Genotypic Characterization of Antimicrobial Resistance- Associated Genes in Citrobacter Freundii Isolated from Patients with Urinary Tract Infection in Al-Najaf Governorate-Iraq

Corresponding Author: Thualfakar Hayder Altoosi Collage University, AlNajaf Governorate, Iraq E-mail: hualfakarhh.edu11p@uokufa.edu.iq ahmedaj.aljanabi@uokufa.edu.iq Abstract: Citrobacter freundii is one of the most important pathogens that cause urinary tract infection in human because it harbors many types of antimicrobials resistance-associated genes. Therefore, this study aimed to evaluate the prevalence of antimicrobial resistance-associated genes citrobacter freundii isolated from patients with urinary tract infection in AlNajaf governorate-Iraq. A total of 30 strains of citrobacter freundii isolated from urine samples of patients with urinary tract infection in Al-Najaf central hospital in Al-Najaf City, Iraq, during April to December 2018. Polymers chain reaction technique was used to detect the presence of antimicrobial resistance-associated genes. From the 30 strains of citrobacter freundii, 15 strains (50%) were positive for bla-tem gene, 16 strains (53.3%) were positive for bla-shv gene, 21 strains (70%) were positive for bla-ctxm gene, 3 strains (10%) were positive for bla-ctxm-1 gene, 6 strains (20%) were positive for bla-ctxm-2 gene. Interestingly, it should be noted that no any strain form the 30 we tested was carrying pse1, bla-ctxm-8, bla-ctxm-9 or bla-ctxm-25 genes. Our data showed that 19 strains (63.3%) were positive for cat1 gene, 9 strains (30%) were positive for cat2 gene and 4 strains (13.3%) were positive for cat3 gene. On the other hand, 17 strains (56.6%) were carrying qnrA gene, 11 strains (36.6%) were carrying qnrB gene and aac(6')Ib-cr gene. Also, the results proved that there were 7 strains (23.3%) being positive for sul1 gene and 11 strains (36.6%) were positive for sul2 gene. We also found 22 strains (73.3%) being positive for strA-strB gene and 4 isolates (13.3%) were positive for aacC1 genes. No strain carrying aacC2, tetA and tetB genes was identified. Citrobacter freundii harbor large numbers of different antimicrobial resistance-associated genes that enable this pathogen to escape the effect of antibiotics which make this bacteria highly resistant causing urinary tract infection.


Introduction
Citrobacter freundii is consider as one of a commensal types of the intestinal tract of humans and different creatures but can also be found in water, sewage, food and hospital settings (Liu et al., 2018). Citrobacter freundii is a Gram-negative, facultative anaerobic, encapsulated motile organism with long rod shape, a typical length of bacterium is 1-5 µm (Hossain et al., 2017;. Citrobacter freundii is often found in clinical specimens as an opportunistic pathogen was implicated with infections that are generally resstricted to infants and immuno-compromised people (Plakkal et al., 2013;Bae et al., 2018). Citrobacter freundii responsible for nosocomial infection, blood stream and wounds infections, gastroenteritis, endocarditis, pneumonia, septicemia, urinary tract infection meningitis and brain abscesses with high mortality and morbidity (Aljanaby and Gafil, 2013). Citrobacter freundii has many types of antimicrobial resistance-associated genes such as shv, tem, ctx-m and others enable this pathogen to escape the effect of various antibiotics, thus became more virulent and cause different types of infection such as urinary tract infection 2018). In Iraq, there are limited studies focusing on the prevalence of antimicrobial resistance-associated genes in Citrobacter freundii isolated from patients with urinary tract infection. Therefore, the aim of this study was to investigate the prevalence of antimicrobial resistanceassociated genes in Citrobacter freundii isolated from patients with urinary tract infection. This study will draw a good genotypic picture of this pathogen.

Study Design and Patients
This is a cross-sectional descriptive study, performed in Al-Najaf central hospital in Al-Najaf City, Iraq, during period from April to December 2018. A total of 461 urine samples were collected from patients infected with urinary tract infection, females (21) and males (9), age groups between 18 to 60 years old.

Eligibility Criteria for Patients
Patients will be considered eligible for registration into this study if they fulfill all the inclusion criteria and none of the exclusion criteria as defined below: 1. Patients (female or male) at least equal or more than 18 years old 2. All infected patients have been diagnosis by physician and primary microscopic characters such as the presence of pus cells in urine samples 3. Patients should have sufficient capacity for informed consent 4. Patients should don't have any other infection 5. Patients should don't take any antibiotics for treatment

Samples Collection, Culture and Bacterial Identification
Ten ml of mid-stream urine samples were collected in sterile disposable containers after cleaned the genitals. All urine samples were centrifuged at 2000 rpm for 5 min, immediately, the sediment incubated aerobically with brain hart infusion broth at 37°C for 24h then streaked with sterile loop on to blood agar and MacConkey agar (Oxoid TM ) plates (Mac Faddin, 2000). Colony Forming Units (CFUs) method was used for growing single and pure bacterial colony; all urine samples containing less than 10 5 CFUs/ml were excluded (Tan et al., 2012). All emerged bacterial isolates were identified according to colony morphology and standard microbiological tests such as; colony morphology, Gram stain, oxidase test, catalase test, imvic test, motility test, coagulase test, growth on MacConkey agar (Oxoid TM ) (Mac Faddin, 2000).
DNA extraction: The previously published protocol (Yang et al., 2008) was used for the extraction of total DNA as follows: Five pure and fresh colonies of citrobacter freundii strains were suspended in 200 µl of sterile deionized water and cells were placed in water bath (Memmert-Germany) at 100°C for 30 min, immediately the solution was placed in ice for 30 min and the other cellular components was removed by centrifugation at 9000 rpm for 15 min. Finally the supernatant was used as the DNA template.
Polymerase Chain Reaction (PCR) detection of antimicrobials resistance-associated genes: Polymerase chain reaction was used to detect 22 antimicrobials resistance-associated genes: All primers used in this study and all PCR thermo cycling conditions are listed in Tables 1 and 2, respectively. All PCR products were loaded on a 1.5% (w/v) agarose gel with 0.5 mg/mL safestain and were analyzed by gel electrophoresis. All primers used in this study and all PCR thermo cycling conditions are listed in Tables 1 and 2, respectively.     Statistical Analysis: Fisher's exact test was used in this study for the comparison between samples by using SPSS version 6 software. P values less than the 0.05 level of significance were considered statistically significant (Aljanaby and Gafil, 2013).

Sulfonamide Resistance Associated Genes
In the current study, genotypic detection of two genes that responsible for encoding sulfonamide resistance associated genes: sul1 and sul2 were detected by Polymerase chain reaction using specific primer sequences which yield products size 668 bp and 707 bp respectively. The results proved that there were 7 isolates (23.3%) positive for sul1 (Fig. 12) and 11 isolates (36.6%) were positive for sul2 gene (Fig. 13).

Streptomycin, Aminoglycoside and Tetracycline Resistance Associated Genes
Different genes were used to detect the prevalence of Streptomycin, aminoglycoside and tetracycline resistance associated genes in 30 isolates of Citrobacter freundii. strA-strB gene was used as a streptomycin resistance associated genes, the results showed that there were 22 isolates (73.3%) were positive for strA-strB gene (Fig. 14). aacC1 and aacC2 genes were used as a aminoglycoside resistance associated genes, the results indicated that there were 4 isolates (13.3%) were positive for aacC1gene ( Fig. 15) while, there was no any isolates was carrying aacC2 gene. tetA and tetB genes were used as a tetracycline resistance associated genes, the results proved that there was no any isolate was carrying these genes. All numbers and percentages of antimicrobials resistance associated genes in Citrobacter freundii isolates are mentioned in Fig. 16 and Table 3.

Discussion
The TEM catalysts was one of the initially stretched out range beta lactamase to be distinguished, first found in 1965. The TEM sort βlactamase was named after the E. coli contaminated patient, Temoneira, in Athens, Greece (Aljanaby and Aljanaby, 2018a). What's more, the bla-tem quality (encoding the protein) is normally found on plasmids (Matsumoto et al., 2014). In current study out of 30 isolates, there were 15 isolates with percentage rate (50%) were positive for bla-tem gene. This result is in agreement with the study by Praharaj et al. (2016) in India, who showed 50% of C. freundii isolated from urinary tract infections samples were positive to bla-tem gene. A similar study by Delgado-Valverde et al. (2016) reported that 65.8% of C. freundii isolates were positive to bla-tem gene. However, other study demonstrated that there was 10.4% of C. freundii isolates were positive to bla-tem gene (Akinduti et al., 2011). Extended spectrum beta lactamase enzymes have been derived from TEM and SHV genes by mutations and have been well described in Enterobacteriaceae family (Liakopoulos et al., 2016).
In the current study, Molecular detection of some antimicrobials resistance genes were detected by PCR using specific primer sequences. In the present study 16 isolates with percentage rate (53.3%) were positive for bla-shv gene. The current study result is in agreement with those previously reported (Sharma et al., 2016) showing 57.5% of C. freundii isolates from urinary tract infection samples were positive to bla-shv gene. Another study in Madagascar by Chereau et al. (2015) showed that there was 66.6% of C. freundii isolates from pregnant women urinary tract samples were positive to bla-shv gene. In the current years, another group of plasmid-interceded ESBLs (extended spectrum beta lactamase) called CTX-M which specially hydrolyze cefotaxime, has risen and the bla-ctxm quality is typically found on plasmids. CTX-M catalysts have for the most part been found in strains of Salmonella enterica serovar Typhimurium and E. coli, however have likewise been depicted in different types of Enterobacteriaceae (Brown et al., 2018). Not at all like different ESBLs sorts, CTX-M family incorporates a complex and non-comparable gathering of catalysts. The primary investigation and arrangement of the amino corrosive groupings of the CTX-M variations ordered these catalysts into five bunches (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) (Aljanaby and Alhasnawi, 2017). CTX-M β-lactamases rose not by changes from prior plasmid intervened catalysts but rather by preparation of chromosomal bla qualities from microscopic organisms are firmly identified with Escherichia coli and discovered around the world, activation of bla-ctxm qualities happened by inclusion successions or by lesser degree bacteriophages. The inclusion succession assumes a part in the over-articulation of bla-ctxm qualities (Tamang et al., 2013). The bla qualities were coordinated into portable hereditary structures and exchanged probably by conjugation into clinical microscopic organisms (Hardiman et al., 2016). These prepared bla-ctxm qualities increment cefotaxime imperviousness to a significantly more prominent degree than imperviousness to ceftazidime (Aljanaby, 2018). The consequences of present study additionally showed that 21 isolates (70%) were positive for bla-ctxm gene, 3 isolates (10%) were positive for bla-ctxm-1 gene, 6 isolates (20%) were positive for bla-ctxm-2 gene, while, there was positive isolate carrying pse1, bla-ctxm-8, bla-ctxm-9 and bla-ctxm-25 genes. This is supportive to previously published study (Castanheira et al., 2017) showing that there was 35.5% of C. frundii isolates were from samples revealed that ESBL (extended spectrum beta lactamase) producing isolates have bla-ctxm genes, these result is not in agreement with this study result. Chloramphenicol is one of the first broad-spectrum antibiotics used to treat grampositive and gram-negative bacterial infections; the major resistance of mechanism to chloramphenicol is enzymatic inactivation by the plasmid transposon-mediated chloramphenicol acetyl transferees cat1 gene and cat2 gene (Huang et al., 2017). Quinolones are expansive range antibacterial specialists, generally utilized for treatment of contaminations therefore, upgraded level of quinolone resistance has happened as of late for instance fluoroquinolones, for example, ciprofloxacin, beforehand appeared to have incredible action against clinical disengages of C. freundii, have turned out to be less successful because of their broad utilize (Aldred et al., 2014). Quinolones are broad-spectrum antibacterial agents, commonly used for treatment of infections as a result, enhanced level of quinolone resistance has occurred in recent years . Mechanisms of Quinolone resistance is mediated by the mutation of chromosomal genes encoding DNA gyrase and/or topoisomerase IV or by the mutation of genes regulating the expression of efflux pumps (Correia et al., 2017). qnr gene found on plasmid, quinolone resistance may also be related to plasmid-mediated quinolone resistance genes, (qnrA, qnrB and aac(6')Ib-cr) (Aljanaby and Medhat, 2017). The qnrA and qnrB genes are coding for proteins belonging to the penta peptide repeat family interacting with DNA gyrase and topoisomerase IV enzymes to prevent quinolone inhibition (Aljanaby and Aljanaby, 2018b). Another plasmid mediated quinolone resistance gene is aac (6')-Ib-cr, which encodes aminoglycoside acetyltransferase enzyme. This enzyme which can diminish a fluoroquinolone activity by adding an acetyl group to this antibiotic (Ramirez and Tolmasky, 2017) Sulfa drugs are an important class of synthetic bacteriostatic antibiotics still used today for the treatment of bacterial infections and those caused by other microorganisms, sulfa drugs achieve this bacteriostatic action by inhibiting the synthesis of folic acid in bacteria (Shama, 2015). They are also known as sulfa drugs and were the main source of therapy against bacterial infections before the introduction of penicillin in 1941. Although sulfonamides have for the most part been replaced by other agents, they still maintain considerable action in certain types of infection, for example in the urinary tract, eye and ear infections (Kapoor et al., 2017). Aminoglycosides are highly potent, broad-spectrum antibiotics with many desirable properties for the treatment of life-threatening infections. Their history begins in 1944 with streptomycin and was thereafter marked by the successive introduction of a series of milestone compounds (kanamycin, gentamicin and tobramycin) which definitively established the usefulness of this class of antibiotics for the treatment of gram-negative bacillary infections (Krause et al., 2016). Streptomycin is an aminoglycoside antibiotic produced by the soil actinomyces Streptomyces griseus. It acts by binding to the 30S ribosomal subunit of susceptible organisms and disrupting the initiation and elongation steps in protein synthesis. It is bactericidal due to effects that are not fully understood (Aljanaby, 2013). The tetracycline which were discovered in the 1940s, are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site. Tetracycline are broadspectrum agents, exhibiting activity against a wide range of gram-positive and gram-negative bacteria (Krause et al., 2016). In USA 2008, the study by Srinivasan et al. (2008) who showed that the strA-strB gene was most prevalence gene in C. freundii isolates with 4.6% rate of, these result is not in agreement with present study. The study by Igbinosa et al. (2018) in Nigeria, showed that the prevalence of multidrug resistance isolates where it contains further antibiotic resistance genes, including those for aminoglycosides (strA and strB), beta-lactams (bla-tem), chloramphenicol (cat2), sulfonamide (sul2), tetracycline (tetA) and trimethoprim (dfrA14). In conclusion: Citrobacter freundii harbor large numbers of different antimicrobial resistance-associated genes that enable this pathogen to be more virulent and make it highly resistant against most antimicrobials and became more dangerous bacteria cause urinary tract infection.