Evaluation of some Virulence Factors and Drug Resistance of Bacteria Isolated from the Urine of Patients with TCC-Bladder Cancer

: Urinary tract infections (UTIs) mean microbial pathogens in the urethra or bladder (lower urinary tract). Important risk factors for recurrent UTI include obstruction of the urinary tract, use of a bladder catheter or a suppressed immune system. This study aims to isolate and identify bacteria from patients with TCC-bladder cancer or patients with a negative cystoscope and estimate antibiotic susceptibility patterns and evaluate some of the virulence factors. From a total of 62 patients with TCC-BC or negative cystoscope, only 35 favorable bacterial growths were obtained, including Escherichia coli (UPEC), a significant bacterial isolate, and Stenotrophomonas maltophilia. The percentage of multi drug-resistance bacteria (MDR) was identified in (62.8%) while the extended drug-resistance bacteria (XDR) was (28.5%). All isolates were producer for biofilm either moderately 18/35 (49%) or strongly 18/35 (51%). Only 25/35 (71%) isolates were produced for siderophore, while 10/35 (29%) isolates were non-produced. Inducing cytochrome P450 expression protein was seen in (14/35) 40% isolates. In conclusion , patients with TCC-BC or negative cystoscope who had a urinary catheter or immune-compromised were at high risk of infecting with nosocomial or opportunistic pathogens, which could be develop antibiotic resistance, the central problem in the cohort of patients undergoing chemotherapy or immune cancer therapy


Introduction:
Urinary tract infections (UTIs) mean microbial pathogens in the urethra or bladder (lower urinary tract), or ureter and pelvis of the kidney (upper urinary tract). In men, the prostate also may be involved 1 . Risk factors for recurrent UTI include obstruction of the urinary tract, use of a bladder catheter, a suppressed immune system, estrogen deficiency, genetic predisposition, and sexual intercourse 2 . Uropathogenic E. coli (UPEC) can invade bladder epithelial cells during UTI and form intracellular bacterial communities (IBC), which can be the cause of UTI recurrence 3 .
Uropathogens bacterial have several virulence determinants necessary for initial adhesion and colonization of host mucosal surfaces, cell and tissue invasion, overcoming the host defense mechanisms, and causing persistent and chronic infections. These microbial virulence determinants include surface factors (fimbriae, adhesins, P pili and type-1 pili) and extracellular factors (toxins, siderophores, enzymes, and biofilm formation) 4 . Transient inflammation is considered part of the body's immune defense against pathogens, while persistent inflammation may promote cancer development 5 . This study was aimed to isolate and identify bacteria from urine samples obtained from patients with transitional cell carcinoma-bladder cancer and patients with negative cystoscope and to detect antibiotic instructions. PCR was performed using a specific primer set for the detection of pap E in bacterial extracted DNA 7 . PCR products were electrophoresed in 1.5% agarose gel. The appearance of a band with a molecular size 321 bp referred to the amplification of papE. Biofilm assay using Tissue culture plate method Overnight Bacterial growth was grown on LB broth at 37 °C for 24hr. The culture was adjusted to 0.01 with McFarland solution. Of bacterial growth,50µl was added to 150 µl of LB broth on tissue culture plate wells and incubated at 37 °C for 24 hrs. The culture was removed carefully, and wells were washed two times with 250 µl distilled water. Then, 250 µl of (0.2%) of Crystal violet was added and incubated for 10 minutes at 25 °C. Wells were washed with distilled water 2-3 times and dried at room temperature. Finally, 200 µl of 95% ethanol was added to wells. Optical density (O.D) was measured at 630 nm. Interpretation producer of results was done 8 .

Siderophore Production
Siderophore production using M9 medium supplemented with glucose 20% and casmino acid 20% was prepared 9 .Bacterial growth turbidity was adjusted to 0.01 with McFarland solution, then cultured in M9 medium supplemented with casmino acid and glucose (2 gm/L each), and incubating at 37 °C for 48hrs. Appearing growth in medium indicates positive results.

Cytochrome P450 production
Detection of cytochrome P450 (P450)producing bacteria was done using an M9 medium containing a P450-inducer as the sole carbon source 2-ethoxyphenol as a carbon source. Bacterial isolates were cultured in tubes contain M9medium incubated at 37C for seven days 10 . Quantification of cytochrome produced by bacteria was done using Modified microplate method 11 .

Results: Bacterial infection (Cystitis) in urine samples
From a total of 62 urine samples, only 32/62(51.6%) samples were positive for bacterial growth, including different species of bacteria. Three patients had co-infection (two bacterial species), so the total number of bacterial isolated was 35 isolates. The vast majority of bacteria isolated was Gram-negative bacteria 30/35(88.5%) while Gram-positive bacteria consisted 5/35 (11.5%).
Uropathic Escherichia coli is the most predominant bacteria isolated from patients with

Antibiotic Susceptibility
Antibiotic susceptibility of different bacterial isolates from urine samples using VITEK 2 compact system are shown in Table 2. E. coli reveals highly sensitivity to imipenem and meropenem while resistance in percentage 100% (7/7) for Ticarcillin, Ticarcillin/Clavulanic Acid, piperacillin, piperacillin/tazobactam, Ceftazidime, Cefepime, aztreonam and Trimethoprim/Sulfamethoxazole Cell wall inhibitor Antibiotic susceptibility of P. aeruginosa and P. fluorescence shown in Table 3. P. fluorescence differ from P. aeruginosa in three antibiotics, aztreonam, minocycline and Trimethoprim/sulfamethoxazole. Only two antibiotics included in VITEK 2 compact system depend on updating for S. maltophilia, which was showed Resistance to Trimethoprim and Trimethoprim/sulfamethoxazole (83%) isolates, Table 4.   Cell wall inhibitor   Note: Bacteria code with a symbol (a) referred to two species of bacteria (dull infection) were isolated from the urine of the same patient, such as 23a was Enterococus faecalis and 23 was Sphingomonas paucimobilis antibiotic pattern in the Table 6. Also, 31a and 40a was Enterococus faecalis while 31, 40 were Escherichia coli antibiotic pattern in Table 2. Identification of uro-pathogenic E.coli using pap E Uro-pathogenic E.coli was first identified as E.coli using VITEK 2 Compact system, then at the molecular level using conventional PCR to detect the presence of papE and the results showed that 7/7 isolates had papE, Fig. 1.

Discussion: Bacterial infection (Cystitis)
The infection of S.maltophilia and P.aeruginosa in patients with a negative cystoscope, may be related to those patients' bloody urine and the nature of these bacteria, especially S.maltophiliawhich need iron as essential nutrients for bacterial survival. Iron was found to play a crucial role in the regulation of numerous virulence factors 12 .
A study done in Egypt in 2015 found that, in 20 patients with TCC-BC, only 15 of them had urinary tract infection, and the most predominant organism isolated was Escherichia coli 13 . A study in the United States at (2013) referred to that endogenous bacteria, including cystitis, caused by bladder bacteria (bladder pathogens) and some intestinal opportunistic Pseudomonas aeruginosa, metabolically activate the bladder procarcinogens 14 , while urinary bladder infection by E. coli plays a significant additive and synergistic roles during bladder carcinogenesis 15 . A study in China in 2019 from a total of 24 urine sample from patients with bladder cancer revealed the abundance of common core bacteria is significantly higher in bladder cancer urine samples, especially Acinetobacter which is much higher in bladder cancer urine samples because of bacterial ability for biofilm formation, adhesion and invasion of epithelial cells 16. A hospital-based comparative crosssectional study in Ethiopia in 2019 included 240 patients with any type of cancer; they found that E. coli (32.1%) was the most common bacteria isolated followed by Klebsiella species (25.0%), Staphylococcus aureus (21.4%), Enterococcus species (10.7%), Serratia species (7.1%), and Enterobacter aerogenes(3.6%) in the urine of patients with any type of cancer 17 . It is essential to focus on urinary tract infection (Inflammation or chronic Inflammation) since that chronic inflammation induced by biological factors associated with increased risk of human cancer at various sites due to Inflammation activates a variety of inflammatory cells, which induce and activate several oxidant-generating, by which these enzymes produce high concentrations of diverse free radicals and oxidants that react with each other to generate other more potent reactive oxygen and nitrogen species which can damage DNA, RNA, lipids, and proteins thus increased mutations and altered functions of enzymes and proteins (e.g., activation of oncogene-products and inhibition of tumor-suppressor proteins) 18 .
A study in Mexico in 2014 included 119 isolates of Stenotrophomonas maltophilia collected between 2006 to 2013, with a resistance rate above 75 % for imipenem, meropenem, ampicillin, aztreonam, gentamicin, and tobramycin, while resistance to trimethoprim-sulfamethoxazole was 32.8% 22 . While in Turkey, a study published in 2016 included 118 isolates of Stenotrophomonas maltophilia collected in a period extended from 2006 to 2012, they found that the resistance rate was 7.6% Levofloxacin. 18.2% chloramphenicol, 20.3% trimethoprim-sulfamethoxazole and 72% ceftazidime 23 .
In 2019 a retrospective cohort study in Hungary extended from 2008 to 2017 included 579 isolates of Stenotrophomonas maltophilia, concluded 5.35% of isolates were multidrugresistant (MDR) while 5.87% were extensively drug-resistant (XDR), that is, in addition to SMX/TMP, they were resistant to amikacin, colistin, Levofloxacin, and tigecycline 24 . In Nigeria 2018, a study included five isolated Pseudomonas aeruginosa, which were Resistance to Ampicillin and Amoxicillin/Clavulanic acid 25 . In Prague, a study extended from 2011 to 2019 included 6897 isolates from total P.aeruginosa form 180 (7.3%) of isolates, which were resistant to ofloxacin and sensitive to colistin 26 .
A study in Duhok in Iraq in 2018 included 371 isolates from a total of 276 (74.4%) E.coli, 12 (2.8) P.aeruginosa, and 9 (2.4) Acinetobacter sp. Which were shows a varies pattern of antibiotic susceptibility also Acinetobacter sp showed resistance (100%) to Aztreonam, Augmentin and Nitrofurantoin while P.aeruginosa resistance (100%) to Augmentin 27 . A study in India in 2018 included 67 isolates of Acinetobacter sp were tested for sensitivity pattern using disk diffusion methods, which were 80.3% of isolates was multi drug resistance but (100%) sensitive to colistin 28 . Case report study in India in 2017 included isolate of Ochrobactrum anthropi from patients with septicemia; antibiotic susceptibility was done using VITEK® 2 Compact system, which was multidrug- resistance, resistance to a wide range of antibioticsceftazidime, cefoperazone, cefepime, chloramphenicol, sulbactam, piperacillin /tazobactam, ciprofloxacin, imipenem, and meropenem while was susceptible to amikacin, tigecycline, cefepime-tazobactam, colistin, cotrimoxazole 29 . A study in Jordon 2017 included four isolates of B.cepacia complex isolates from the urine; antibiotic susceptibility was done using disk diffusion methods, which were resistant to lincomycin, nalidixic acid, oxacillin and penicillin G and sensitive to ceftazidime, ciprofloxacin, gentamicin, imipenem, and Levofloxacin 30 . One year prospective study in India 2018 included 43 isolates of B.cepacia complex isolates from blood and sputum, antibiotic susceptibility was done using VITEK 2 Compact system, showed Maximum Resistance with β-lactamase inhibitor drugs (83.7%) 31 . Case report study 2017 in Malaysia isolates Ralstonia mannitolilytica from the blood of 65 years female with underlying hypertension, diabetes mellitus and ischaemic heart disease as well as received regular renal dialysis culture showed sensitivity to ceftazidime and piperacillin/ tazobactam while resistant to amikacin, gentamicin, meropenem and polymyxin 32 .
Another case report study (2019) in Italy studying Antibiotic susceptibility of Ralstonia mannitolilytica isolated from the blood of 46 years female with underlying diseases using the broth micro dilution method which resisted to a wide range of antibiotic including Amikacin, Aztreonam, cefepime, Ceftazidime, Ertapenem, gentamycin, imipenem, and meropenem 33 . While in China (2019) antibiotic susceptibility was studied using the VITEK 2 Compact system on two isolates of Ralstonia mannitolilytica isolated from blood indicated resistance to aminoglycosides, β-lactams, and polymyxin B 34 . Another case study in India 2014 included isolate of Brevundimonas vesicularis antibiotic susceptibility was done using disc diffusion methods, which was susceptible to piperacillin-tazobactam, minocycline, and cotrimoxazole, while resisting to amikacin, gentamicin, tobramycin, netilmicin, amoxicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, cefoperazone, ceftazidime, cefoperazone-sulbactam, imipenem, meropenem, ertapenem, aztreonam, norfloxacin, Levofloxacin and colistin 35 .

Identification of uropathogenicE.coli using papE gene
In this study, uropathogenic Escherichia coli (UPEC) is the most predominant bacteria isolated from bladder cancer patients. A study in Egypt in 2015 referred to that E. coli is the most common uropathogenic bacteria, which infects bladder cancer patients. Surface virulence factors (adhesins) are significance virulence factors of UPEC as the primary attachment factor, P fimbriae are associated with pyelonephritis and are encoded by pap genes(Pyelonephritis-Associated Pili), allowing them to colonize host mucosal surfaces and invade the normally sterile urinary tract 39 .A study in Al-Kufa, Iraq (2015) investigated the severity of urinary tract infections in 48 patients with TCC and 20 patients with non-TCC as a negative control group. They obtained bacterial growth from 15 cultured urine samples. Among them, 41.67% were Gram-positive bacteria, and 27.78% was Gram-negative bacteria 40 . A prospective descriptive (cross-sectional) Iraqi study in 2020 included 170 specimens (100 urine and 70 stool specimens) screened for papE gene using conventional PCR. They indicated that (29/100) isolates were identified as UPEC 41 . In Iran, Rahdar et al. (2015) referred to that from 100 isolates of E.coli isolated from the urine of patients with UTI, only 57% was harbor papE detected using PCR 42 . In Egypt, a study in 2019 included 173 isolates of UPEC and diarrheagenicE.coli(DEC) investigated for phylogenetic typing and urovirulence genes using PCR, results 16.5% of UPEC was harbor papE but not present in DEC 43 .

Virulence Factors of Isolated BacteriaBiofilm formation
In this study, all isolates were produced biofilm either moderate 17/35 (51%) or strong producer 17/35 (49%). The quantification test of biofilm was proved to be useful in detecting biofilm production by the clinical isolates (44). Urinary tract infections significantly associated with microbial biofilms, developed on catheters which conclude a high percentage of all nosocomial infections and are the most prevalent source of Gram-negative bacteremia in hospitalized patients 45 . Biofilm formation is also considered a virulence determinant responsible for the long-lasting persistence of bacteria in the genitourinary tract 46 . Escherichia coli (UPEC) form biofilms on urinary catheters, as well as within bladder epithelial cells, which protects UPEC from environmental conditions, antimicrobial therapy, ultraviolet radiation, oxidizing biocides, and host immune defenses 47 . Iranian study in 2018 included 100 isolates of UPEC was screened ability for producing biofilm using microtiter plate methods (TCP) indicated 36/100 was strong producer, 48/100 was moderate producer, and 10/100 was the weak producer 48 . Another study in India in 2019 included 100 isolates of E.coli isolated from patients suffering from UTI, was studied the ability to produce biofilm their results indicated 69% of isolates were producers of biofilm 49 . Hungarian study in 2020 on 250 isolates of E.coli from patients with UTI screened ability for producing biofilm using crystal violet tube-adherence method their results indicated that 119 (47.6%) were positive for biofilm formation 50 22 .
Pseudomonas aeruginosa produce biofilm is an essential mechanism for survival, and its relationship with antimicrobial-resistance represents a challenge for patient therapeutics, especially in nosocomial infections of immune-compromised patients 51 . A study in Brazil in 2018 compromised 40 clinical isolates of P.aeruginosa has studied ability to produce biofilm using TCP method their results indicate 77.5% of isolates were biofilm producer 52 . A study in Belgium in 2014 in six isolates of Burkholderiaspp studied their ability to produce biofilm using TCP, indicated all isolates were able to produce biofilm but in different percentage 53 . Egyptian study in 2017 included 90 clinical isolates of Enterococcus faecalis studied their ability to produce biofilm using two methods Congo-Red agar (CRA) biofilm assay and TCP method. In (CRA) five potent biofilm-producing isolates 81 isolates ranged between moderate and weak, and 4 non-biofilm-producing isolates while in TCP methods only 5/90 (5.5%) were classified as strong biofilm-formers; 38/90 isolates (42%) were moderate; 43/90 were weak biofilm-formers (48%); and 4/90 (4.5%) could not form biofilm 54 . A study in china 2018 included 113 isolates of E.faecalis from urine of patients with UTI, studied the ability to produce biofilm using TCP methods, indicated that (59.7)% as a strong biofilm producer while (30.6 %) as non-biofilm producer 55 .

Siderophore production
In the present study, 25/35 (71%) isolates were producer while 10/35 (29%) isolates were non producer. A study in India in 2017 included 200 isolates of UPEC isolated from patients with UTI, screened for siderophore production using Chrome azurol assay (CAZ) noticed that (95%) of isolates were siderophore production indicated that siderophores or Iron acquisition constitute a significance virulence factor requisite for UropathogenicE.coli in the pathogenesis of UTI 56 . Another study in al-Kufa in Iraq in 2017 included 50 isolates of E.coli from different infections, screened for virulence factors including siderophore production using M9 medium supplemented with 2,2ʼ-dipyridyl, their results indicated (100%) of isolates were siderophore producer 57 . A study in Argentina in 2012 included 31 clinical isolates of S.maltophilia screened for siderophore production using (CAZ) assay indicated that all isolated(100%) were siderophore production 58 .

Cytochrome P450
In the present study, the inducing of cytochrome P450 expression protein was seen in (14/35) 40% isolates as induced, while not induced in (21/35) 60% isolates. Many types of research indicated that microorganisms such as Bacteria were shown to express cytochrome CYP-like genes (these genes in microbial organisms differ extensively even between species of the same genus) even though individual organisms having no CYP genes present (e.g., Escherichia coli), the considerable metabolic activity of the microbe is associated to its abundant collect of CYP enzymes 59 . This reason could explain the failure to induce (8) isolates of UPEC to express cytochrome P450. It was also reported that Bacterial cytochrome P450s were characterized in their high expression level in many microorganisms 60 .
Microbial P450s have diverse catalytic functions. For example, in Sphingomonas paucimobilis had fatty acid α-hyroxylase (H2O2 dependent peroxygenase), and in Pseudomonas sp function as α-terpineol hydroxylase activity. Besides Microbial P450s have roles in the degradation of toxic compounds 61 . It is crucial to study microbes' ability to express cytochrome P450 proteins (specifically quantity) in the cell wall because Pseudmonas aeruginosa would metabolically activate the bladder procarcinogens, which is achieved by the presence of cytochrome P450 14