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Prevalence and Distribution of Staphylococcus aureus Virulence Enzymes Isolated from Clinical and Environmental Sources

_ ABSTRACT The goal of this study was to isolate and identify Staphylococcus aureus from clinical and environmental sources. This study also looked at the prevalence and distribution of different virulence enzymes among its isolates. For this objective, 65 samples were collected and dispersed among 50 distinct clinical samples and 15 environmental (soil, water, and air) samples. The results revealed that 20 bacterial isolates were S. aureus . The isolation rate was 30.76% from all samples, with 13 isolates 20% from a clinical source and 7 isolates 10.76% from an environmental source. The phenotypic detection of virulence enzymes revealed that all isolates, including environmental isolates, produced virulence enzymes (between two and six enzymes), indicating the clinical importance. The isolates produced virulence enzymes at different rates. The results revealed a clear rise in the rate of production of the enzymes: lecithinase and lipase producing at a rate of 95%, protease at 90%, urease at 80%, hemolysin at 60%, and beta-lactamase at 55%.


INTRODUCTION
Staphylococcus aureus is a common, ubiquitous bacterium that colonises the skin, nose, and throat. Human ailments caused by it include food poisoning, pharyngitis, meningitis, pneumonia, bacteremia, wounds, and post-operative infections [1]. Furthermore, S. aureus can also be detected in the atmosphere, soil, and water [2]. S. aureus clinical is owing to the presence of a collection of virulence factors, some of which are naturally present in bacteria and others that are released by them [3]. Extracellular enzymes are secreted by these microorganisms. The most important enzyme is beta-lactamase, which is responsible for betalactam antigen resistance. It is encoded by the blaZ gene, which is found on a chromosome or plasmid [4]. S. aureus also secretes the hemolysin enzyme, which breaks down red blood cells, gives iron to germs, and interferes with host cell membranes. Lecithinase is a protein that degrades phosphorylated lipids, causing tissue deterioration and death. Lipase penetrates fatty skin tissues and subcutaneous fat to break it down [5]. S. aureus also produces proteases that destroy host proteins such as human neutrophil protein, antimicrobial platelet protein, and complement protein [6].
Because virulence enzymes are important in the clinical of S. aureus, this study was carried out to isolate and diagnose bacteria from clinical and environmental sources, as well as to investigate the prevalence and distribution of various virulence enzymes among its isolates.

MATERIALS AND METHODS
In this study, 65 samples were collected from different sources in sterilized conditions. 50 from clinical sources (sputum, urine, skin, and blood swabs) and 50 from ambient sources (15 samples that included soil, water and air samples) [7]. The samples were cultured on Mannitol Salt Agar (Neogen / UK) plates for 24 hours at 37°C.
Bacterial isolates emerged on mannitol salt agar as yellow fermented colonies of mannitol sugar. The streak approach was used to select and reseed bacteria on the purification medium [8].

Microscopic Examination
Swabs were obtained from forming colonies and stained with Gram stain on a glass slide. To assess the type and color of cells, the bacteria were viewed under a microscope using an oil lens.

Biochemical Test
The tests were conducted according to the conventional method mentioned in [9]. In this test include catalase test, oxidase test, and coagulase test which was conducted in two ways: Slide Coagulase Test and Tube Coagulase Test.

Diagnostics with the Vitek-2 System
The isolates were diagnosed with the Vitek-2 device in Al-Mansour laboratory in Mosul city. This new instrument can easy detect the specific bacteria in any type of isolates.

Phenotypic
Investigation of Virulence Enzymes in S. aureus

Detection beta-lactamase in vitro
100 μl of penicillin G solution at a concentration of 6 g was poured in test tubes and added to the bacterial suspension before incubating. After mixing, the tubes were kept in the incubation chamber for an hour at 37ºC for 18-24 hrs. The solution was then dropped into each tube, followed by a drop of iodine solution, and the tubes were shaken vigorously and allowed for 5 minutes at the laboratory temperature before the results were read [10].

Detection beta-lactamase enzymes using nitrosphine disk
One of the reagents on bacterial growth is the nitrosphine disk. After being moistened with a drop of sterile physiological saline, the nitrosphine disk becomes yellow. The juvenile colony was then moved and placed on the nitrosphene disk. The colour of the disc changed to pink, suggesting the synthesis of betalactamase [11].

Detection urease
A young developing colony of S. aureus was injected into the prepared blood agar medium (Neogen/UK), and the plates were incubated at 37°C for 24 hours. The emergence of a change in the color of the medium from yellow to pink is a favorable result and a sign of urease enzyme secretion [12].

Detection of hemolysin
The prepared blood agar medium (Neogen/UK) was injected with a young developing colony of S. aureus, and the plates were incubated at 37°C for 24 hours. The presence of translucent patches around the colonies implies that the bacteria can examine blood [13].

Detection of lecithinase & lipase
Egg Yolk Agar (Neogen/UK) was inoculated with a young developing colony of S. aureus and incubated for 24 hours at 37°C. The translucent area around the growth after incubation signifies a positive result for lecithinase synthesis. To examine the lipase enzyme, bacteria were added to egg agar and the plate was incubated for 24 hours. The plate was immersed in a 20% saturated copper sulfate CuSO4 solution for 20 minutes. The surplus solution was removed, and the plate was dried in the incubator for 30 minutes. Changing the hue of the colonies to a greenish blue yields a good result, indicating that the enzyme is being secreted [14].

Detection of protease
Skim Milk Agar (Microxpress / Spain) medium was inoculated with a young developing colony of the bacteria to be studied and then incubated for 24 hours at 37 °C. The emergence of a translucent region around the bacterial colonies after the incubation period reflects a positive result [14].

RESULTS AND DISCUSSION
65 samples were gathered from various clinical and environmental sources for this study. In the initial isolation process, mannitol medium was based on salt agar and as an electoral source for S. aureus. Mannitol sugar, salt, and phenol red index are the key components of the medium. Because the bacteria ferment the mannitol sugar, the colonies appear golden yellow in it.
Because S. aureus is resistant to salinity and multiplies in the presence of sodium chloride, the colour of the red phenol index was modified from pink to yellow as a signal of low PH [15].
Gram stain microscopy was used as one of the diagnostic procedures because its cells showed in a spherical shape organized as grapes cluster under a light microscope, Fig. 1A. S. aureus was also replanted in salt and mannitol agar to observe the aforementioned agronomic features, Fig. 1B. Some biochemical tests were also performed, and all of the S. aureus isolates tested positive for the catalase that breaks down hydrogen peroxide H 2 O 2 into oxygen gas O 2 and H 2 O, which shows as gas bubbles, Fig. 2A [10]. A 100% positive result appeared for the coagulase enzyme, which is one of the most important diagnostic tests, Fig. 2B [16]. The Vitek-2 was used to confirm the diagnosis of all isolates under examination, Fig. 3.
Based on the aforementioned diagnostic processes, 20 isolates of S. aureus were obtained, with an isolation rate of 30.76% of all samples analyzed, Table 1. The isolates were divided into 13 isolates 20% from a clinical source and 7 isolates 10.76% from an environmental source. Isolates were recovered from all clinical tissues examined. The same is true for many environmental samples such as soil, water, and air, Table 2.  Toxic shock syndrome is caused by the release of toxins into the body [17,18]. Other compounds, in addition to enzymes, toxins, and surface proteins, can assist bacteria invade the body's tissues [19]. Toxins A, B, and D are the most deadly, causing food poisoning [20]. Furthermore, S. aureus has the ability to spread through water, causing health issues in humans as well as economically significant aquatic species like fish [21].

Prevalence and Distribution of Virulence Enzymes
S. aureus has a set of virulence factors that are either contained in the cell structure or produced as enzymes, allowing the bacterium to avoid the immune system and infiltrate human tissues [22]. According to the data indicated in Table 3, all of the isolates investigated with diverse sources of isolation produced virulence enzymes (2-6), including the environmental isolates. This confirms the relevance of this bacteria from a clinical standpoint, regardless of the source of its isolation [23].
The rate of creation of each type of virulence enzyme varied depending on the isolate, however the current investigation found a clear rise in the rate of production of these enzymes. As indicated in Fig. 4, the isolates produced the most lecithinase and lipase 95%, whereas protease was 90%, urease 80%, hemolysin 60%, and beta-lactamase 55%.
The results showed that the isolates produced 95% of the lecithinase as shown in Fig. 5. S. aureus secretes an enzyme called lecithinase, which disturbs host tissues by dissolving phospholipids in eukaryotic cell membranes, allowing germs to proliferate and access nutrition [24]. The current study's findings also revealed that the rate of production lipase was high 95% as shown in Fig. 6. The lipase enzyme breaks down fats and allows them to linger in fatty tissues, damaging the host tissues. The enzyme also plays a crucial role in the production of biofilms and cysts [25].

Fig. 4. Production rates of virulence enzymes of S. aureus isolates
The activity of the protease production was studied, as the rate of its production approached 90% as shown in Fig. 7. It plays a significant role in bacterial clinical because it breaks the peptide bonds that bind amino acids, destroying proteins and regulating the immune response. It causing necrosis and damage to host tissues. In addition to obtaining nutrients essential for bacterial development from hydrolyzed proteins derived from host tissues such as collagen [26].
The recent investigation found that 80 percent of the isolates producing urease, Fig. 8. The urease enzyme is important in the clinical of S. aureus because it decomposes urea into carbon dioxide and ammonia during the colonization stage. S. aureus is at the forefront of pathogens that cause urinary tract infections and kidney stones, hence the urease enzyme protects bacteria from excessive urea concentrations [27].
The results revealed that the concentration of hemolysin enzyme was 60%, Fig. 9. One of the key virulence factors in S. aureus is the development of hemolysin enzyme, which destroys erythrocytes by dissolving the cell membrane enclosing the globule, resulting in anemia and a weakening of the immune response. This also aids in the provision of iron, which is required for bacterial metabolic processes. Hemolysin also damages leukocytes and macrophages, as well as attacking sphingomyelin in animal cell membranes, allowing bacteria to infiltrate and spread within the host [28].
Two approaches were used to investigate betalactamase synthesis. The in-tube approach was utilized, with penicillin G as the foundation of the examination and nitrosphene disk as illustrated in the Fig. 10. According to the current study's findings, the percentage of isolates producing beta-lactamase by both methods was 55%. S. aureus has numerous antibiotic resistance mechanisms, particularly against penicillin and cephalosporin. One of the most prevalent is the creation of beta-lactamase, which breaks the beta-lactam ring in antibiotic manufacture, resulting in resistance [29].
The findings of our current study on the production rates of virulence enzymes investigated by S. aureus accord with the findings of many other studies, including [5,30, 31 and 32]. On the other hand, the production rates of virulence enzymes in the current study differed with the results of other studies [14,24, and 33]. This disparity in results could be attributed to the different isolates, their sources of isolation, the kind of assays used, and the conditions under which these tests were performed.

CONCLUSIONS
The phenotypic investigation of virulence enzymes revealed that all investigated isolates from various sources of isolation, including environmental isolates, produced virulence enzymes. This indicates the relevance of S. aureus in clinical, independent of the source of isolation. The isolates' production rates of each type of virulence enzyme varied, and the results demonstrated a definite increase in the production rate of these enzymes.