Prevalence of Biofilm Formation in Salmonella typhi Isolated from Enteritis Patients in Al-Najaf-Iraq

Several types of bacteria enhance their survival by attaching to non-living surfaces or tissues, presenting them as multicellular communities covered by a protective extracellular matrix called biofilm. There has been a clear interest in assessing the relationship between antibiotic resistance phenotype and biofilm production. The aim of this paper was to present additional experimental results on this topic and to test the ability of Salmonella typhi isolates to biofilm formation using in vitro methods in the context of their antibiotic resistance. In the result of the study, 28 isolates of S. typhi were included. All 28 clinical specimens 100% showed strong biofilm formation and all clinical specimens showed the presence of pml gene by clear bands in gel electrophoresis. The results of the antibiotic sensitivity test of S. typhi isolates by disk diffusion method against 18 types of commonly used antibiotics showed a large variation in their rates of antibiotic resistance, as the highest percentage of resistance to the antibiotic to S . typhi resistance to the most common antibiotics used in treatment. The highest rate of resistance was seen with


INTRODUCTION
Salmonella is a serious food-borne illness that affects people all over the world and causes serious morbidity, mortality, and financial loss (Siddiky et al., 2021). A leading cause of foodborne illness around the globe, Salmonella is estimated to have a 3.7-billion-dollar annual economic cost burden. One of the most prevalent foodborne organisms, Salmonella, is responsible for outbreaks of foodborne illness around the world (Shamma et al., 2016). The most important genus in the Enterobacteriaceae family is Salmonella. The Salmonella genus is a group of Gram-negative, facultative anaerobes that may live on a wide range of hosts, including humans, animals, poultry, pigs, and the environment (Appling et al., 2018).
Salmonella species may be pathogenic to domestic or wild humans and animals not producing spores, mostly motile, rod-shaped and flagellated (peritrichous flagella-all around the cell body) with cell lengths ranging from 2.0 to 5.0 m and diameters between 0.7 and 1.5 m (Abdul-Hassan et al., 2020). The major ways that these bacteria are disseminated are through tainted food and water People are more likely to get typhoid fever in heavily populated places When someone has typhoid fever, these bacteria live in their blood and intestinal tract. They enter the body through contaminated food and water, reproduce, and spread throughout the bloodstream (Rana et al., 2022). According to O and H antigens, Kauffman-White proposed a classification based on the one serotype-one species rule. Through this classification, the Salmonella genus is divided into more than 2500 species. These days, there are two species of Salmonella: enterica and bongori. S. enterica has six subspecies: enterica, salamae, arizonae, diarizonae, houtenae, and indica, based on an examination of the genome and biochemical traits. Based on antigenic traits, the subspecies are split into serogroups (O antigen) and serovars (H antigen) (Popa and Popa, 2021).
The two most prevalent serotypes of Salmonella, Typhimurium and S. enterica serovar Enteritidis, can infect a variety of hosts, including people and birds. The primary cause of acute gastroenteritis in humans is S. Enteritidis, while S. Typhimurium produces a systemic sickness similar to typhoid (Wang et al., 2020). Thus, the main aim of this study was to present results that show the association between the ability of S. typhi to produce biofilms and antibiotic resistance phenotypically and genotypically using in vitro experiments.

MATERIALS AND METHODS
The study was conducted at Bacteriology and Molecular Laboratories in Biology Department/Sciences Faculty/Kufa University/Iraq.

Isolation:
Blood samples were collected from patients who attended consultation clinics in Al-Najaf hospitals who suffer from enteric fever. Each blood specimen (2-3 ml) was added to 10-15 ml of brain heart infusion (BHI) broth. The bottle of BHI broth was incubated at 37°C for 1-2 days. Each BHI was subcultured on SS agar and then incubated at 37°C for 24 hr to give the microorganism more chance to grow.

Identification of S. typhi:
The identification of S. typhi from blood samples was done with the Gram stain, IMViC tests and then was confirmed by using Vitek-2 compact system (Al Naiemi et al., 2008;Christner et al., 2010).

Antibiotic Profile of S. typhi Isolates:
The disk diffusion method, recommended by the Clinical and Laboratory Standards was used to determine the susceptibility of all S. typhi isolates to different antibiotics (Bioanalyse Company, Turkey).
Overnight-grown cultures of each isolate in brain heart infusion (BHI) broth were prepared. The turbidity of the broth was checked to 0.5 McFarland standard tube. Each isolate was spread on the surface of Mueller-Hinton (MH) agar with a sterile cotton swab and after 15 minutes, antibiotic discs were placed on plates (the distance between discs was 15 mm at least), then incubated the plates at 37°C for 18-20 hrs. The zone diameter around each disc was measured and compared with CLSI guidelines to interpret the results.

DNA Extraction:
Using the boiling technique, genomic DNA was successfully recovered from S. typhi isolates. The RNA/DNA spectrophotometer (Biodrop) instrument directly evaluated the concentration and purity of extracted DNA; extracted DNA purity ranged between (1.8-2). Gel electrophoresis was used to confirm and analyze the extracted DNA.

Detection of biofilm formation:
Semi-quantitative measurements of biofilm formation were determined using tissue culture-treated, 96-well polystyrene plates (microtiter plates MTP), based on the methods of Lizcano et al., (2010).

Primer type of pml:
Primer pml Sequence was 5՛-3՛ F: ACTCAGGCTTCCCGTAACGC, R: GGCTAGTATTGTCCTTATCGG in the Product size 563 bp (Abbas et al., 2015) The condition of the primer was included: Initial Denaturation 94\2m, followed 35 cycles of (Denaturation 95\30 sec., Annealing 52\30 sec and Extension 72 \30 sec), finally the Final Extension was 72\7 min. The reaction mixture was held at 4°C until use while the final extension step took place at 72°C for about 10 minutes (Abbas et al., 2015). Each and every PCR amplification was performed using a Verity Thermal Cycler (Agilent, UK). Then, 1% agarose gel electrophoresis was used to analyze all of the PCR products, and they were all stained with red ethidium bromide dye. Finally, the gel documentation system was used to identify the electrophoresis results.

Statistical Analysis
Experimental data were presented in terms of observed numbers and percentage frequencies; SPSS (Statistical Package for Social Science) program version was used. Regarding other data were analyzed Correlation.

RESULTS AND DISCUSSION 1. Isolation and Identification of S. typhi:
During the collection period, a sum of 100 samples was gathered from the patients with suspected enteric fever from the main hospitals in Al-Najaf Al-Ashraf. After observing the culture and morphological characteristics of bacterial isolates and performing the classical IMViC tests, then confirmed by the Vitek-2 compact system. A total of 28 (28%) isolates had been identified as S. typhi.
The Gram-negative bacilli bacteria S. typhi can be found in food and water. Despite the fact that the disease has been linked to a wide range of food sources, poultry, in particular, has been identified as the sole major cause of salmonellosis in humans. In addition to harming the poultry business, avian salmonellosis can also infect people and is brought on by eating tainted chicken products like meat and eggs (Oscar, 2021). One of the most dangerous zoonotic and foodborne infections that endanger people's overall health and well-being is Salmonella. Despite this, Salmonella continues to be a significant human disease and a significant global public health concern Salmonellosis, often known as food poisoning, is an illness brought on by Salmonella and typically presents as moderate diarrhea (Balasubramanian et al., 2019).

Antimicrobial Susceptibility of S. typhi:
All of the 28 S. typhi isolates were evaluated for susceptibility to 18 different antibiotics A summary of susceptibility rates (according to (CLSI, 2021) guidelines as resistant, intermediate resistant, and susceptible) for all antibiotics against S. typhi is given in the following Table (1). Patients with shock and enteritis should be treated with comprehensive antibiotic coverage for gram-negative, grampositive, and gas-producing bacteria at first, and then deescalate when cultures and sensitivities improve. Patients with positive results of cultures for S. typhi were studied by von Graevenitz et al., (2010) who discovered that both species were sensitive to tetracycline, colistin, piperacillin/ tazobactam, higher generation cephalosporins, aminoglycosides, carbapenems, and polymyxin. However, research has revealed that multidrug-resistant carbapenem species exist (Kim et al., 2012).
Aggressive source control, in addition to antibiotics, was discovered to be important for successful therapy in these individuals (Tram-Anh Duong et al., 2019).

Detection of Biofilm Formation:
The ability of all isolates of S. typhi 28 to form biofilm was detected by using microtiter plates (MTP). Biofilms were measured by quantifying the absorbance of stained biofilms at 630 nm with a microtiter plate reader. The results in this study were indicated according to Salwa et al., (2011) in which 28 (100%) of S. typhi e isolates appeared high biofilm formation (strong positive adherence) (Fig. 1). The MTP method is a precise and reproducible method used to screen and determine biofilm production Therefore, we used this method in our study. Bacteria utilize an assortment of methods to continue in their specific niche in the host (Munǒz-Elías et al., 2002). Microorganisms form biofilms that are present in the environment, whether on living animals or inanimate objects, the bacteria attach to these surfaces, such as aquatic systems and soil, and have been recorded in the human literature developing on medical equipment, within surgical implants, middle ear, lungs, external ear, heart valves, and tooth enamel (Singh et al., 2013). Biofilm production in G-ve bacteria happens when bacterial cells first swim along a surface, utilizing flagellarmediated motility until their attachment is initially reversible at a specific site (O'Toole, 2000). Correlation between Biofilm and Antibiotic Resistance in S. typhi: Figure (2) revealed a positive significant moderate correlation (0.569**) in S. typhi isolates between the number of antibiotic resistance and the mean of biofilm formation. To date, it has been demonstrated that some correlations exist between antibiotic resistance and the biofilm-forming ability of S. typhi isolates. There is an association between biofilm production with persistent infection and antibiotic failure. Gilbert et al., (2002) reported that biofilm producers were to be 10-1000 times less susceptible towards antibiotics than are the equivalent cells growing planktonically. Curtin et al., (2003) revealed that biofilm hampered the penetration of antimicrobials and the concentrations required to eradicate biofilm-producing bacteria are higher than those required to eradicate strains that did not produce biofilm. Also, Keren et al., (2004) explained this issue as bacterial populations produce persister cells that neither grow nor die in the presence of antibiotics and that persisters are largely responsible for high levels of biofilm tolerance to antimicrobials.
So, the process of biofilm formation is particularly relevant for clinicians because biofilm-associated microorganisms exhibit dramatically decreased susceptibility to antimicrobial agents (de-Silva et al., 2002). There are many virulence factors in Enterobacteriacae strains associated with its pathogenicity. Among these factors is the capacity of Enterobacteriacae strains to form a biofilm that protects it from the host immune response as well as from antibiotics (Bandeira et al., 2014;Chung and Rimal, 2016).

Molecular Study of Biofilm Formation by the Detection of pml Gene:
The molecular detection of pm1 gene by using a specific primer for S. typhi isolates revealed positive amplification for 100% as shown in Figure (3).
The results of the study showed that 100% of the isolates belong to the PM1 bacteria strain, which is characterized by its distinctive phenotypic characteristics and its possession of all virulence factors that enable it to cause infection (Abbas et al., 2015).
Al-Dahhan, (2017) confirmed that the PM1 strain is characterized by the homogeneity of its colonies on the solid media and the lack of clumping growth in the liquid culture media (Brooks et al., 2007), and they also indicated Fusco et al., (2017) the high ability of the PM1 isolate to adhesion in the epithelial cells. And the formation of the biofilm on the biological surfaces after 2 hours of incubation, up to the full phenotypic characteristics of the biofilm after 6 hours of incubation through its high ability to swarm and thus using this strain to create treatments targeting specific pathways of virulence factors.
According to the study's findings, all of the isolates are members of the PM1 bacteria strain, which is distinguished by its unique phenotypic traits and by its possession of all the virulence factors necessary to spread infection. The study's findings revealed that 100% of isolates carried the mrpA and pm1 genes, indicating the prevalence of the PM1 S. typhi strain enteritis. According to Abbas et al., (2015), the gene was present in 47% of all S. typhi bacterial isolates, the PM1 isolate has a high capacity for adhesion in epithelial cells and is distinguished by the homogeneity of its colonies on solid media and the absence of clumping growth in liquid culture media (Brooks et al., 2007).

Conclusion:
This study demonstrated that Salmonella typhi was one of the important pathogens causing enteric fever, enterocolitis, and other types of invading Salmonella infections, such as septicemia, it accounted for 19.5% of bacterial isolates. It was highly resistant to many common antibiotics, particularly ß-lactam. All bacterial isolates have the ability to produce strong biofilm. The study found a positive correlation between study interesting genes, biofilm formation and antibiotics in S. typhi.