Molecular Profiling of Class I Integron Gene in MDR Salmonella typhi isolates

Typhoid fever is a paramount reason for horribleness that more mortal sin “around the sum ages aggregations clinched alongside iraq it initiated by salmonella typhi. Salmonella typhi is diagnosed serologically by the Widal test and confirmed by vitek and using polymerase chain reaction (PCR) based amplification of DNA from the bacterial samples of typhoid fever patients. The present study was designed to detect class I integron gene encoding antimicrobial of S. typhi using appropriate primers by PCR. These isolates of this study were collected from postgraduate laboratories (Prepared samples in vitro prepared diagnostics), they were a previous collected from carried out in Al Najaf provenance, throughout those period from July 2018 on March 2019 including 231 cases from blood, stool samples collected from patients suffering from typhoid fever were attended to Al-Sader Medical City and AlHakim General Hospital in Al-Najaf province. Biochemically tests and monovalent antisera gave 117 (50.64%) positive result S. typhi isolates and confirmed by Vitek system and PCR which showed positive result 59 (50.42%). Fifty nine isolates of S. typhi, were collected from patients with typhoid fever that distributed to 40/59 (34 %) from blood , 19/59 (15.1%) stool. Molecular detection revealed that most isolates of S. typhi were positive results to (intI) gene 43/59 isolate (the specific primer (intI) gene for S. typhi bacteria was designed in this study by using bioinformatics programs with NCBI website). According to the different diagnostic above, Vitek and PCR method were more sensitivity technique for S. typhi detection among typhoid patients. The results of virulence factors of S.typhi isolates were negative results for gelatinase, hemolysin, protease and capsulated. Multidrug resistance (MDR) of S. typhi isolates were represented by 18 antibiotics resistance to class and sub class of antibiotic. All S. typhi isolates appeared high resistance 100% to Aztreonam (AZM15), Nitrofurantion (F), Amoxicillin/ clavulanicacid (AMC30), (PY25), Clarithromycin (CLR), Cefoxitin (FOX30), Penecillin(P10), Cefotaxime (CTX30), ampicillin (AMP), Meropenem (MEM), Tetracycline(TE30). Also resistance of isolates that revealed 91% to Impinem (IP ), 88% Ampicillin (AM10), 85%Amoxillin (AX), 81% Gentamicin (CN10), 80% Chloramphenicol (C30), 74% Cefpirome (CPR) and 68% Carbenicillin (CB). Keyword: Salmonella typhi, Typhoid fever, PCR, integron class I, intI gene, Virulence Factors


INTRODuCTION
Typhoid fever is an acute illness, potentially fatal systemic caused by Salmonella typhi and Salmonella paratyphi, pathogen only specific to humans. It has been shown that typhoid occurs in many developing countries, which it still remains an important cause of mortality and morbidity 1 . Typhoid fever is still a major health concern in the developing world as it is found endemically throughout Asia Africa, particularly in South Asia,East Asia and South America. In a broader sense, typhoid fever is categorized under enteric fever which is described as a systemic illness described by abdominal pain, fever and other symptoms as vomiting, nausea, anorexia and headache 2 . When enteric fever is caused by Salmonella typhi, it is known as typhoid fever and when it is due to Salmonella Paratyphi A, B, or C, it is called paratyphoid fever.
Salmonella typhi bacterium are an obligate parasite with no other known reservoir outside of humans. A 100,000 organisms of Salmonella typhi make up an infectious dose, and the disease is typically spread through feces and urine of infected people in contaminated food and water 2 . Typhoid is transmitted by the fecal-oral route, in most cases an asymptomatic carrier of S.typhi, or an individual who has recently recovered from the infection, continues to excrete large number of organisms in the stool and contaminates food or water, either through direct food handling, through transfer of bacteria by flies and other insects, or by contamination of poTable water 3 . Approximately 10% of patients recovering from typhoid fever excrete S.typhi in the stool for three months, and in the 2-3% became permanent carriers 4 .
Salmonella typhi contains an endotoxin typical of gram negative organisms, as well as the Vi antigen which is thought to increase virulence. Many of the S.typhi virulence factors, such as toxin genes invasion and adhesion are clustered in certain areas of the chromosome known as Salmonella pathogenicity islands 5 . Virulence factors present on the surface of Salmonella typhi such as lipopolysaccharides, capsule and fimbriae can affect the virulence of the bacterium, and also are key targets of the host immune system 6 . Endotoxin play an important role in the pathogenesis of Salmonella typhi infection during the bacteremia stages of typhoid fever that produced as a result of the acting endotoxin directly and indirectly through the release of endogenous pyrogens from leukocytes 7 .
Most Salmonella strains carry a large number of plasmids that contains virulence genes that are essential to trigger systemic disease, their involvement in the enteric stage of the infection is unknown 8 . Antimicrobials is important for the control and treatment of Salmonella spp. However, antimicrobial-and multidrug-resistant Salmonella strains have emerged, leading to treatment failure 9 . Many Strains of Salmonella typhi resistant to all three first-line drugs were in existence,with the development of quinolone resistance, third-generation cephalosporin's were used for treatment, but sporadic reports of resistance to them also followed 10,11 .
Multidrug resistance is an increasing threat to global health. There are > 14 million cases of typhoid fever every year and > 135,000 deaths, the disease is primarily controlled by antimicrobial treatment, but this is becoming increasingly difficult due to MDR 11 .Multidrugresistant typhoid fever is defined as typhoid fever caused by Salmonella typhi are resistant to all the three first-line recommended drugs for treatment, including cotrimoxazole ampicillin and chloramphenicol 12 . ESBL-producing Salmonella isolates resistant to several antibiotic agents,thirdgeneration cephalosporin's referred to extended spectrum cephalosporin's were effective against ampicillin hydrolyzing β-lactamases and gained widespread use in clinical use. They include ceftriaxone, ceftazidime, cefotaxime and others. Treatment of Salmonella typhi with thirdgeneration cephalosporin, azithromycin and fluoroquinolones drugs are the main treatment 13 .
Integron are genetic elements that contain the genetic determinant for the components of asite-specific recombination system that recognizes and captures mobile gene cassettes. An integron is a gene capture system found on plasmids, on chromosome and in transposon, platforms that incorporate exogenous open reading frames by site-specific recombination and convert them to functional genes by ensuring their correct expression 14 . Integron can be divided into two major group : resistance integrons and superintegrons Although not independently mobile, integrons are widespread versatile DNA elements and can be divided into two distinct subsets: the mobile integrons and the chromosomal integron 15 .
Integrons are classified based on sequence similarity, there are at least five classes of integrons with class I integrons being the most studied and characterized 16 . Class 1 integrons are the most common and widely distributed among Gram-negative bacteria and are associated with functional and nonfunctional transposons 17,18 . Class 1 integron is composed of the(3"CS) and including 2 (ORFs)open reading frames. QacE1 gene, it confers resist to qac quaternary ammonium compound; and Soul gene is confer resist to sulphonamides. There are responsible for the mobility of transposons and also found in Salmonella typhi 19 . PCR is rapid and sensitive assay used to detect microbial pathogens in clinical samples. PCR method had diagnostics value for detection of Salmonella among all the diagnostic tests [20][21][22] . The present study carried out to achieve the following objects: 1-Isolation and identification of Salmonella typhi collected from patients with typhoid fever from blood and stool by Vetik and PCR diagnosis . 2-Determenation of the virulence factors gelatin and protease among clinical isolates. 3-determination of MDR of the antimicrobial susceptibility in S.typhi isolates. 4-Detection of the Class I integron (intI) gene in S.typhi isolates.

MATERIAL AND METHODS Samples collection
These isolates of this study were collected from postgraduate laboratories(Prepared samples in vitro prepared diagnostics), they were a previous collected from 231 samples were involved 122 stool samples and 109 blood samples. Those period from July 2018 on March 2019 were attended to Al-Hakim General Hospital and Al-Sader Medical City Hospital in Al-Najaf province. It collected from patients were suffering from typhoid fever and swabbed onto Xylose-Lysine-Desoxycholate Agar (XLD) and MacConkey agar media were incubated overnight.  isolates were inoculated the Skim agar medium, it was incubated for 24-48hr,at 37˚C. the positive result it was observed when clear zone was appeared around the colony while the negative results weren't that show 28 . • Detection of gelatin production: The specific media for detection gelatinase liquefaction was inoculated with a single colony of overnight culture from nutrient medium and incubated at 37 C°, for 24 hr of incubation period by placing the tube in refrigerator for (30)min. A positive result was indicates if the media was liquid which refer to ability of bacteria to produced gelatinase and negative results was solidify because of no ability to produced it 29 .

Statistical Analysis
In the current study using the program of SPSS computing (version 24) for the data analysis.

Identification of Salmonella typhi isolates
During the current study period were collected 231 clinical samples from (122) blood samples and, (109) stool samples from patients with typhoid disease. Identification of Salmonella typhi isolates have been depending on initial identification the morphological, biochemical tests, and microscopically. Microscopically identification of S.typhi was appeared gram negative bacilli singly or in pairs, peritrichous flagellated, motile, nonspore forming bacteria, and non-capsulated. While the Morphological of S.typhi isolates were used the specific media such as XLD agar and MacConkey agar, appeared on culture medium once appeared the typical characteristics at 37°C after 18-24h, on blood agar S.typhi colonies appeared smooth, rounded,convex, and nonhemolytic and grey white color. But on MacConkey agar were appeared pale yellow ( non-lactose ferment ), 1-3mm in diameter and after 18-24 hour at 37°C. as well as, good growth of S.typhi colonies on XLD agar were appeared gray color with black center colonies because of it was ability to produce the H2S, as showed in [Fig. 1].
Biochemically tests results of S.typhi isolates were revealed on TSI, Sugars, Oxidase, Indole, Ureases and simmone citrate test. In  Fig. 1. Salmonella typhi colonies on XLD agar TSI slants test, the slant and butt turned into AKL/ACID red and yellow colour respectively indicating to non-fermentation of glucose on slant and production of acid with H2S in the bottom. Glucose, mannitol, L-arabinose and sorbitol sugars were fermented by all isolates. Other tests of S.typhi isolates were appeared negative result for oxidase test, indole production, urease production and citrate utilization as showed in Table [Table.1].
VITEK@2GNID Cards System results of S.typhi isolates, the identification were included many biochemical tests. The results revealed S.typhi were revealed with cards ID the ranging excellent of isolates (percentage% from 95 to 99%). The 59 Salmonella typhi colonies on MacConkey agar.

Detection virulence factors of Salmonella typhi
The ability of (59) S. typhi to produce many virulence factors. All isolates of S.typhi have been produce the non-hemolytic on blood medium and non-capsulated. Likewise S.typhi isolates have no ability to produce of Gelatinase on specific gelatin media as well as it has nonproduced of Proteinase on Skim agar medium.

Molecular Characteristics Salmonella typhi isolates Characteristics of Class I Integron intI Gene of S.typhi isolates
The present investigation indicates that the vicinity about a integron might have been altogether connected with 43(73 %) MDR isolates. Those distinguished genes encodes  Fig. 3].

DISCuSSION
The current study identified of Salmonella typhi isolates were depended on, microscopically, the morphology, biochemical tests and initial identification. Salmonella typhi colonies on XLD agar were appeared gray color with black center colonies. Results from previous studies suggested that iNTS disease and Typhoid fever were major causes of invasive bacterial febrile illness in the sampled locations, most commonly aff ecting child in both high and low population density 30 . Typhoid fever infection was caused by S.typhi and was transmitted through the oral route by the contaminated poultry, meat,, milk and eggs. Disease is most common in young child and elderly with peak incidence in summer and fall rainy season 31 .
Biochemically tests results of Salmonella typhi were observed negative result to simmons citrate, indole production, urease oxidase and H 2 Sproduction and TSI was ALK/ACID for all isolates. It was founded S. typhi isolates couldn't produce Urease enzyme. All biochemically result were agreed with [32][33][34] .
All results of virulence factors of Salmonella typhi showed that all isolates didn't produced genelatinase, hemolytics and proteinase enzymes. Thus, entero-chelin secretion in vivo could be a vital step in the stages of the infection cycle in which intestine -population, penetrate, the mucous layer covered the intestinal, translocate out of intestinal lumen through the epithelial cells, and finally step spread to organs and they may survive 35 .
M D R r e s u l t s i n t h i s s t u d y b y susceptibility of (18) antibiotics, including as high resistance of Salmonella typhi isolates 100% to Aztreonam(AZM 15 ), Nitrofurantion(F), Amoxicillin/ clavulanicacid (AMC 30 ), (PY 25 ), Clarithromycin (CLR), Cefoxitin(FOX 30 ), Penecillin (P 10 ), Cefotaxime (CTX 30 ), ampicillin (AMP), Meropenem (MEM), Tetracycline (TE 30 ). Antibiotics resistance in S.typhi is a threat to human public health 36 . In previously studies, MDRSalmonella bacteria were frequently observed among pigs,chickens and ducks. In addition, MDR Salmonella was serotype dependent 37 . As well as Salmonella enteritidis and Salmonella typhimurium were strongly association with MDR antibiotics phenotypes. MDR isolates could transfer to humans via animal or animal production and a great risk to public health 38 .
While, these isolates gave high resistance to other antimicrobial such as 91% to Impinem(IP), 88% Ampicillin(AM 10 ), 85%Amoxillin(AX), 81% Gentamicin (CN 10 ), 80% Chloramphenicol(C 30 ), 74% Cefpirome(CPR) and 68% Carbenicillin(CB).as mentioned in [Fig.2]. Other studies mentioned that the 41 Salmonella typhi screened for antibiotics susceptibility, 25 (61.0%) were resistant to five or more antibiotics. Also, 84% of bacteria were isolated from patients who didn't respond to initial treatments. The high percentage of MDRSalmonella typhi revealed was worrying and quite revealing 39 . MDR Salmonella typhi in Nigeria, Lagos. Periodic review of antimicrobial used in hospitals was essential as the efficacies of cotrimoxazole chloramphenicol and ampicillin were now doubtful. ofloxacin and Ciprofloxacin were effective antibiotics for treatment of typhoid infection, but with current trends of drug in Nigeria, resistance was likely to development. Therefore, provide an early warning signal for the prudent use of fluoroquinolone DRUGS to preserve their usefulness 39 .
Our results of molecular profiling of Class I Integron gene in S.typhi isolates, the present investigation indicates that the vicinity about a integron might have been altogether connected with 43/59 (73%) MDR isolates. Those distinguished genes encodes (integrase) from claiming class 1 integron. Integron were universal Around medication of Gram-negative clinical segregates. In addition, A percentage combinations for gene cassettes appear to be should need a nearby distribution, inasmuch as others appear will have a broad appropriation. Class 1 integron need aid spotted every now and again on plasmids that could make exchanged by conjugation. Those integron-carrying segregates would generally multidrug resistant, while those integron-lacking detaches would additional defenseless with drugs, Anyhow Likewise over other cases, not the sum safety determinant were spotted on Integrons 40 .

CONCLuSION
The conclusion of this study included into 1-Salmonella typhi isolates have been a big role in causing typhoid disease among clinical isolates. 2-Virulence factors of Salmonella typhi isolates have no ability to produce the gelatinase, proteinase and non-hemolytic among clinical isolates. 3-Genotypic detection by PCR revealed the presence of Class1 integron gene that have are relationships with MDR. 4-the current study revealed high resistance of S.typhi isolates have a reach to resistance 100% to Aztreonam (AZM 15 ), Nitrofurantion (F), Amoxicillin/ clavulanicacid (AMC 30