Iraqi J Pharm Sci, Vol.31( 2 ) 2022 Quinolone-resistance genes in Salmonella enterica Serotype Typhi DOI: https://doi.org/10.31351/vol31iss2pp218-222 218 Occurrence of Quinolone-Resistance Genes in Ciprofloxacin-Resistant Salmonella Enterica Serotype Typhi Isolated form Blood Sample of Patients with Typhoid Fever. Jabbar S. Hassan *,1, Bushra J. Al-Tamimi** and Fuad Ghazi Hassan *** *College of Medicine, Al-Nahrain University, Baghdad, Iraq **College of Medicine, University of Babylon, Babylon, Iraq ***Department of Medical Laboratories Techniques,Immunity, Al-Mustaqbal University College, Babylon, Iraq Abstract Salmonella is approved as a common foodborne pathogen, causing major health problems throughout the world particularly in low‐ and middle‐income countries. Low-level fluoroquinolone resistance is conferred by both chromosomal and plasmid-encoded resistance, this research was carried out look into the occurrence rate of qnrA,qnrB and qnrS genes in Salmonella enterica serotype Typhi Cipr ofloxacin-resistant insulate from blood samples of patients with typhoid fever. Fifteen Salmonella enterica serotype Typhi isolated previously from patients with typhoid fever were included in this study. All bacterial isolates were confirmed to have ciprofloxacin resistant by VITEK 2 microbial identification system; after plasmid DNA extraction; multiplex-PCR was done with primer sequences intended to plasmid-mediated quinolone-resistance genes which is qnrA, qnrB, and qnrS. In this study; it was 21 qur genes amongst 15 isolates. The qnrS gene was the commonest (10/21, 47.6%) followed by qnrA (6/21, 28.5%), whereas only 4 isolates were positive for qnrB (5/21, 23.8%). Some isolates had more than one qnr genes. So, Ciprofloxacin-resistant Salmonella typhi can have more than one gene at the same time; and the most occurrence rate in regards to qnr gene in this study was qnrS compared to qnrA and qnrB Keywords: Salmonella enterica serotype Typhi , Typhoid fever, Fluoroquinolone, Ciprofloxacin, qnr gene. وجود الجينات المقاومة للكوينولون في بكتريا السالمونيال المعوية المقاومة للسيبروفلوكساسين النمط للمرضى المصابين بالحمى التيفوئيديةالمصلي تايفي المعزولة من عينات الدم ***فؤاد غازي حسن و ** بشرى جبار التميمي حسان، 1*،جبار سلمان ، بغداد ، العراق كلية الطب جامعة النهرين فرع االحياء المجهرية * ، بابل ، العراق كلية الطب جامعة بابل فرع االحياء المجهرية** ، بابل ، العراق الطبية المختبرات تقنياتكلية المستقبل الجامعة قسم *** الخالصة تعتبر بكتريا السالمونيال أحد مسببات األمراض الشائعة المنقولة عن طريق األغذية ، والتي تسبب مشكلة صحية كبيرة في جميع أنحاء تحدث مقاومة الفلوروكينولون في هذه البكتريا عن طريق المقاومة المشفرة بالكروموسومات . العالم وخاصة في البلدان المنخفضة والمتوسطة الدخل ، أجريت هذه الدراسة للتحقق من معدل حدوث جينات في سالمونيال التيفوئيد المقاومة للسيبروفلوكساسين qnrSو qnrBو qnrAوالبالزميد سابقا من مرضى تم استخدام خمسة عشر عزلة من السالمونيال التيفية التي تم عزلها. ئيدالمعزولة من عينات دم المرضى المصابين بحمى التيفو في هذه الدراسة تم التأكد من أن جميع العزالت البكتيرية لديها مقاومة للسيبروفلوكساسين بواسطة نظام التعرف الميكروبي . مصابين بحمى التيفوئيد VITEK 2 .زميدي تم إجراء بعد استخراج الحمض النووي البالMultiplex-PCR باستخدام بادئات تم تصميمها من اجل ان تستهدف جينات كان جين . عزلة 15من بين qurجين 21في هذه الدراسة؛ كان هناك . qnrSو qnrBو qnrAمقاومة الكينولون بوساطة البالزميد بما في ذلك qnrS يليه ( ٪47.6، 10/21)هو األكثر شيوًعاqnrA (6/21 ،28.5٪ ) عزالت فقط موجبة لـ 4، بينما كانتqnrB (5/21 ،23.8٪ .) بعض وكان . المقاومة للسيبروفلوكساسين على أكثر من جين واحد في نفس الوقت هذة البكتريا لذلك يمكن أن تحتوي . qnrالعزالت لديها أكثر من جين qnrو qnrAمقارنة بـ qnrSفي هذه الدراسة هو qnrأعلى معدل حدوث لجين السالمونيال التيفية ، حمى التيفوئيد ، الفلوروكينولون ، سيبروفلوكساسين : الكلمات المفتاحية Introductions Salmonella enterica serotypes is a Gram- negative, rod shape, flagellated and aerobic bacteria; it is posing a great danger to human health, most notably in low‐ and middle‐income countries (1). For a long time, the first medication for Salmonellosis infections included chloramphenicol and trimethoprim in addition to penicillin, but the increase in resistance to such treatments prompted most doctors and with the emergence of newer types of antibiotics to use new antibiotics particularly Fluroquinolone groups (2). Fluoroquinolone un-responsiveness are primarily caused by two mechanisms: chromosomally mediated mutations in topoisomerase's quinolone resistance determining regions (QRDR) and quinolone resistance determining region mutations; resistance genes belong to qnr groups plasmids mediated play a role in Fluoroquinolone resistant (3). 1Corresponding author E-mail: jabbarsalman30@yahoo.com Received: 12/9 /2021 Accepted:19 /1 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31iss2pp218-222 Iraqi J Pharm Sci, Vol.31(2) 2022 Quinolone-resistance genes in Salmonella enterica Serotype Typhi 219 Low-level fluoroquinolone resistance is conferred by both chromosomal and plasmid- encoded fluoroquinolone resistance (4). Determinants belong to qnr have been found in a numeral of enterobacterial species from different parts of the world, including America, Europe, and Asia (5). So far, six variants (qnrA1 to qnrA6) have been discovered. Quinolones produced by other plasmids qnrB (qnrB1 to qnrB5) and qnrS (qnrS1 and qnrS2) resistance determinants outlined in enterobacterial species (6). The current study aimed to investigate occurrence rate of qnrA, qnrB and qnrS genes in ciprofloxacin-resistant Salmonella typhi (S. typhi) insulate from blood samples of patients with typhoid fever. Material and Methods Bacterial strain A retrospective study of archived isolates, including 15 Salmonella enterica serotype Typhi isolates which was previously recovered from blood samples of patients with typhoid fever were used in this study. Resistance to ciprofloxacin for all bacterial isolates that included in the current study was detected by using of VITEK 2 microbial identification system, the detection was carried out according to manufacturing company (bio- Merieux). The bacterial isolates were diagnosed and classified in medical microbiology department at the faculty of medicine, AL -Nahrain University Plasmid DNA extraction Protocol Salmonella enterica serotype Typhi was harvested by using Luria-Bertani broth media, after centrifugation (8,000) rpm for two minutes; the supernatant was discarded and the pellet was collected. Plasmid extraction of the Salmonella enterica serotype Typhi was performed as described by company instruction (Wizard® Plus Minipreps DNA Purification System, Promega) Multiplex-PCR was carried out with primer sequences specific for plasmid-mediated quinolone- resistance genes (Table 1), which is qnrA, qnrB, and qnrS. The settings of the PCR as follows: after initial denaturation at 94°C for 7 min, the 35-cycle amplification profile consisting of 94°C for 30 s, 62°C for 30 s, and 72°C for 1 min using a cleaver scientific thermal cycler (TC 32/80-UK). The last elongation was place at 72°C for 10 minutes. For 1.5 hours, using 2% agarose at 7 V/cm (Merck- Germany) PCR product was identified. Concomitantly, a molecular marker (1-kb DNA ladder; Bioneer) was used. After the gel was stained with ethidium bromide, DNA bands were seen and photographed under UV light. Table 1. Primer nucleotide for identification of plasmid-mediated quinolone-resistance genes Qnr genes Nucleotide sequences (5' 3') Products bp References qnrA F GATAAAGTTTTTCAGCAAGAGG ATCCAGATCGGCAAAGGTTA 593 7 R qnrB F GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC 469 8 R qnrS F TGGAAACCTACAATCATACATATCG TTAGTCAGGATAAACAACAATACCC 656 9 R Results Multiplex PCR was used to check for the presence of the plasmid-mediated quinolone resistance genes qnrA, qnrB, and qnrS in 15 isolate of Salmonella typhi that were resistant to Ciprofloxacin; since amplicons product by PCR with the expected amplification product size qnrA gene (593 bp), qnrB (469 bp) and qnrS (656 bp) respectively (Figure 1). Over 15 Salmonella typhi isolate; 21 qur genes were detected; and the qnrS gene was the most common (10/21, 47.6%) followed by qnrA (6/21, 28.5%), whereas only 4 isolates were positive for qnrB (5/21, 23.8%). Some isolates had more than one qnr genes (Table 2). Iraqi J Pharm Sci, Vol.31(2) 2022 Quinolone-resistance genes in Salmonella enterica Serotype Typhi 220 Table 2. Distribution of qnr genes through Salmonella enterica serotype Typhi isolates Ciprofloxacin resistance. Isolates qnrS qnrA qnrB Total genes 1 + - - 1 2 - - + 1 3 + + + 3 4 + - - 1 5 + + + 3 6 - + - 1 7 + - - 1 8 + - + 1 9 - - + 1 10 - + - 1 11 + + - 2 12 + - - 1 13 + - - 1 14 - + - 1 15 + - - 1 Figure 1. Gel electrophoresis of Multiplex PCR products (2% agarose, 7 v/cm2,1.5hrs) for qnrA, qnrB, qnrS of Salmonella enterica serotype Typhi positive isolates. lanes 7, 9, 10,11, 12 and 13 qnrA gene (593 bp) positive isolates; lane 3,4,6,9,11,14: qnrB (469 bp) positive isolate; lanes 5,6,8,11,13 qnrS (656 bp) positive isolates. Discussion Previous studies have documented that Salmonella disease in humans can vary from self- limited gastroenteritis usually connected with non- typhoidal Salmonella (NTS) to typhoid fever with obstacles such as a fatal intestinal perforation; this bacterium has a propensity to acquire resistance to multiple classes of antimicrobial agents, and eradication of infection by highly resistant Salmonella typhi can be mostly difficult (10). This study tested the occurrence of qnr genes among Ciprofloxacin resistance Salmonella enterica serotype Typhi isolated from the blood of patients with typhoid fever. Out of 15 Salmonella enterica serotype Typhi isolates;( 21) qnr genes were detected. Two isolates harbored the three qnr genes qnrA, qnrB, qnrS whereas two isolates harbored two qnr genes in which one isolates harbored qnrA and qnrS while the second isolates have qnrA, qnrB. Quinolone resistance is initiated mostly through chromosomal mutations. Quinolone resistance caused by plasmids has been reported in numerous places of the world in the last few years. This type of resistance is caused by plasmid- mediated qnrA, qnrB, or qnrS genes (11). Quinolone resistance at low levels has been linked to DNA from transferrable plasmids. Several investigations have found that qnr determinants are widely distributed among bacterial isolates all over the world. Quinolones are antibacterial agents with a broad spectrum of action that are commonly utilized in both human and veterinary medicine. Their widespread use has been linked to an increase in quinolone resistance. (11). Hopkins et al., mentioned that qnr genes contributed to high-level Ciprofloxacin resistance in Salmonella species in chromosomal and plasmid mediated (12). There are many articles that reported an increase in non-susceptible bacterial strain to Iraqi J Pharm Sci, Vol.31(2) 2022 Quinolone-resistance genes in Salmonella enterica Serotype Typhi 221 fluroquinolone agents due to harboring the qnr genes may be contributed to using of such antimicrobials in food-producing animals (13,14). Interestingly; in the current study; the qnrS gene was more prevalent (47.6%) than qnrA and qnrB which were (28.5%) and (23.8%) respectively. qnrS gene can increased selective pressure on the drugs and subsequently contributes to resistance even in the absence of mutations and its it is more easily transmitted from animals to humans (15). The prevalence rate of qnr genes among Gram negative bacteria varies depending on sample type and locational area; Most studies reported that; the regional distribution of qnrA genes is known to be wide (15). Many Enterobacteriaceae species have been found to have qnrA-like determinants, and six variants have been identified in qnrA and qnrB which is (qnrA1 to qnrA6) and (qnrB1 to qnrB6) while qnrS (qnrS1 and qnrS2), genes for plasmid mediated quinolone resistance (PMQR) have been found on many bacteria in addition to Enterobacteriaceae such as pseudomonas species with varying in size and incompatibility specificity (16). Cameron-Veas et al. mentioned that 15% of Salmonella enterica in Brazil which have been isolated from pig harbored qnrB, and none was carrying qnrA and qnrS (17). While in China Lin D et al. reported that (66%) Salmonella species carrying qnrS (18). It's worth noting that the transfer of resistance genes among Enterobacteriaceae bacteria, such as quinolones genes, is a complicated process involving a variety of mechanisms, such as plasmid- mediated resistance gene transfer and chromosomal alterations. Types of clinical isolates, geographic location, and antibiotic usage rates in each country can all influence these pathways (19). Many individuals in our country randomly use Ciprofloxacin without following clinicians' prescriptions in self-medication. In addition, the use of this type of antibiotic in the treatment of animals could result in greater selective pressure on such groups of antibiotics, which could lead to resistance through the different types of qnr genes. Conclusions This study reported that ciprofloxacin- resistant Salmonella enterica serotype Typhi may harbor more than one gene at the same time; and the most prevalent qnr gene in this study was qnrS compared to qnrA and qnrB. 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