رسائل ماجيستير
English
2017
Molecular Identification and Sequence Based Typing of Legionella pneumophila in Environmental and ClinicalSamples in Palestine
دينا البيطار, محمود جميل اسحق عمرو
كلية الصيدلة-جامعة القدس · فلسطين
Legionellae are gram-negative bacteria, rod shaped, strictly aerobic and nutritionallyfastidious. Legionella species are implicated in two clinical syndromes: Legionnaires’ Disease(LD), and Pontiac fever, which are collectively known as legionellosis. Among the 56 speciesand 70 serogroups of Legionella species, Legionella pneumophila is the major cause ofsporadic 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 inlarge buildings like hospitals and hotels. The contamination of hospitals' water systems withLegionella is high risk for patients with various diseases, especially immunocompromised andthose who may stay hospitalized for long period of time. LD is acquired by inhalation ofaerosols contaminated with Legionella spp. or less commonly by aspiration of contaminateddrinking water.Previous work in the Microbiology Research Laboratory at AQU has shown high prevalenceof Legionella spp. in the water and biofilm samples collected from eight hospitals in the WestBank over a two-year period December 2012- December 2014, by using culture method and16S rRNA-based PCR. Moreover study of the prevalence of L. pneumophila in 195respiratory samples (sputum or Broncho alveolar lavage (BAL)) by culture yielded only onepositive. 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 SequenceBased 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 absenceof isolates. This high-resolution molecular typing tool is recommended by the EuropeanWorking Group for Legionella Infections (EWGLI).The previous results in the Microbiology Research Laboratory entitled the use of NPSBT to beable to do epidemiological typing of the respiratory samples in the absence of isolates and torelate the ST’s of environmental samples previously collected from the same hospital with theST 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 positiverespiratory samples by NPSBT method. Also to determine the ST’s of the environmentalsamples obtained from the same hospital ward.Our sample study included a subset (34 samples) out of the 44 respiratory samples previouslytested positive by PCR targeting 16S rRNA for L. pneumophila. These thirty-four positivesamples were further subtyped by NPSBT method. Also DNA previously extracted from 15biofilm 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 samplesshowed 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 < 4alleles 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 revealedfourteen 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 STSequence 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 restof samples (28.5%, 4/14) were unspecified Sequence Types.Thus ST1 is the most prevalent sequence type in both the respiratory samples and theenvironmental samples representing 29.1% and 28.5% respectively. The other ST’s wereunique to each type of sample. ST1 is also the most prevalent ST worldwide in clinical andenvironmental samplesST 461 (25%, 6/24), ST 1037 (4%, 1/24), and (41.9%, 10/24) gave incomplete profile.