الكشف الجزيئي Molecular Identification والتصنيف القائم على التسلسل الجيني Sequence Based Typing لبكتيريا الليجيونيلا نيوموفيلا Legionella pneumophila في العينات البيئية والسريرية في فلسطين
Date
2017-05-08
Authors
محمود جميل اسحق عمرو
mahmod jameel ishaq amro
Journal Title
Journal ISSN
Volume Title
Publisher
AL-Quds University
جامعة القدس
جامعة القدس
Abstract
Legionellae are gram-negative bacteria, rod shaped, strictly aerobic and nutritionally
fastidious. Legionella species are implicated in two clinical syndromes: Legionnaires’ Disease
(LD), and Pontiac fever, which are collectively known as legionellosis. Among the 56 species
and 70 serogroups of Legionella species, Legionella pneumophila is the major cause of
sporadic and outbreak legionellosis (91.5%), and serogroup 1 is the predominant serotype
(84.2%).
Many studies have demonstrated that the main source for LD is the potable water systems in
large buildings like hospitals and hotels. The contamination of hospitals' water systems with
Legionella is high risk for patients with various diseases, especially immunocompromised and
those who may stay hospitalized for long period of time. LD is acquired by inhalation of
aerosols contaminated with Legionella spp. or less commonly by aspiration of contaminated
drinking water.
Previous work in the Microbiology Research Laboratory at AQU has shown high prevalence
of Legionella spp. in the water and biofilm samples collected from eight hospitals in the West
Bank over a two-year period December 2012- December 2014, by using culture method and
16S rRNA-based PCR. Moreover study of the prevalence of L. pneumophila in 195
respiratory samples (sputum or Broncho alveolar lavage (BAL)) by culture yielded only one
positive. However, by PCR, 23% (44/195) of the respiratory samples were positive for L.
pneumophila. BAL presented a higher percentage 35% (26/74) than sputum 15% (18 /121).
Molecular diagnosis of L. pneumophila is well established and adopted worldwide especiallythat culture methods are time consuming and less efficient. Furthermore, genotyping of L.
pneumophila is important for epidemiological investigation and control of legionellosisoutbreaks. The current gold standard for L. pneumophila genotyping is Nested PCR Sequence
Based Typing (NPSBT), based on the sequence of seven loci (flaA, pilE, asd, mip, mompS,
proA and neuA). NPSBT allows the Sequence Typing (ST) of L. pneumophila in the absence
of isolates. This high-resolution molecular typing tool is recommended by the European
Working Group for Legionella Infections (EWGLI).
The previous results in the Microbiology Research Laboratory entitled the use of NPSBT to be
able to do epidemiological typing of the respiratory samples in the absence of isolates and to
relate the ST’s of environmental samples previously collected from the same hospital with the
ST of the respiratory samples to evaluate possible nosocomial infection.
The overall goal of this study was to determine the Sequence types (ST’s) of the PCR positive
respiratory samples by NPSBT method. Also to determine the ST’s of the environmental
samples obtained from the same hospital ward.
Our sample study included a subset (34 samples) out of the 44 respiratory samples previously
tested positive by PCR targeting 16S rRNA for L. pneumophila. These thirty-four positive
samples were further subtyped by NPSBT method. Also DNA previously extracted from 15
biofilm samples previously collected from Makassed hospital wards and tested positive for L.
pneumophila was also analyzed by NPSBT.
Analysis of the seven allele profiles for the NPSBT of the 34 selected respiratory samples
showed a full 7-allele profile for 3 /34 (8.8 %) specimens, a further 18/34 (52.9%) gave 5- or6-allele profiles (sufficient to identify the strain as one or two sequence types (ST’s), 6/34
(17.6%) gave 3- or 4-allele profiles (usually enough to differentiate different profiles), and 5
/34 (14.7%) gave 1- or 2-allele profiles (sufficient to distinguish strains). Overall, 24/34
(70%) samples gave ≥ 4 alleles profiles (4, 5, 6 and 7 alleles). However, 10 samples gave < 4
alleles profiles, these samples were excluded from sequencing in order to identify the ST.
Analysis of the seven alleles’ products of the selected fifteen environmental samples revealed
fourteen samples positive for six to seven alleles and one sample was positive for one allele,
this sample was excluded from sequencing in order to identify the ST
Sequence analysis showed the following ST’s in the 24 respiratory samples: ST1 (29.1%,
7/24On the other hand, 14 environmental samples typing showed: ST 1(28.6%, 4/14), ST 187
(21.4%, 3/14), one sample of each ST 2070, ST 461 and ST 187 (7.1 %, 1/14), while the rest
of samples (28.5%, 4/14) were unspecified Sequence Types.
Thus ST1 is the most prevalent sequence type in both the respiratory samples and the
environmental samples representing 29.1% and 28.5% respectively. The other ST’s were
unique to each type of sample. ST1 is also the most prevalent ST worldwide in clinical and
environmental samplesST 461 (25%, 6/24), ST 1037 (4%, 1/24), and (41.9%, 10/24) gave incomplete profile.
Description
Keywords
الكيمياء الحيوية والاحياء الجزيئية , Biochemistry & Molecular Biology