Detection the Spa type of methicillin-resistant Staphylococcus aureus isolated from local Basturma in Mosul city, Iraq

1. Isolation and identification of MRSA.
2. Detection of same gene (nuc, mecA, and spa genes).
3. Make the phylogenetic analysis tree to find the relationship between S. aureus isolates.

Introduction

Spa typing is one of the most popular MRSA typing techniques. It is used to characterize the distribution and evolution of MRSA clones and to detect MRSA transmission, which depends on the sequencing of the spa gene (1,2). In addition to ensuring consistent and updated nomenclature, the 2003 installation of the Ridom StaphTyper software made it possible for researchers, groups of disease control, and doctors to exchange and liken information (3). Spa sequence typing was recently used for S. aureus molecular typing (4). The polymorphism in the X region of the Staphylococcus Protein A (spa) gene is determined by sequencing (5). S. aureus is a pathogen responsible for many infectious disorders, including skin and soft tissue infections, toxin-mediated illnesses, including pneumonia and bacteremia, are caused by the bacteria S. aureus up to 50% of people on the planet may also have S. aureus in their natural flora (6). Penicillin-binding protein 2a (PBP2a), translated by the mecA gene, is the mechanism through which S. aureus develops methicillin resistance (7,8). Quickly later, methicillin was prescribed as the medicament used for treating S. aureus, which is resistant to penicillin; MRSA was first identified in 1961 (9). Since then, MRSA cases have been documented in hospitals and community settings throughout the world, beginning in the early 1990s. When testing for staphylococci, oxacillin and cefoxitin were chosen over methicillin in the early 1990s, although the acronym MRSA is still used for historical reasons (10). It's critical to identify MRSA carriers to prevent and monitor these infections. There is rising concern about MRSA infections contracted outside of hospitals as well as those obtained there. (11). Foodborne infections are a significant public health issue worldwide. As per the World Health Organization, foodborne disease (FBD) can be described as an infectious or toxic circumstance brought on by food or water consumption or has been suggested to be caused by it (12,13). A prominent problem in global public health programs is foodborne illness initiated by S. aureus, one of the most common types of FBD. It's among the most often cited reasons for FBD in American reports (14). S. aureus generates a diverse range of toxins. There are nine main serological types of heat-resistant enterotoxins in the group of Staphylococcal enterotoxins (SEs) (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, and SEJ) (15). Foods frequently related to SFD included food-producing animals and food such as meat, egg products, milk and its products, salads, and cakes (16). Iraq produces a lot of basturma, a meat product, throughout the nation, but primarily in Mosul City (Nineveh governorate). They should only be consumed after heat processing (frying or grilling) and may be partially dried or smoked (17). meat and its byproducts food products are frequently contaminated during manufacture, storage, or sale, either directly from unsanitary conditions or indirectly via contaminated food animals (18).

The present investigation aimed to isolate and characterize MRSA from basturma, identify the risks linked to raw cured fermented meats and other primary product categories, and identify the spa gene using PCR technique to determine the correlation between the MRSA isolated in this study and MRSA isolated globally.

Materials and methods

Ethical approval

All samples were gathered with owner consent and used according to the ethical guidelines provided by the Institutional Animal Care and Use Committee at Mosul University's College of Veterinary Medicine, with an approved ID of UM. Vet. 2023.057.

Samples collection

Between April 2023 and June 2023, 45 samples of regional sausages were collected from various shops across several areas, including 8 samples of basturma taken from each of the following areas: Al-Noor, First Qadsia, Ras Al-Jada, Bab Al-Tob, and Al-Zahra, and 5 samples from Al-Tahrer in Mosul city/Iraq. The basturma from the area were gathered in sterile containers and put in containers with peptone water, and all samples were delivered to the Research Centre and Laboratories at Mosul University in an icebox. All samples were incubated for 18 to 24 hours at 37°C for pre-enrichment. Afterward, these samples were streaked over 7.5% Mannitol salt media and Blood media plates, which were incubated for a full day at 37°C.

1. aureus isolation and characterization

Gram staining, conventional biochemical methods, such as catalase and coagulase tests, and morphological evaluation were used to examine the distinctive S. aureus colonies (19).

Isolation of DNA

The positive isolates were cultivated on a mannitol-salt medium at 37°C for 24 h, allowing the genomic DNA extraction of S. aureus. The DNeasy Blood and Tissue Kit (Qiagen, Germany) was utilized for DNA isolation, following the constructor's Gram-positive bacteria-specific methodology. After being measured with Nanodrop (Biodrop, UK), the extracted DNA was kept at -20°C.

Reaction of PCR

Three genes were identified using the PCR assay: the nuc gene, the mecA gene, and the spa gene of MRSA. The nuc gene has a molecular weight of 166 bp (20), the mecA gene is 147 bp (21), and the spa gene's molecular weight varies (22). The mixture was comprised of This process was carried out in a 200 μl tube (Biozym, Oldenhorf, Germany). A total of 25 μl was used for the PCR reaction. The reaction mixture was composed of 12.5 μl of 2× GoTaq Green Mix-Master from Promega Corporation (USA), 1 μl of primer 1, 1 μl of primer 2, 6.5 μl of DNeasy-free water from Promega Corporation (USA), and 4 μl of the S. aureus DNA template and MRSA DNA template. Gel electrophoresis on a 2 % agarose gel (Peqlab, Germany) with a 100 bp ladder as a reference was used to analyze the resulting amplicons.

Protein A (spa) typing

Spa typing was carried out with specific primers; Table 1 provides further information. After amplifying the specific repeat area of Staphylococcal Protein A, PCR was used to sequence the DNA (22). The MegAlign software was used to assign all spa repeats and kinds. The Ridom Spa Server website (www.spaserver.ridom.de) was rendered, and numerical codes for spa typing and spa types were ascertained.

Table 1: Sequences of Primers and PCR programs to detect nuc, mecA, and spa of S. aureus and MRSA

PCR program: I: 35 times (94°C – 30s, 55°C – 30s, 72°C – 30s), II: 35 times (94°C – 30s, 54°C – 30s, 72°C – 30s)

DNA sequencing

The samples were sent to Macrogen, a South Korean commercial sequencing business, to purify and sequence three PCR amplicons obtained from basturma isolates, which had all been previously determined to be S. aureus-positive by traditional PCR. The spa gene was the intended target for sequencing. The resulting spa gene sequences were compared using the NCBI BLASTn tool, edited at http://www.ncbi.nlm.nih.gov, against previously published S. aureus sequences available on GenBank. Using CLUSTALW from GenomeNet, an online multiple sequence alignment tool, the alignment and comparability of these sequences were further investigated. The Neighbor-Joining (NJ) program and the identical GenomeNet tool CLUSTALW were utilized to create phylogenetic trees. To improve robustness, five hundred replicates of the S. aureus spa gene sequences were used as an outgroup while building the phylogenetic tree. This all-encompassing strategy aimed to better comprehend the phylogenetic context of the S. aureus isolates from basturma by utilizing purification, sequencing, and subsequent bioinformatics analysis to clarify the genetic relationships among the isolates.

Results

Based on the appearance of colonies on Mannitol salt agar, the colonies of S. aureus that yielded positive results exhibited a golden-yellowish color. Furthermore, affirmative outcomes were achieved for specific biochemical examinations like the coagulase and catalase tests, which substantiated the presence of positive S. aureus isolates. Our investigation demonstrated that among 45 isolates of S. aureus, 3 (6.7%) were identified as S. aureus, as depicted in table 2 and figure 1. Additionally, the results of the PCR assay disclosed the occurrence of the mecA gene in S. aureus. This confirmed that all the isolated S. aureus strains were MRSA, 12.5% (1/8) found in First Qadsia and 25% (2/8) found in Ras Al-Jada (Table 2 and Figure 2). As seen in Figure 3, the protein's X region found a gene spa in each isolate, and no discernible variations in the sizes of the amplified products were seen. Table 3 shows that one spa type was t213 (n = 3), which was created from the S. aureus isolates (Table 3 and Figure 3). In addition, three unique spa-type gene sequences were found from basturma. Individual sequencing analysis (BLASTn) was used to analyze the sequencing data. As indicated in table 4, the S. aureus sequences in the NCBI Genbank are indexed underneath the previous accession numbers: PP388962.1, PP388963.1, and PP388964.1. Additionally, local spa-type gene sequences indicated a similarity between the S. aureus isolates in this study and those available in the GenBank database that were previously reported, according to a phylogenetic tree analysis performed in MegAlign software using the maximum likelihood technique. The sequence types obtained in this study (PP388962.1, PP388963.1, and PP388964.1) and the sequence types AP027135.1 Japan and EF203503.1 Australia had a 96.2% close relationship (Figure 4).

Table 2: The number of samples and percentage of positive S. aureus and MRSA isolates

Table 3: Spa types' frequency of S. aureus isolates examined

Table 4: The NCBI GenBank accession numbers for the S. aureus sequences in basturma

Figure 1 displays a 2% agarose gel electrophoresis image demonstrating the characteristic amplified product of the nuc gene. The amplification of DNA appears as a ladder-like pattern. Lane 1 is the positive control, and Lanes 2 – 4 represent positive isolates. Lanes 5 and 6 represent negative isolates. Lane 7 is the negative control, and Lane M is the DNA Marker 100 bp ladder (Biozym Diagnostic).

Figure 2: Illustrates a 2% agarose gel electrophoresis depiction presenting the characteristic amplified product of the mecA gene. The amplification of DNA appears as a ladder-like pattern. Lane 1 is the positive control, Lanes 2 – 4 represent positive isolates, Lanes 5 and 6 represent negative isolates, Lane 7 is the negative control, and Lane M is the DNA Marker 100 bp ladder (Biozym Diagnostic).

Figure 3: Illustrates a 2% agarose gel electrophoresis depiction presenting the characteristic amplified product of the protein A gene spa product. The amplification of DNA appears as a ladder-like pattern. Lanes 1 – 3 represent positive isolates, 4 - 6 represent negative isolates, Lane 7 is the negative control, and Lane M is the DNA Marker 100 bp ladder (Biozym Diagnostic).

Figure 4 shows the phylogenetic tree for the partial spa gene sequence of the S. aureus local sequences available in the NCBI GenBank. Bootstrap supports (500 replications) are indicated by the numbers at the branches.

Figure 5: DNASTAR-calculated the spa gene sequence similarity and divergence of each pair for S. aureus. Sequence pair comparisons with the tree produced by MegAlign DNASTAR are used to calculate a percentage divergence. By utilizing % similarity, sequences are directly compared without considering evolutionary ties.

Discussion

The World Health Organization (WHO) defines foodborne diseases (FBDs) as infectious or toxic illnesses brought on by or supposed to be taken on by consuming food or water. Many different disease-causing microorganisms that can contaminate food can cause more than 250 FBD, including food poisoning in humans (23). S. aureus is a cause of foodborne infections in the majority of countries worldwide (24). Traditional methods in several countries involve dry-curing, rinsing, pressing, and drying whole-muscle beef chunks before coating them with a spice paste. This product is known as basturma. Unfortunately, S. aureus occurred in basturma due to several factors, including contaminated water used for washing and rinsing carcasses and intestinal contents during evisceration or poor hygiene during transportation and slaughter (25). Increased cross-contamination between workers and meat can occur from improper personal hygiene practices during meat processing. For example, forgetting to wear gloves, wearing dirty clothes, and not washing your hands can all lead to increased levels of S. aureus contamination (26). Nonetheless, S. aureus and other dangerous germs can be removed from butcher shops by following proper sanitation procedures and cleaning procedures for tools and equipment before and after handling meat (27).

This investigation used PCR against the nuc gene to identify S. aureus isolates and the mecA gene to detect MRSA. PCR is a highly sensitive and accurate method for differentiating S. aureus from other Staphylococci species, as demonstrated by numerous research (28,29). For the quick identification of MRSA in food samples, the nuc and mecA gene PCR amplification strategy is very successful and regarded as the gold standard method for MRSA identification (30). In this study, 3 (6.7%) of the 45 basturma samples tested positive for S. aureus isolates with the nuc and mecA genes. The occurrence of S. aureus in this investigation was lower than that of prior research, which reported 30% in Saudi Arabia (31), 50% in Morocco (32), and 42.3% in the United States of America (33). The results of this study indicated that the occurrence of S. aureus was almost in agreement with studies that found 5.6% in Iraq (34) and 6.9% in Iowa (35). Differences between the current study's results and those from the previously stated research may be attributed to variations in the food's source, additional components, preparation methods, and laboratory techniques.

MRSA poses a severe risk to human and animal public health (31). In recent years, the death rate from MRSA infections has remained high. Numerous earlier investigations have documented the isolation of MRSA from animals (36). Based on the geographic distribution, MRSA can be isolated and identified differently from meat and its products in different parts of the same nation and between different countries. Furthermore, out of 45 basturma samples, 3 (6.7%) had MRSA identified. The previous studies showed that 2.9% of samples tested positive for MRSA during a one-year survey conducted among 3520 retail meats in the USA (37). Meanwhile, an Italian study discovered that MRSA was present in 6 out of 160 (3.75%) foods derived from animals (38). However, 3.6% (15/421) of retail meat in Korea included MRSA (39).

In the protein, the X region in each isolate, a gene spa was detected; the size of the amplified products detected was spa type t213. The previous study in Bangladesh between January 2021 and December 2022 showed that all S. aureus isolated from the inpatients or outpatients at the Hospital showed clinical signs; the t213 was discovered in all S. aureus isolates (40). Furthermore, between December 2008 and December 2009, t213 was the spa type of MRSA that was recovered from nasal swabs of patients in the Thames Valley Primary Care Research Partnership (41). Moreover, 212 rodents were found near a house in Guangzhou, Southern China, on the lawn and in the trash can. The spa type of MRSA isolated from them was t213 (42-46).

Moreover, the phylogenetic tree of the spa gene sequences in S. aureus isolated from Mosul city revealed a tight link between S. aureus obtained for this study. Sequence S. aureus, PP388962.1, PP388963.1, and PP388964.1, as well as AP027135.1 Japan and EF203503.1 Australia, were my sequence kinds, exhibited a 96.2% significant connection. These molecular targets are effective genotyping techniques for the spa genus of the S. aureus isolates. The previous studies identified S. aureus species based on the spa gene, further supported by the strong similarity results with this study.

Conclusion

The principal foodborne pathogen responsible for producing several types of staphylococcal enterotoxins, resulting in food poisoning in humans, was discussed in the study. Butcher shops can use knives, hooks, tables, machinery, and staff hands to contaminate meat products with S. aureus. These items are important in the contamination and spread of S. aureus to foods. One meat product that could be contaminated with S. aureus or other foodborne pathogens is basturma. Additionally, the PCR method is a useful molecular diagnostic tool for detecting the characterization and identification of S. aureus isolates from basturma, opening up new avenues for research and shedding light on the potential implications of S. aureus in foods of animal origin.

Acknowledgment

We would like to express our gratitude to the University of Mosul's College of Veterinary Medicine for providing the necessary tools for conducting this study.

Conflict of interest

The manuscript's author has confirmed that no conflicts of interest arose during the writing or data analysis phases.