Identification and Characterization of Staphylococcus aureus 16s rRNA Gene Isolate from Environmental and Clinical Sources

One of the most well-known and widespread bacterial pathogens, Staphylococcus aureus, causes hundreds of thousands to millions of more harmful, invasive infections each year, in addition to an unusual number of uncomplicated skin infections (Mohamed et al., 2019). Rasigade et al., 2014). The global spread of infectious bacteria that are resistant to many first-line medications is highly concerning and might have negative effects on people’s health, the economy, and society (Al-Hamdani et al., 2024; Ventola, 2015). As a result, the World Health Organization (WHO) has designated Staphylococcus aureus as a high-priority antibiotic-resistant pathogen for the purpose of prioritizing the research and development of new antibiotics. S. aureus has an annual downfall of 20-50 cases/100,000 individuals and a mortality rate of 10-30% (Kumari et al., 2024); (Nirwan et al., 2022). This is more fatal than AIDS, TB, and viral hepatitis combined (Klevens, 2007; van Hal et al., 2012). The anterior nares and vestibules contain the most enormous reservoir (Sakr et al., 2018). Nasal colonization was previously assumed to be the cause of bacteremia in 30% of people (Tong et al., 2015). In numerous in vitro and in vivo infection models, it has been demonstrated how important these characteristics of virulence are during infection (Cheung et al., 2021; Lindsay, 2019). The synthesis of multiple distinct virulence factors that accomplish the same objective can be a waste of scarce resources and detrimental to survival since pathogenic S. aureus is typically prevalent in environments with restricted natural resources (Tam and Torres, 2019). However, should one of the virulence variables become inefficient, this redundancy can guarantee the bacteria’s survival. Alternately, the bacteria may have gained certain virulence characteristics that appear to be redundant during the history of their evolution and can more effectively adjust to different diseases or colonization areas. The wastewater that is discharged to wastewater treatment plants (WWTPs), particularly those from hospitals and areas where intensive livestock farming is conducted, can include these bacteria (Al-Jadir et al., 2022; Al-Musawy et al., 2021). Due to their exposure at work, there is a significant risk that WWTP employees will contract S. aureus and transmit antibiotic resistance in the environment, which poses a serious threat to public health (Ferguson et al., 2016; Kozajda and Jeżak, 2020). The strongly conserved 16s rRNA gene is the most widely used gene marker for genus and species identification, in addition to its taxonomic significance in bacteria and archaea (Callahan et al., 2017; Kim and Chun, 2014). The information found in the variable regions of 16S rRNA differs between species, which makes the detection particular (Dias et al., 2020).

In order to distinguish between Staphylococcus aureus isolates, this study will use 16S rRNA gene sequencing.

One hundred sixty-eight samples were obtained from various sewage treatment facilities in Baghdad utilizing sterile techniques, including submersion of gloved hands in 250 mL screw-top, sterilized wide-mouth vials. All samples were processed within 24 hours of collection or immediately following arrival at the laboratory.

Clinical samples from Centric Kid’s Technical Hospital The collection of specimens was accomplished by rotating a sterile swab in (Nasal, hands) and then placed in the transport media and collected in an ice box until transported to the laboratory between November (2021) and January (2022). (Thapaliya et al., 2017) and then Identification of S. aureus by VITEK 2 Compact system GP. All experiments were conducted at the laboratories of the Ministry of Science and Technology in Baghdad-Iraq.

The susceptibility to antibiotics was examined in all verified S. aureus. The Kirby-Bauer method was applied in accordance with the Clinical and Laboratory Standards Institute’s recommendations, involving standard disk diffusion on Mueller-Hinton agar incubated at 37°C for 24 hours (James et al., 2024), Cefoxitin, Erythromycin, Amikacin, Azithromycin, Chloramphenicol, Ciprofloxacin, Clindamycin, Penicillin, Gentamicin, Linezolid, Nitrofurantoin, Rifampin, Tetracycline, Tigecycline, Tobramycin and Trimethoprim/Sulphathiazole were among the 16 antimicrobial disks used to test the susceptibility of S. aureus isolates the National Committee Clinical for Laboratory Standards standard recommendations for recording experiment findings were followed.

Positive samples of Staphylococcus species based on morphological and biochemical assays were subsequently handled for  molecular  analysis using a PCR-based test; genomic DNA was obtained using the Wizard Genomic DNA construction (Promega, Madison, WI). Following the extraction of genomic DNA from bacterial colonies, the PCR  method-which is far more sensitive than the most widely used microbial culture and stainingtechniques-is used for recognizing the nuc gene and 16S rRNA gene techniques-is used for recognizing the nuc gene and 16S rRNA gene.

Then, PCR was performed utilizing universal 16SrRNA oligonucleotide primers: R- 5-AGA CCC GGG AAC GTA TTC AC -3' and F-5- GTG CCA GCA GCC GCG GTA A -3' and also primer R- 5 -CGT AAA TGC ACT TGC TTC AAG -3' and F- 5' -TCA GCA AAT GCA TCA CAA ACA G -3 for nuc gene (Haque et al., 2017). In the finished volume of 25 ml, the reaction combination comprises 1 ml of bacterial DNA, 9.5 ml of nuclease-free and 1 ml of each primer. The PCR master mix had this combination added to it in line with the production handbook (GoTaq, Promega, USA).Use a thermal cycler (Lab Net, USA) to do the following procedure: A thermocycler was used to complete 30 cycles, with each cycle consisting of three steps: denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min and final extension at 72°C for 10 min.

Conventional electrophoresis was used to assess amplified products and a Gel documentation image analysis system was used to identify bands (Bio-Rad, USA). The universal primer-amped products had sizes of 1000 bp, which suggests that the bands were made of the 16S rRNA gene.

Utilizing an automated DNA sequencer ABI3730XL, the PCR product was transferred to Macrogen Corporation-Korea for Sanger sequencing of the 16S rRNA gene. After receiving the findings via email, we analyzed them using specialist tools.

First, traditional techniques were used to identify clinical isolates. All isolated strains of Gram-positive cocci were sub-cultivated on nutrient agar (HiMedia Laboratories Pvt.Ltd), and biochemical tests were then investigated overnight at 37°C before being promptly cultured on both Manitol salt agar and blood agar plates by direct streaking procedures (Gajdács and Urbán, 2019).

One hundred clinical samples, consisting of 45 nasal swabs and 55 hand swabs, were taken from healthcare workers and patients (male and female) of various ages and conditions, including chronic otitis media and wounds.

A 250 mL autoclaved screw-cap jar containing 50 mL of Baird Parker broth was supplemented with water samples and incubated at 37°C for 18 to 24 hours. Following a 24-hour incubation period, 50 ul of broth was placed onto Baird Parker agar (BPA) with egg yolk tellurite enrichment (BD Difco TM, Becton, Dickinson and Company, Sparks, MD). Following 24-48 hours of culture at 37°C, the inoculum was examined for the occurrence of bacterial development (Thapaliya et al., 2017). S. aureus indicated (black colonies with clear shadows on Baird Parker Aagar). S. aureus was confirmed by the catalase examination, the sliding coagulase test, and the preservation of S. aureus isolates at 80°C.

S. aureus was confirmed using the mannitol fermentation method, colony morphology, Gram stain, Catalase, coagulase, and DNase tests. The isolates that were determined to be S. aureus were then used in the investigation. Following that, species were determined to use the VITEK 2 (BioMérieux, France) Automation Technology with probable S. aureus isolates. 60 S. aureus from clinical and water samples were found using conventional methods and automated system results, (Fig 1).



For clinical isolates 100% (40/40) of isolates were resistant to penicillin, 97% (39/40) to erythromycin, 65% (26/40) to tetracyclin, 45% (16/40) to Cefoxitin and Methprim, 55% (22/40) to Azithromycin, 62.5% (25/40) to tobramycin, 40% (16/40) to Clindamycin, 32.5% (13/40) to Rifampin, 25% (10/40) to Linezolidin, 22.5% (9/40) to Gentamicin, 15% (6/40) to Amikacin, 7.5% (3/40) to Ciprofloxacin and Nitrofuratoin, 5% (2/40) to Chloramphenicol, and 2.5% (1/40) to Tigecycline. (Fig 3). For environmental isolates, 100% (20/20) of isolates were resistant to penicillin, 65 % (13/20) to erythromycin, 45% (9/20) to Rifampin, Azithromycin and Clidamycin, 40% (8/20) to Cefoxitin, 30% (6/20) to Linezolidin, 20% (4/20) to Ciprofloxacin and Nitrofuratoin, 15% (3/20) to tetracycline and Tobramycin, 10% (2/20) of isolates were resistant to Tigecycline, Chloramphenicol, and Gentamicin; all isolates were sensitivity to Ciprofloxacin. (Fig 2).





In this study, drug resistance to three or more antimicrobials was defined as multiple drug resistance. Relatively fewer environmental isolates than clinical isolates were present. it was revealed that isolates had 80% resistance to at least three antibiotics and 100% at least resistance to one antibiotic, as illustrated in (Table 1).



The nuc gene was determined by agarose gel electrophoresis and imaged under an ultraviolet (UV) trans-illuminator, as shown in (Fig 4). The amplification indicated a product of 300bp. We used PCR primers targeted to this gene, as explained by McClure et al., (2017). Each isolate was also confirmed at molecular levels.      

           

For this aim, the 16SrRNA gene, one of the S. aureus-specific genes, was detected using a single-target PCR assay. Results from gel electrophoresis revealed that every single one of the 60 S. aureus isolates contained the 16SrRNA gene (Fig 5). The 16SrRNA gene was utilized to compare with the type strains identified by 16SrRNA gene sequencing at GenBank using Geneious software for the purpose of identification. The identified isolates’ nitrogen bases matched those of the closest isolates. novel local isolates were obtained from clinical and environmental samples, and two isolates were registered in GenBank. Distance Tree Using the Blast Tool in Geneious Software (Fig 6).





From clinical sources, a new local strain of S. aureus was identified. (MIM2022) were genetically homologous with a new local strain of S. aureus, which was isolated from environmental sources (MIM2023) with a percentage of homology 100%, where the results show that the sample isolates belonged to a monophyletic group with other S. aureus strains, with the closest genetic distance between the two isolates being 0.00. Clinical isolate (MIM2022) has the greatest relative distance to environmental isolate (MIM2023) based on genetic distance, suggesting that   clinical isolates of S. aureus are likely to be the same strain as environmental isolates.

The aim of the study is to analyze the evolutionary relationships between Staphylococcus aureus strains isolated from ecological niches and those from clinical sources. This study attempts to show a possible connection between environmental reservoirs and clinical strains by comparing the genetic features of these isolates, especially the unique local strains MIM2023 (environmental source) and MIM2022 (clinical source). This link may shed light on the spread routes, evolutionary dynamics, and the involvement of environmental strains in clinical illnesses.                         

Clinical and environmental isolates showed high resistance to Penicillin, clindamycin, erythromycin, and azithromycin, and approximately all isolates were multi-drug resistant; these results agreed with the study of Akya et al., (2020). Additionally, numerous studies have demonstrated that antibiotic-resistance genes are frequently found in wastewater treatment facilities (Bergeron et al., 2016). (Everage et al., 2014). (Naquin et al., 2015). One of the main elements contributing to the spread of antimicrobial resistance in the environment is horizontal gene transfer (Kristiansson et al., 2011). Horizontal gene transfer is regarded as a non-random approach to genome innovation because it heavily depends on internal and external variables (D’Costa et al., 2011). According to (Kateete et al., 2010 and Gumaa et al., 2021); the identification of S. aureus using PCR amplification of the nuc gene and 16S rRNA is considered a gold standard method. The novel local strains (MIM2022) and (MIM2023) support a previously published study from Sudan that discovered a genetic similarity between various S. aureus strains around the world and isolated strains (Ali et al., 2019). 16S rRNA analysis is regarded as an effective discrimination method for differentiating unrelated isolates (Al-Obaidi et al., 2018). A host is anticipated to switch from interacting with humans to environmental factors under the strain of adjusting after a protracted engagement with them. The environment and people being in close proximity can encourage host-switching incidents (Haag et al., 2019). The risk to public health posed by some pathogens’ capacity to spread from one niche to another is significant (Richardson et al., 2018). According to the genetic profile, there is a clear genetic link between environmental and clinical S. aureus isolates, and the direction of S. aureus transmission in this study is most likely from patients to the environment. The isolate from clinical sources was genetically closely linked to one S. aureus isolate from environmental sources.

Prokaryotes, including S. aureus, use the 16S rRNA gene to identify species and build phylogenetic connections because of its extremely conservative sequencing and minimal discrimination against closely related species (Vìtrovsky and Baldrian, 2013). When identified using visual and biochemical characteristics, organisms that resemble S. aureus present numerous challenges for species identification. However, the sequencing of the 16S rRNA region is a viable tool for species identification.

The study of 16S rRNA gene sequences to ascertain bacterial phylogeny and taxonomy has been the most popular housekeeping genetic marker for a number of reasons. The function of the 16S rRNA gene has not modified across time, suggesting that random sequence changes are a more precise indicator of time. These factors include (i) its existence in practically all bacteria and (ii) the fact that it frequently exists as a multi-gene family or operons (evolution).

All clinical isolates were multidrug resistant. Clinical isolates were highly resistant toward penicillin and Erythromycin and low resistant to Ciprofloxacin, Nitrofurantoin, Chloramphenicol, and Tigecycline, while all the environmental isolates were sensitive to Ciprofloxacin. The study got a novel local strain of S. aureus that was isolated from clinical sources (MIM2022) and was genetically 100% homologous with a novel local strain of S. aureus that was isolated from environmental sources (MIM2023), which demonstrates S. aureus isolates’ capacity to jump from one niche to another which means poses a significant hazard to public health.

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided but do not accept any liability for any  content direct or indirect losses resulting from the use of
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.