isolates appeared intermediate result.

In contrast, frequency of positive results was higher than intermediate with high significant (p≤0.01).

41(87.2%) of 47 isolates appeared negative results and frequency of this result was higher than positive and intermediate with high significant (p≤0.01).

No significant differences were appeared between denture and orthodontic results for each test (positive, intermediate and negative) .

TCP method was recovered in denture, frequency of weak positive results [29(61.7%) of 47 isolate] appeared to be higher than high [8(17.02%) of 47 isolates] and moderate [7(14.9%) of 47 isolates] positive results with high significant difference (p≤0.01) , 3(6.4%) of 47 isolates showed negative results .

However , the frequency of positive isolates 44(94%) of 47 isolates (high, moderate ,and weak ) was higher (p≤0.01) than negative results.

The TCP method for orthodontic, recovered that the weak positive [9(19.2%) of 47 isolates] result is higher than positive[2(4.3%) of 47 isolates] with high significant difference (p≤0.01).

Furthermore, 36(76.6%) of 47 isolates showed negative results with high significant (p≤0.01) than positive (high, moderate and weak) results .

However, frequency of positive result (high, moderate and weak ) in denture was higher (p≤0.01) than in orthodontic .

Following, the frequency of negative result in orthodontic is higher (p≤0.01) than in denture .

icaAD gene method for isolates of denture showed that the present of icaA [37(78.7%) of 47 isolates] results was higher than the absent of icaA [10(21.3%) of 47 isolates] gene with high significant difference (p≤0.01).

icaD gene was recovered 40(85.1%) of 47 isolates as positive results with high significant difference (p≤0.01) than negative results 7(14.9%) of 47 isolates.

In orthodontic, the frequency of icaA[2(4.3%) of 47 isolates] gene negative results was higher than icaA[45 (95.8%) of 47 isolates] gene positive results with high significant difference (p≤0.01).

While, the icaD gene was recovered 24(51.1%) of 47 isolates as positive results and 23(48.9%) of 47 isolates as negative results but without significant difference .

Comparing ,in denture, TCP 44(93.6) and icaA and /or icaD gene 47(100%) are the best methods for detection of biofilm formation (no significant differences between these results), on the other hands, icaA and /or icaD gene were only the best method for detection biofilm formation in orthodontic.

The frequency of bacteria was higher (p≤0.01) with patients aged >35 (70.1%), denture washing (in day) < 2 time (61.7%), without tonsillitis (100%), without gingivitis (100%), no cigarettes smoking (95.7%) and without dental caries (95.7%), but no significant difference with date of denture wearer (year).

In orthodontic , the frequency of bacteria was higher (p≤0.01) in patients aged <18 (78.7%) , orthodontic washing > 2(68.1%),tonsillitis (100%) , gingivitis (70.2%) , no cigarettes smocking (100%),without dental caries (100%), but no significant differences in patients with date of orthodontic insertion factor.

Main Subjects

Biology

No. of Pages

246

Table of Contents

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Language

English

Data Type

Arab Theses

Record ID

BIM-743822