Bacteriological Study of Escherichia coli Isolated from different Infections in Diyala

The goal of present study was to Isola and Identifi--cation of "Escherichia coli" from different Infections and detection the sensitivity and resistance of Ecoli to antimicrobials Additionally detection of virulence factors . This study was conducted from the period from 1 / 6 / 2016 to 1/ 9 / 2016 in Baquba city in Iraq.. It included; Fiftin samples were collected from different infections from Baquba General Hospital and AL-Batool Hospital. Twenty isolates were found to be Escherichia coli .The susceptibility test was applied on these isolates against deferent antibiotics. The results revealed that the highest resistances were for Piperacilline(93.3%) and highest Sensitive Imipenem with 100%, While the lowest resistance were for Tobramycin (9%) . Moreover the results of virulence factors that had E. coli showed possession of all isolates many virulence factors and a high production of which increases the pathoginicity of it. All isolates were unable to produce urease and gelatinase, but heamolycin (35%) . As well as, Tow isolate (10%) were able to production Extended SpectrumβLactamases enzyme. Furthermore, four isolate (20%) were able to production metalloβLactamases Finally, six isolate(30%) were able to production Bacteriocin. Bacteriological Study of Escherichia coli Isolated from different Infections in Diyala Eman Abass Ali , Ibtihal Hameed Mohsin and Izdehar M. Jasim 250 Vol: 13 No:3 , July 2017 DOI : http://dx.doi.org/10.24237/djps.1303.315C P-ISSN: 2222-8373 E-ISSN: 2518-9255 Conclusion: E. coli isolates highest resistances were for Piperacilline and highest Sensitive Imipenem with 100%, While the lowest resistance were for Tobramycin . additionally some of isolates production Extended Spectrumβ-Lactamases enzyme, metallo β-Lactamases and Bacteriocin. Keywards: Escherichia coli , Extended Spectrumβ-Lactamases, Metallo βLactamases,Bacteriocin , Antibiotics ايرتكبل ةيجولويرتكب ةسارد E. coli ةنيدم يف ةفلتخم جامخأ نم ةلوزعملا ىلايد يلع سابع ناميأ 1 , نسحم ديمح لاهتبأ 2 و مساج دمحم راهدزا 3 1,1 ةايحلا مولع مسق مولعلا ةيلك ىلايد ةعماج 3 ةيئايحلآا ةناقتلا مسق مولعلا ةيلك ىلايد ةعماج

/ 6 / 2016 to 1/ 9 / 2016 in Baquba city in Iraq..It included; Fiftin samples were collected from different infections from Baquba General Hospital and AL-Batool Hospital.Twenty isolates were found to be Escherichia coli .The susceptibility test was applied on these isolates against deferent antibiotics.The results revealed that the highest resistances were for Piperacilline(93.3%) and highest Sensitive Imipenem with 100%, While the lowest resistance were for Tobramycin (9%) .Moreover the results of virulence factors that had E. coli showed possession of all isolates many virulence factors and a high production of which increases the pathoginicity of it.All isolates were unable to produce urease and gelatinase, but heamolycin (35%) .As well as, Tow isolate (10%) were able to production Extended Spectrumβ-Lactamases enzyme.Furthermore, four isolate (20%) were able to production metalloβ-Lactamases Finally, six isolate(30%) were able to production Bacteriocin.

Introduction
Escherichia iloc (E.ioli) bacteria usually live in the entrails of community and animals.mainly E. iloc are not dangerous and in fact are an essential part of a healthy human intestinal region.However, some E. iloc are pathogenic, importance they can foundation sickness. (1) iloc is the mainly prevalent infecting individual in the family of gram--negative bacteria identified as Enteroba-cteriaceae. (2) E. iloc bacteria were revealed in the "human colon in 1885 by German bacteriologist " Theodor-Escherich.(3) E. iloc is a facultative "aerobic and anaerobic growth" gram-negative organism, stick formed, may or may not be motile.a little rods are flagellated and a few are not). (4)Bacteria that can be usually develop greatest at 37 C. E. iloc is a Gram-negative that cannot sporulate.for that reason, it is simple to destroy by easy boiling or essential sterilization.(5) E. iloc has single one circular-chromosome, several beside with a circula-r plasmid.(6) . E ioc is a main human pathogen that occurs in numerous unlike types of infections of the human body, this suitable to E iloc have different virulence factors as producing a extensive" variety of enzymes and toxins" .(7) Nearly all strains of E iloc top secret a group of" enzymes which includes heamolycin, Extended Spectrum β-Lactamases enzyme,Metallo β-Lactamases enzyme" ,"Bacteriocin, Catalase enzyme.The main function of these proteins may be alter limited host tissues into nutrients obligatory for bacterial increase".Extended-spectrum β-lactamases (ESBLs) are a group of plasmid-mediated, diverse, composite and fast evolving enzymes that are posing a main' therapeutic challenge today in the treatment of hospitalized and community-based patients.these enzymes share the ability to hydrolyze third-cohort cephalosporins and aztreonam and yet are reticent by clavulanic acid".(8) In accumulation, "ESBL-producing organisms exhibit co-resistance to many other classes of antibiotics", follow-on in restriction of therapeutic option.due to inoculum effect and-substrate specificity, their discovery is also a most important challenge.(9) (10) .The everincreasing and quick "spread of metallo-beta-lactamase (MBL) producing Enterobacteriaceae", mostly Escherichia iloc, Metallo--β-lactamases era a varied set of enzymes that catalyze the hydrolysis of a broad range of β-lactam drugs including carbapenems.This multiplicity is reflected in the inspection that the enzyme mechanisms disagree based on whether 1 or 2 zincs are bounce in the lively site which, in turn, is dependent on the subclass of β-lactamase .(11) The dissemination of the genes encoding these enzymes amongst Gram--"negative bacteria has made them an important cause of resistance".
In addition, there rre currently on clinically accessible inhibitors to block metallo-β-lactamase action . (12)

Samples collection
Fifteen different clinical samples were collected from patients and carriers in Baquba General Hospital and Al-Batool Hospital over period from 1/6/2016 to 1/9/2016.The samples were included (14 from urin, 9 from umbilical cord, 15 from wound and 12 from burn).

Isolation and Identification of Escherichia coli
The collected samples were inoculated on the blood agar, incubated at 37ºC for 24 hours.The isolates were examined for their shape, size, colour, pigments, and haemolytic activity.Then transferred and streaked on MacConky agar for detecting the ability of each isolate to ferment lactose.every one plates erew incubated at 37ºC for 1day then a single pure then transferred to Eosin methylene blue agar (EMB) appearing as a metallic green sheen.pure isolated colony was transferred to Nutrient broth medium for the preservation and to carry out other biochemical tests that confirmed the identification of isolates.The isolate erew recognized according to the "Bergey's Manual'. (15), (16) As the following: gram stain and biochemical tests Standard biochemical tests were used for detecting Ecoli strains.

Antimicrobial susceptibility test
The sensitivity and resistance of Ecoli to antimicrobials was tested by the disc diffusion method on Muellar-Hinton agar using antibiotic "discs according" to medical and Lab-oratory standard association (CLSI) liuidelines. (17)Twelve antibiotics were tested: Ampicillin inhibition zones was carried out based on the manufactures and CLSI guidelines . (18)Then the plates are incubate during the night at 37 oC , and the region of reticence of bacterial growth is use-d sa-a calculate of defenselessness, "where big zones of inhibition signify that the organism is susceptible", while little or on region of inhibition indicate confrontation."An analysis of intermediate is particular for zones which fall between the established cutoffs for the other interpretations". (19)

Detection of virulence factors &Biochemical test
The Ecoli ability to produce some of virulence factors (enzymes and toxins) were recognized and tests were applied on 20 isolates that identified.it included: Haemolysin production, urease production and .identified by Biochemical test such as Triple Sugar Iron Agar (TSI) , IMViC examination "indole, methyl red, Voges-Proskauer, dan citrat". (20)

Detection of Extended-spectrum β-lactamases (ESBLs)
Extended-spectrum β-lactamases production saw tested by compact disk estimate.using double disc synergy" test.Briefly", a disinfected Mueller-Hinton agar was prepared and" a 0.5 MacFarland equivalent standard of the test organisms was streaked on the surface of the agar with a sterile loop and permitted for 15-20 mins to pre-diffuse".An Augmentin which is a combination of clavulanic acid 20 (μg) and amoxicillin (10 μg) was placed at the center of the petri-dish and cefotaxime (30 μg), "ceftaxidime (30 μg), aztreonam (30 μg) ciprofloxacin (30 μg) were placed15mm apart center to centre on the plates with a sterile forceps".These erew incubate at 35oC for18-24 h.An superior zone of reserve from 5 mm beyond in the attendance of Augmentin -is regarded as positive for phenotypic production of (ESBL) enzyme. (21)DTA disc compared to that of the imipenem disc alone was considered positive for MBL production". (22)

Detection of Bacteriocin (colicin)
The occurrence of colicin production saw strong-minded using the agar overlay method with pointer strain E. iloc CL173.momentarily, agar plates erew stab inoculated with the test strains and incubated overnight at 37°C.Colonies were lysed for 15 min using cellulose pads impregnated with chloroform."To get rid of residual chloroform vapour the plates were then showing to air and overlaid with soft agar containing an indicator strain and incubated overnight at 37°C". (23)

Isolation and Identification
Fifteen samples were collected from patients and carriers, the samples comprised from (urine, umbilical cord , wound and burn).Twenty isolates (25%) have the ability to grow on the MacConky agar which "considered choosy and discrepancy media for gram off-putting bacteria " ( 24 ) .All 20 isolates had ability to ferment Lactose and form large Pink colonies, smooth.They are grow on "Eosin methylene blue--agar "(EMB) "choosy and degree of difference media" appearing as a metallic green sheen .Microscopic examination was used to all 20 isolates after staining by gram stain and cells appeared as Gram-negative rods.For further identification some of the biochemical tests was performed on 20 isolates, included: catalase test was all 20 isolated gave positive results ,While 20 isolates gave the negative result for all of oxidase test, H2S production test and Gelatin laquification test .Also all 20 isolates were positive to Indol test and Methyl red but Negative result for Voges-Proskauer test and Citrate Utilization Test .Additionally , nitrate reduction test was not applied for further identification because the Ecoli often unable reduce nitrate to nitrite. (25)7(35%) isolated can be produced Haemolysin.Ecoli can be production haemolysin that enzyme imported play role in against immune cells for host. (26)ol: 13 No:

Susceptibility test of Ecoli:
The sensitivity of 20 isolates were tested against 12 antibiotics.The susceptibility test was applied according to the Kirby-Baure Method (antibiotic disc diffusion method).The results in table (1) showed that isolates were resistance for β-lactamases antibiotic suchas: Piperacillin (93.3%),Ampicillin (67.5%) ,Cefixime (66.66%) ,Cefotaxime (40%),ceftazidime ( 46.6%) this results was agreed with studies (27)   , who reported (61.1%) to Ampicillin and (42.8%) to Cefotaxime, (28) but disagrees with the work of Bonomo etal.(2003) for the resistance of Cefotaxime (13.3%) . (29)" because of modify in incursion of external cell membrane because has protein called burin the cell wall is enclosed with an outer film that establishes a permeability barrier against the antibiotic". (30)While aminogiycosides antibiotics group such as Gentoamycin(9%),Tobramycin (13.33%) this result Gentoamycin agreed with studies conducted in Ethiopia (57.8%). (31)Because this antibiotics can able inhibition of syntheses protein by linked with small ribosome unite (30S). (32)Quinoloes antibiotics such as Ciprofloxacin (33.3%)this result agreed with study by Mavroidi.etal.(2012)   . (33)Because this antibiotics can able inhibition of DNA syntheses and super coiling. (34)ported result Resistance for Co-trimoxazole (75%) ,Augmentin (73.3%) this study agreed with the work of Drawz and Bonomo(2010). (35)Furthermore, Resistance result of Nitrofourantion higher (86.66%) this result agreed with study by Gums (2005). (36)While imipenem sensitive higher 100% this result agreed with Livadariu etal.(2006). (37)In general the resistance to different antibiotics may be due to the type of antibiotics and how much that used among the patients in the community.In addition to that the resistance to ward any antibiotics was depended on the amount of PBP2a or β-Lactamase enzyme that produced by each strain of Ecoli .All these reasons could create variations in the rate of resistance.The results in table (3) showed that 2 (10%) isolates were production for Extended-spectrum β-lactamases , in this study also agreed with the findinds of Babypadmini, and Appalaraju (2004). (38)Who reported 41% ESBL positivity E. coli and 40% was reported by Jayapradha etal.(2007) . (39)Isolates can be explained in most cases to production of β-Lactamase enzyme that destroyed the β-Lactam ring and inactivated the penicillin and this enzyme was encoded by plasmid that easy to transfer among strain.While 4(20%) isolated nac be produced of " Metallo β-lactamase"s,this product agreed with numerous recent studies from eroth parts of Asia such sa (18.98%) about this" study Khanal etal.(2013)" (40) Also demonstrated rising incidence fo M.B.L invention ni Enterobacteriaceae isolate. (41). (42)ol pattern and the prevalence pace may diverge greatly in different geographical areas and as of institute to institution". (43)6 (30%) isolated can be produced Bacteriocin (colicin) , As noted previously, variable levels of susceptibility to colicins could be due to variability in the number of colicin receptors per cell or due to shielding of receptors by the lipopolysaccharide O-antigenic. (44)