3.1 Biotyping by Vitek 2 Identification System
VITEK 2 can be defined as an automatized fungi and bacterial recognition apparatus which produces consistent and repeatable results, as evidenced by clinical studies. With its colorimetric reagent cards and related software and hardware advancements, the VITEK 2 provides a state-of-the-art evolution program for phenotypic discovery procedures The present study revealed that the accuracy of the VITEK 2 system for a direct identification and susceptibility testing in blood cultures of Grampositive rods and Gram-negative cocci varied according to the type and species of bacteria as well as the antimicrobial screened. [9]. out of 365 strains of the E. coli that have been obtained from 3 hospitals in Duhok- Iraq 68 strains were identified from males (18.68%) and 296 were isolated from females (81.3%). Females were more susceptible to UTIs compared to males, according to the findings.
3.2 Molecular Investigations
The average concentration of genomic DNA extracted using a commercial kit was 115.25ng/lµl, with an extremely high purity of 1.8 The commercially available kit approach was discovered to be an extremely effective approach for: suitable for PCR amplification, rapid yielding of DNA, high purity, and the extraction of a large number of samples. [10].
3.3 E. coli Species-Specific PCR Amplification
All E. coli isolates (100) have been amplified effectively in repeated tests, resulting in a single band regarding the uidA as species-specific locus in all strains with molecular weight of roughly 657bp, as can be seen in Fig. (1). The effective amplification regarding uidA amplicon in all of the chosen samples has been validated at the molecular level, confirming that all of the strains have been E. coli. This phase, according to several research, is a prerequisite for any subsequent molecular investigation [11]. Yet, with the use of the same primer sequence as Adamus-Bialek et al., (2009), such molecular weight was shown to be different. This variation could be explained by the fact that genes often include multiple coding mini- and microsatellite repeats, which are extremely dynamic genomic components. As a result of recombination events within such tandem repeats, the repeat numbers vary, causing the sequences to change.
This variety might give functional diversity and/or the illusion of the host immune system, allowing for quick adaptability The obtained result was in agreement with a study conducted in Duhok Province using the same primer (uidA gene) in Fig. 1 for the identification of E. coli which produced the same molecular weight [12]. Due to the fact that uidA gene is considered as a housekeeping gene, it encodes the B-D-glucuronidase enzyme [13], which is produced by up to 97% of E. coli [14]. As a result, numerous investigations have employed the gene as one of the molecular markers for the identification of E. coli However, this molecular weight was found different from that obtained by by using the same primer sequence. This variation may be attributed to the fact that genes usually contain multiple coding mini- and microsatellite repeats that are highly dynamic components of genomes. Therefore, recombination events within these tandem repeats lead to changes in repeat numbers, which in turn alters the sequences. (15, 11). This approach could deliver speedy and reliable findings in a short amount of time for E. coli specific-species identification, as well as pave the path for more detailed molecular applications This variation may provide the functional diversity and allows rapid adaptation to the environment and/or illusion of the host immune system Since uidA gene is a housekeeping gene encoded B-D-glucuronidase enzyme and up to 97% of E. coli produce this enzyme therefore, many studies used this gene as a molecular marker for the identification of E. coli This method may provide rapid and robust results in a short time for the confirmation of specific-species identification of E. coli and may also pave the way for further and more details molecular applications.
3.4 Prevalence and distribution of virulence and Antibiotic Resistance genes among E. coli strain
All E. coli isolates (100) were exposed to PCR methods to evaluate the prevalence rates regarding virulence as well as antibiotic resistance-associated genes such as the hyl show in Fig. 2 and pai markers, as well as their distribution throughout phylogenetic groupings. The findings of such tests are detailed in detail for each city independently.
These rates are consistent with a number of other researches. For instance, Oliveira and colleagues (2011) discovered that 28% of E. coli strains lacked the virulence and antibiotic resistance genes. Rasol (2013) found that virulence gene prevalence amongst isolated of E. coli from the urine was 38% and 67%, respectively, in another work done in Duhok province. Karimian and associates (2012) investigated the virulence factors regarding E. coli isolated from urine and discovered that hyl and pai had varying prevalence rates of 50.4% and 50.4%, respectively.
In the current investigation, the high prevalence of pai as a (PAI) marker that might carry virulence genes amongst the strains of E.coli has been also consistent with other recent researches. Brzuszkiewicz and collaborators (2006), for example, found that genomic differences between the strains of E. Coli were mostly limited to large (PAIs). In another research, Navidinia and colleagues (2012) found that (PAIs) have been enriched among the isolates of E. coli and that the prevalence regarding 8 PAI markers in the strains of E. coli isolated from the urine of children with UTI was confirmed. Furthermore, the high incidence of both hly genes correlating to the incidence of pai marker found in this work could indicate a strong association between such genes, which are frequently connected and known to carry PAIs.
Differences in the city of E. coli virulence and Antibiotic resistance genes as a result of the geographical area has been reported as well by Rasool., 2013 in Duhok governorage and by Karimian and coworkers (2012) in Iran who had suggested that climate of every region, food, customs, public health levels and hospital hygiene can be considered as factors that are attributed to presence of variations in rates of prevalence of the virulence genes of the strains of E. coli amongst various regions. The high incidence of both hlyA, gene corresponding with incidence of pai marker obtained in this study may reflect the fact that there is a high association between these genes which are often linked and known to carry PAIs. Similar studies have been reported reflecting this type of association (10). Furthermore, (13) suggested that sfa genes are also present on PAI in E. coli strains. Thus; PAIs may contribute to urovirulence and may potentially serve as targets for interventions.