Ministry of Higher Education and Scientific Research University of Babylon College of Science Department of Biology Phenotyping and Molecular Characterization of Proteus vulgaris Isolated from Patients with Urinary Tract Infections Submitted to the Council of the College of Science, University of Babylon, in a Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Microbiology By Ghaida’a Jihadi Mohammed M.Sc./Medical Microbiology/ 2008 Supervised by Prof. Dr. Abdallah K. Hindi Assist. Prof. Dr. Wang Yanchang 2014 AD 1435 AH Summary In this study, a total of 150 urine specimens, only 43 isolates of Proteus species are isolated and from these only 28 isolates were identified as Proteus vulgaris. It was showed that all isolates (100%) possess haemolysin gene, urease gene, fllagellin gene, adhesive factors genes (MR/P, MR/K, UCA and ATF), and Beta-lactamase gene. Whereas, 90% of the isolates gave positive result for metalloprotease ZapA gene and 50% for quinolones resistant gene. The swarming activity was detected in all isolates(28) and the anti- swarming agents (resveratrol, p-nitrophenyl glycerol, fatty acids, urea ,sodium azide and ethanol) were used to show their ability to overcome this activity .It was found that there was diminishing in swarming activity when these agents are added to the culture media separately. Also it has been investigated the ability of resveratrol and p-nitrophenyl glycerol to inhibit production of haemolysin and urease in addition to swarming inhibition. Furthermore, the susceptibility of Proteus vulgaris isolates to a variety of antibiotics has been investigated. It has been found that all the isolates were resistance (100%) to ampicillin, amoxicillin, cephalothin, chloramphenicol and ceftazidime, and some isolates have shown lower frequencies of resistance to ceftriaxone (89%), ciprofloxacin (82%) and cotrimazole (92.8%).Whereas, all the isolates were sensitive 100% to amikacin, gentamycin, nitrofurantion, imipenem, meropenem, and norfloxacin. The detection of quorum sensing produced by Proteus vulgaris was also studied. It was found that there was an aggregation of the bacterial cells after addition of the supernatant that likely occurs as a result of the presence of homoserine lactone. The best interval for accumulation of homoserine lactone was after 4 hours of incubation in which the homoserine lactone reached at maximum concentration. Proteus isolates were subjected for genotyping by using specific primers for genes (16SrRNA and rpoB). The examination of Proteus isolates by 16SrRNA sequencing showed between 3 -8% nucleotide differences and it was shown from the constructed phylogenetic tree that Proteus penneri and Proteus mirabilis have related one to another where both of them have the same branch in the phylogeny tree. Whereas, Proteus vulgaris was found to be located in another branch of the tree. In contrast to rpoB gene sequencing which was showed between 5 and 13% nucleotide differences and it was found that clinical strains were generally closely related to their respective type and type strain of Proteus penneri were close somewhat to Proteus vulgaris in phylogeny tree than to Proteus mirabilis. 1.1. Introduction Urinary tract infections (UTIs) are considered as one of the most common groups of infections in humans and affect either the upper (kidneys-pyelonephritis) or the lower (bladder-cystitis) part of the urinary tract. The gastrointestinal tract is a reservoir from which uropathogens emerge. Reflecting this, Enterobacteriaceae are the most important cause of UTI in all population groups, accounting for more than 95% of all UTIs. Among these microbes, E. coli is by far the most common invader, causing some 90% of UTIs in outpatients and approximately 50% in hospitalized patients. Proteus species is a common cause of urinary tract infection (UTI) in catheterized patients and those with urinary tract abnormalities. It shows a predilection for the upper urinary tract where it can cause serious kidney damage, acute pyelonephritis, bladder or renal stones, fever and bacteraemia. Proteus vulgaris belongs to family Enterobacteriaceae and is a rod- shaped, Gram negative bacterium that inhabits the intestinal tracts of humans and animals. It can also be found in soil, water and fecal matter. It is an opportunistic pathogen of human, it is known to cause urinary tract infections and wound infections. Several potential virulence factors, including adherence to the uroepithelium mediated by fimbriae, urease production, invasion of eukaryotic cells, cleavage of IgG and IgA by a proteolytic enzyme, haemolysin production, and swarming motility dependent on flagella may be responsible for the pathogenicity of P. vulgaris. The adaptation of microorganisms to antibiotics causes proliferation and persistence of drug resistance, currently a major public health problem, therefore it is urgent to discover new drugs endowed with antimicrobial activity. In recent years, an increasing interest has been biologically active compounds including antioxidants from plants and other natural sources. Molecular phylogenetic is the branch of phylogeny that analyses hereditary molecular differences, mainly in DNA sequences, to gain information on an organism's evolutionary relationships. The sequence of the 16SrRNA gene has been widely used as a molecular clock to estimate relationships among bacteria (phylogeny), but more recently it has also become important as a means to identify an unknown bacterium up to the genus or species level. Advances have been made in automating and minimizing the detection times using biochemical methods, however, biochemical identification is not accurate for determining the genotypic differences of microorganisms. The16SrRNA has properties, which predestine it as a universal phylogenetic marker. There are regions on the 16SrRNA that are quite conserved and others, which are variable. Comparing the differences in the base sequence of this 16SrRNA gene is, therefore, an excellent means to study evolutionary changes and phylogenetic relatedness of organisms. The gene coding for the Beta subunit of the RNA polymerase, rpoB has been proposed as an alternative biomarker for microbial community studies. This gene is described as possessing the same key attributes as 16SrRNA, in that it is common to all bacteria and is a mosaic of conserved as well as variable sequence domains. Most importantly, the rpoB gene exists as a single copy in bacterial genomes. The rpoB gene, has emerged as a core gene candidate for phylogenetic analyses and identification of bacteria, especially when studying closely related isolates. Sequencing of rpoB enables efficient estimation of bacterial G+C% content, DNA–DNA hybridization value and average nucleotide identity (percentage of the total genomic sequence shared between two strains) when taxonomic relationships have been firmly established. Together with the 16S rRNA gene, rpoB has helped to delineate new bacterial species and refine bacterial community analysis. The aim of this study: To characterize the clinical isolates of Proteus vulgaris in terms of the molecular and phenotypic. This study has been carried out to achieve the following objectives: 1-Collection of urine samples from patients with UTI and indwelling materials. 2-Isolation and identification of Proteus vulgaris by biochemical tests. 3-Detection of some virulence factors genes. 4-Detection of adhesive factors genes such as mrp, mrkA, uca and atf fimbrial genes using genetic markers. 5-Study the effect of some chemicals on P. vulgaris swarming and some virulence factors. 6-Study the effect of some antibiotics on P. vulgaris isolates. 7-Detection of quorum sensing in P. vulgaris isolates. 8-Genotyping of Proteus strains by PCR using genetic markers such as: 16SrRNA and rpoB. 1.2. Literatures review 1.2.1. Urinary tract infections Urinary tract infections (UTIs) are among the most frequently occurring human bacterial infections, accounting for about 20% of all infections acquired outside the hospital. Almost 90% of UTIs are ascending, with bacteria gaining access to the urinary tract via the urethra to the bladder and then to the upper part of the urinary tract (Negut and Buiuc, 2008). The organism causing a UTI usually originates from the patient’s own bowel flora. The most frequent etiological agents causing UTIs are gram-negative bacteria belonging to the Enterobacteriaceae family (Gupta et al., 2001 ; Stickler et al., 2003). Proteus bacilli play a particularly important role in urinary tract infections (UTI), which can be subdivided into two categories: hematogenous infections and ascending infections (Abraham et al., 2001). The second type of UTI is more common to Proteus strains. Proteus mirabilis is one of the most common causes of UTIs in individuals with long-term indwelling catheters or complicated UTIs and of bacteremia among the elderly. UTI are also caused by the two other species, P. vulgaris and P. penneri (Stickler et al., 2003 ; Sosa and Zunino, 2009; Stickler and Feneley, 2010). 1.2.2. Classification and environmental distribution of Proteus spp. The genus Proteus, which was described for the first time by Hauser in 1885, belongs to the Enterobacteriaceae family. In this family it is placed in the tribe Proteeae, together with the genera Morganella and Providencia (Rozalski and Staczek, 2011). Proteus spp. consist of gram-negative, motile, aerobic rod-shaped bacilli generally range from 0.3 to 1.0 µm in width and 0.6 to 6.0 µm in length (Abbott