Journal of Pioneering Medical Sciences10.61091/jpms202413522Research ArticlePrevalence of Clostridium Perfringens Spores in Selected Regions of Saudi ArabiaBanawasSaeed S.KronmillerBrent A.AbdelghanyMohamed G.AlqarawiAli A.SarkerMahfuzur R.Department of Medical LaboratoriesCollege of Applied Medical Scienceand Health and Basic Sciences Research CenterMajmaah UniversityAlMajmaah11952Saudi Arabia.Center for Genome Research and BiocomputingOregon State UniversityCorvallisOR97331USA.Microbiology DepartmentFaculty of ScienceAin- Shams UniversityAbbaseyaCairo11566Egypt.Department of Clinical LaboratoriesCollege of Applied Medical ScienceHail UniversityHail2440Saudi Arabia.Departments of Biomedical Sciencesand MicrobiologyOregon State UniversityCorvallisOR97331USA.The prevalence of Clostridium perfringens spores in the environment is crucial for the pathogenesis of this bacterium because these dormant spores, upon contact with a suitable host, can return to active growth to cause disease.We evaluated the prevalence of C. perfringens spores in the Hail and Qassim regions of Saudi Arabia.Methods: A plating method was used to identify C. perfringens. PCR analyses and DNA sequencing were performed for genotypic characterization of the newly isolated C. perfringens.25 of 300 collected samples were identified as C. perfringens by selecting black colonies on selective media and monitoring b-hemolysis on blood agar plate. PCR analyses showed that all 25 isolates carry a-toxin gene (plc), but not the enterotoxin gene (cpe), further confirming that these isolates are indeed cpe-negative. Genome sequencing found that Saudi isolates are genotypically similar to the American and European isolates; no homologues of toxinotyping genes encoding b-, i-, e- and NetB-toxin were detected in 22 Saudi isolates, with the exception of 3 isolates that carry only e-toxin gene etx. Collectively, our findings suggest that C. perfringens are highly prevalent in the Hail and Qassim environment, with 22 (88%) of 25 isolates are type A and remaining 3 (12%) are type D.C. perfringenssporesC. perfringens enterotoxincpe\(\alpha\)-toxinplcfood poisoning206202420820243082024This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.1. Introduction

Clostridium perfringens is an anaerobic, gram-positive, spore-forming, enteric bacterial pathogen that causes a wide range of human and animal diseases owing to its prolific toxin-producing capability [1-7]. C. perfringens can be classified into seven types (A through G), based on the presence of genes encoding six major toxins (\(\alpha\), \(\beta\), \(\epsilon\), \(\tau\), C. perfringens enterotoxin (CPE), and C. perfringens necrotic enteritis \(\beta\)-like toxin (NetB)) [1,8]. CPE, the medically important toxin produced by C. perfringens type F, is the major virulence factor for C. perfringens type F food poisoning (FP) and non-foodborne (NFB) gastrointestinal (GI) diseases [9]. C. perfringens type F FP is the third most commonly reported foodborne disease in the United States. The annual cost of illness is estimated to be more than $300 million [10-12]. Interestingly, in C. perfringens type F isolates, the CPE-encoding gene (cpe) can be located either on the chromosome or on a plasmid. In general, chromosomal cpe isolates are generally linked to FP, whereas plasmid-borne cpe isolates are associated with NFB GI diseases [13-15]. Nevertheless, some studies have found that plasmid-borne cpe isolates can also be causative agents for C. perfringens type F FP [15-18]. Numerous studies have attempted to understand why type F isolates carrying a chromosomal cpe gene are more likely to be associated with C. perfringens FP outbreaks [14,16,18,19]. A survey reported that  1.7% of raw meat, fish, and poultry items sold in retail food stores contain type F isolates carrying chromosomal cpe [20]. Interestingly, this survey noted the absence of type F plasmid-borne cpe isolates in retail foods. These findings suggest that meat, seafood, and poultry, common food vehicles for C. perfringens FP in the United States and Europe can be contaminated with type F chromosomal-cpe isolates by the time of retail purchase. However, these survey results do not preclude the possibility of food contamination in food processing environments. C. perfringens spores are more resistant to a number of lethal factors than their vegetative forms [15,21,22]. Spores, especially spores of FP strain (21), can survive thermal processing and sanitizing treatments employed in the food industry. In addition, they are highly hydrophobic, complicating their removal when they are attached to food contact surfaces [23-26]. A potential source of pathogen transmission to food products is the contamination of food contact surfaces during food processing, catering, and in domestic environments [21,26]. Another possibility is that the food items might become contaminated with type F chromosomal-cpe isolates residing in environmental niches, such as the soil or home kitchen surfaces [27,28]. A study that surveyed different soils and home kitchen surfaces in Pittsburgh, PA, did not detect C. perfringens isolates from home kitchens, while most of the soil samples tested positive for this bacterial isolate. The soil isolates were predominantly type A, although types C, D, E and F were also identified. All cpe-positive soil isolates were genotyped as type F, harboring cpe genes on a plasmid [27]. Although Clostridium spore-mediated disease outbreaks, such as FP and Clostridioides difficile infection (CDI), are common in the USA and Europe [21,29], no such outbreaks have been systematically documented in Middle Eastern countries, including the Kingdom of Saudi Arabia (KSA). The absence of disease monitoring and control systems implies that the outbreaks normally occur in Saudi Arabia, but have not yet been reported. The KSA has no data on the incidence or prevalence of Clostridium-associated diseases, except for a limited study in the Dhahran region [30]. Therefore, the purpose of this study was to determine the prevalence of C. perfringens spores in the Hail and Qassim regions of the KSA by: 1) isolating C. perfringens bacteria from various samples obtained from soil, food, and hospital floors; 2) evaluating the presence of plc (encoding \(\beta\)-toxin) and cpe in newly-isolated C. perfringens strains using polymerase chain reaction (PCR); and 3) examining genome organization by determining the DNA sequences of the representative Saudi isolates. Our results suggest that C. perfringens spores are highly prevalent in the Hail and Qassim regions, KSA.

2. Materials and Methods

A. Survey of soil, food, and hospital floors for the presence of C. perfringens spores We selected three hospitals from both Qassim (hospitals A-C) and Hail (hospitals D-F) regions of KSA for our study. We collected soil samples from the hospital surroundings, swab samples from hospital floors, and food samples, such as various raw meats, including ground beef, and chicken supplied to hospitals. Collectively, 300 soil, swab, and food samples were collected from different places. B. Isolation of C. perfringens from soil samples Soil (1.0 g) was collected in a 15-ml sterile plastic tube and then 2.0 ml sterile tryptone glucose yeast (TGY) broth (3% trypticase [Difco, BD Diagnostic Systems, Sparks, MD, USA], 2% glucose [Sigma-Aldrich, USA], 1% yeast extract [Difco, BD Diagnostic Systems, Sparks, MD, USA], and 0.1% L-cysteine [Sigma-Aldrich, USA]) was added and mixed vigorously. An aliquot (0.1 ml) of suspension was cultured onto TSC (tryptose-sulfite-cycloserine, a relatively selective medium for Clostridium isolates) (Millipore, Burlington, MA, USA) agar plates and incubated in anaerobic jars containing GasPak (BD EZ anaerobe container system. Becton, Dickinson and company spark, Maryland USA) at 37 ºC for 24 h. For enrichment,   1.0 ml aliquot of TGY-soil suspension was added to each of two tubes containing 9 ml of sterile TGY. One tube was incubated at 37 ºC overnight ( 18 h) to grow vegetative cells. The other tube was heat-shocked at 75 ºC for 20 min, then anaerobically incubated at 37 ºC overnight ( 18 h), allowing spores to germinate and grow. If there was growth in a TGY tube, an aliquot (0.1 ml) of TGY grown culture was plated onto the TSC plate and anaerobically incubated at 37 ºC for 24 h. Three black colonies of the TSC plates were selected and allowed to grow in TGY at 37 ºC for 18 h. These TGY-grown cultures were then streaked onto sheep/horse blood agar plates and anaerobically incubated at 37 ºC for 24-48 h. The culture that produced a clear double zone of \(\alpha\)-hemolysis, with the inner zone (complete hemolysis) caused by perfringolysin O and the outer zone (partial hemolysis) caused by \(\beta\)-toxin, was considered as C. perfringens. C. perfringens cultures were stored as glycerol stock at -80 ºC freezer until used. C. Isolation from raw meat samples Twenty-five grams of each raw meat or meat product was suspended in 225 ml 0.1% peptone (Difco, BD Diagnostic Systems, Sparks, MD, USA) and the resultant mixture was homogenized for 1–2 min, at low speed, in a sterile blender jar. The blended solution was then serially diluted from 10-1 to 10-8. Thereafter, 0.1 ml of each dilution was plated onto TSC agar plates and anaerobically incubated at 37 °C. After 24–48 h incubation, colonies showing morphology consistent with Clostridium isolates (i.e., black color) were selected for further testing to confirm their identity as C. perfringens as described above. D. Screening for the presence of plc and cpe genes in C. perfringens isolates Total C. perfringens DNA was isolated from the overnight TGY medium cultures, using the Wizard® Genomic DNA Purification Kit (Promega) and then subjected to PCR analysis using primers specific to each of the cpe and plc genes. The design of the primers was based on the C. perfringens strain SM101 genome sequence [8]. These PCR analyses utilized 100 ng template DNA, 25 pmol of each primer, 200 \(\mu\)M deoxynucleoside triphosphates (dNTPs) (Roche), 2.5 mM MgCl2, and 1 U Taq DNA polymerase (Fermentas) in a total volume of 50 \(\mu\)l. The reaction mixture was placed in a thermal cycler (Techne) for an initial period of 2 min at 94 °C, then 35 cycles, each 1 min at 94 °C, 1 min at 47 °C, 1 min at 72 °C, followed by an extension period of 10 min at 72 °C. The presence of a PCR amplified product was examined by subjecting an aliquot of each PCR sample to agarose (1.0%) gel electrophoresis, followed by ethidium bromide staining and photographing under UV light. The following primers were used to detect toxin genes: Forward plc primer: (5’-GATGGAAAAATTGATGGAACAGGAACT-3’), Reverse plc primer: (5’-CATGTAGTAGTCATCATCTGTTCCAGCATC-3’), Forward cpe primer: (5’-GGAGATGGTTGGTTGGATATTAGGGG-3’), and Reverse cpe primer: (5’-CTTCCAAGTCACATCTTTCGTCAG-3’) E. DNA extractions, library preps, sequencing Samples were prepared for sequencing with the Nextera DNA library prep. Twenty-five samples of C. perfringens isolates were multiplexed onto a single lane, 51 bp single end HiSeq3000 run, at the Center for Genome Research and Biocomputing (CGRB) at Oregon State University, Corvallis, OR, USA. F. Bioinformatics: Assembly, Alignment to SM101 and genes, sequence logos of gene regions Each sample was individually assembled with SPAdes [31]. SPAdes was run with default K-mers and the careful pipeline option to reduce mismatches and short indels. SPAdes assembly output scaffold files were compared against the C. perfringens SM101 genome [32], using nucmer [33], delta, and show-coords from MUMer [34]. Each sample was compared to eighteen C. perfringens genes as follows: sleC (ABG87393.1), virS (ABG86783.1), sigF (ABG87692.1), sigG (ABG86124.1), sigE (ABG85707.1), plc (ABG86694.1), cpe (ABG85760.1), cspB (ABG86463.1), gerKB (ABG85755.1), gerKA (ABG86956.1), gerKC (ABG86274.1), gerAA (ABG86934.1), spo0A (ABG85493.1), cpb (WP_003453250.1), etx (WP_164789292.1), iap (WP_003463422.1), ibp (BAK40944.1) and netB (WP_110003253.1). BlastN [35] was used to identify the general genomic region of the gene in the assembly. When necessary, custom Perl scripts were used to extract and reverse-complement the gene sequence. Promer and show-snps from MUMer were used to identify translated amino acid single nucleotide polymorphisms (SNPs) between the assembled sample and the gene reference sequence. WebLogo [36] was used to evaluate the gene regions across all samples.

3. Results and Discussion

A. Isolation of C. perfringens Our survey could detect C. perfringens isolates from all the targeted locations, i.e., the soil, floor, and food samples from selected hospitals (Table 1). Based on their characteristic, black colonies appearance on TSC plates (Table 1), the isolates were putatively identified as C. perfringens. The black colonies on TSC plate are a result of sulfite reduction by C. perfringens [37]. The soil samples obtained from all six hospital areas showed characteristic appearance of black colonies on TSC plate, albeit to varying degrees (Table 1). However, floor samples from only two of the six hospitals (Qassim hospital B and C) produced black colonies on TSC plate. Similarly, the food samples from only three of the six hospitals (Qassim hospitals A and B; Hail hospital D) produced the characteristic black colonies on the TSC plates (Table 1). When these isolates were subjected to Clostridium-specific biochemical tests, they fermented lactose, produced acid and gas, reduced nitrates to nitrites, and liquified gelatin within 48 h. Collectively, these results demonstrated that our survey successfully isolated Clostridium species from Hail and Qassim environments. Table 1: Isolation of Clostridium perfringens from various samples collected from the Hail and Qassim regions   Soil (%)# Floor (%) Food (%) Qassim hospital A 73.3 0 7 Qassim hospital B 45.8 53.3 4.6 Qassim hospital C 37.5 6 0 Hail hospital D 8 0 38.4 Hail hospital E 33.3 0 0 Hail hospital F 25 0 0 B. Differentiating C. perfringens from other Clostridium species by monitoring PLC and PFO phenotypes on blood agar plates When newly-isolated Clostridium cultures were anaerobically grown on sheep blood agar plates, 25 out of 54 cultures produced a clear double (PFO-mediated inner and the \(\beta\)-toxin-mediated outer) zone of \(\alpha\)-hemolysis on the plates (Table 2). These results confirmed that these 25 cultures are C. perfringens isolates. However, the remaining 29 cultures, that were identified as Clostridium species by TSC plating and biochemical tests, failed to produce \(\alpha\)-hemolysis; no inner or outer zone of \(\alpha\)-hemolysis was detected on blood agar plates. These results suggest that other Clostridium species can be detected in soil, food, and hospital floor samples. However, further studies are required to support this hypothesis. Table 2: Formation of double zones of \(\beta\)-hemolysis on sheep blood agar plates by C. perfringens isolates   Soil Floor Food Qassim hospital A 0 0 0 Qassim hospital B 0 0 10 Qassim hospital C 0 0 0 Hail hospital D 0 0 4 Hail hospital E 8 0 3 Hail hospital F 0 0 0 Total 8 0 17 C. Detection of the plc and cpe genes in C. perfringens isolates by PCR To confirm the presence of the plc gene in our newly-isolated C. perfringens, which produced PLC, we performed PCR analyses, using plc-specific primers. As a positive control, plc PCR analyses amplified the  900 bp plc-specific band from wild-type strains, SM101 and F4969. As expected from our blood agar plate phenotypic results, plc-specific PCR-amplified bands were obtained from DNA of all 25 surveyed isolates (Figure 1 A). As CPE is the main toxin responsible for C. perfringens type F FP, we examined whether our isolated C. perfringens carry cpe gene. Our PCR analyses, using cpe-specific primers, amplified the  1500 bp cpe-specific band from DNA of the reference strains SM101 and F4969. However, when DNA of our newly-isolated 25 strains were subjected to similar PCR analyses, no cpe-specific band was amplified from any DNA sample (Figure 1 B). Kuske et al. [38] had the same result; none of their surveyed C. perfringens soil isolates were cpe-positive. In contrast, Li et al [27] reported cpe-positive soil isolates harboring cpe gene on the plasmid. These findings support the possibility that soil may not be the major reservoir for C. perfringens chromosomal cpe isolates. Figure 1: Identification of plc and cpe genes in Clostridium perfringens strains, isolated from Hail and Qassim, KSA. Total DNA, isolated from wild-type (SM101 and F4969) and KSA strains, was subjected to PCR analyses using primers specific for plc (A) and cpe (B), as described in the Material and Methods. Lane M represents the molecular size marker (base pairs) D. Comparing C. perfringens nucleotide sequences isolated from the KSA, USA, and Europe We hypothesized that there might be differences in nucleotide sequence between the American/European and KSA strains, as FP outbreaks in the US and Europe are more frequent than those in the Middle East and KSA. To prove our hypothesis, we compared the whole genome sequence of Saudi ‘s C. perfringens strains with that of the American/European’s C. perfringens strains. SPAdes yielded de novo assemblies with a wide range of values; number of scaffolds ranging from 337–27220, assembly lengths ranging from 3.1–22.8 kb, and N50 ranging from 974–63344 bp (Table 3). When compared to the C. perfringens reference genome, most of the reference genome was found in the assemblies, excluding samples s007 and s008. On average, 87% of the C. perfringens genome was assembled in each sample. Samples s007 and s008 did not sequence well; s007 represented 53% of the C. perfringens genome and s008 only 9% (Table [t4]). Thirteen proteins were compared to the assembled samples. As previously mentioned, s007 and s008 were poor-quality assemblies that did not align well with the proteins. Sample s006 also showed a poor alignment with the protein sequences, along with many low-quality errors, due to low coverage in the assembly. Overall, the proteins and assembled contigs were well-matched, with only SNP differences. In the remaining 22 samples, the number of amino acid SNPs ranged from 0–89 (Table 1S), and the average across each gene was aligned to 22 genomes ranging from 0–44.5 SNPs (Table 4). cpe (ABG85760.1) was not found in any of the genome assemblies, which confirmed our PCR results (Figure 1B) that none of the 25 samples isolated from different places contained the cpe gene. gerAA (ABG86934.1) did not align with sample s001. plc (ABG86694.1) was aligned to all tested genome samples with average SNPs of 10.95 (Table 4). etx (WP_164789292.1) aligned to samples s019, s021, s022 only. Each KSA sample shared the same single SNP (Table 4). cpb (WP_003453250.1), iap (WP_003463422.1), ibp (BAK40944.1), and netB (WP_110003253.1) were not found when aligned to the KSA isolates. Table 3: Assembly of SPAdes yielded de novo assemblies of all Saudi Arabian samples Samples Contigs Assembly length Longest contig N50 s001 5441 4394834 83758 8892 s002 1140 3695605 91700 27495 s003 1076 3726820 101287 28498 s004 721 3115795 101405 36366 s005 542 3148052 136630 30348 s006 14321 12326586 28624 2726 s007 6419 14213758 264909 12641 s008 1009 3736936 142751 57391 s009 519 3319337 110603 30553 s010 443 3318911 151873 40772 s011 6168 8501859 170643 3915 s012 594 3115402 191201 37292 s013 555 3408298 235785 40516 s014 409 3291632 128382 37491 s015 379 2986986 248334 43576 s016 20168 12912111 73446 974 s017 8044 8489997 110019 2939 s018 27220 22812868 187921 1722 s019 734 3509100 188089 44804 s020 350 3342300 262729 77505 s021 488 3376723 201291 52706 s022 488 3372578 202330 58206 s023 1924 7208676 191597 17641 s024 1727 7238402 160484 22289 s025 337 3309147 192708 63344 Table 4: Genome size of C. perfringens, and the predicted percentage of Saudi Arabian samples Samples Assembly size Percent ID Contigs in alignment Percent of SM101 covered by assembly s001 2633034 96.93% 904 88.95% s002 2628043 96.74% 374 88.78% s003 2611250 96.54% 315 88.22% s004 2557608 96.46% 230 86.40% s005 2549355 96.58% 246 86.12% s006 2674583 97.12% 1770 90.35% s007 1561619 89.82% 1451 52.76% s008 270015 94.73% 484 9.12% s009 2567660 96.29% 265 86.74% s010 2563778 96.31% 207 86.61% s011 2559681 96.19% 219 86.47% s012 2556930 96.68% 243 86.38% s013 2571706 96.41% 212 86.88% s014 2563279 96.38% 214 86.59% s015 2482422 95.08% 189 83.86% s016 2532848 96.48% 459 85.57% s017 2555097 96.61% 225 86.32% s018 2528230 96.42% 201 85.41% s019 2580073 96.55% 234 87.16% s020 2559669 96.44% 167 86.47% s021 2533240 96.52% 192 85.58% s022 2532439 96.56% 178 85.55% s023 2522041 95.42% 271 85.20% s024 2521730 95.47% 262 85.19% s025 2557348 96.47% 158 86.39%   Table 5: Alignment of C. perfringens genes with Saudi samples and missing data, including missing sequences Genes Average SNPs Amt missing Alignments sleC (ABG85493.1) 4 0 sigE (ABG85707.1) 0 0 gerKB (ABG85755.1) 17.86363636 0 cpe (ABG85760.1) 0 25 sigG (ABG86124.1) 1.136363636 0 gerKC (ABG86274.1) 7 0 cspB (ABG86463.1) 39.13636364 0 plc (ABG86694.1) 10.95454545 0 virS (ABG86783.1) 24.77272727 0 gerAA (ABG86934.1) 3.363636364 1 gerKA (ABG86956.1) 8.590909091 0 sleC (ABG87393.1) 44.45454545 0 sigF (ABG87692.1) 0.227272727 0 etx (WP_164789292.1) 1 22 Based on the data listed in Table [t6], proteins, that result from translating nucleotides with significant sequence differences between Saudi Arabian and American standard samples, may give rise to a similar protein pattern for both. For example, SigE (ABG85707.1) and SigF (ABG87692.1) have significantly different nucleotide sequences, but upon translation, there was no difference in protein patterns between the Saudi Arabian isolates and the American reference sample, SM101. Interestingly, all KSA strains, with the exception of sample numbers S23 and S24, had two proteins that differed from the reference sample SM101 in the SigF gene. SigE in all KSA strains showed no significant difference in amino acid sequence, indicating minimal change in protein function compared with that of SM101. Both nucleotide and protein sequences showed that the protein sequence was not affected by codons changing nucleotides (Table [t7]). This suggest that the underlying protein (and therefore protein function) is conserved while mutations occur. In other cases, the protein sequence did change, indicating that the amino acids at those positions were not conserved, which probably resulted in altered protein function (Table [t6]). Table 6: Alignments of translated nucleotide sequences of Saudi Arabian isolates showing possibilities of proteins with conserved and non-conserved amino acid sequences, suggesting the possibility of differences in functionality   S 001 S 002 S 003 S 004 S 005 S 006 S 007 S 008 S 009 S 010 S 011 S 012 S 013 S 014 S 015 S 016 S 017 S 018 S 019 S 020 S 021 S 022 S 023 S 024 S 025 sleC (ABG87393.1) 49 46 48 49 48 -10 89 -10 40 40 40 47 40 50 47 51 40 47 40 40 40 40 48 48 40 virS (ABG86783.1) 33 26 28 27 25 -10 -10 -10 30 30 30 28 25 10 0 27 27 25 25 24 25 25 25 25 25 sigF (ABG87692.1) 0 0 0 0 0 0 -10 -10 0 0 0 1 0 0 0 0 0 0 0 0 0 0 2 2 0 sigG (ABG86124.1) 1 1 1 1 1 -10 -10 -10 1 1 1 1 2 1 1 1 1 1 1 1 1 1 2 2 1 sigE (ABG85707.1) 0 0 0 0 0 0 -10 -10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 plc (ABG86694.1) 13 10 14 11 14 -10 82 -10 11 11 11 11 11 10 11 14 9 11 8 14 8 8 10 10 11 cpe (ABG85760.1) -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 -10 cspB (ABG86463.1) 0 40 40 50 40 -10 -10 -10 39 39 39 40 41 40 49 40 40 38 41 40 41 41 41 41 41 gerKB (ABG85755.1) 17 15 15 18 15 16 -10 -10 20 20 20 19 17 17 23 17 14 18 17 16 17 17 22 22 17 gerKA (ABG86956.1) 8 8 8 8 8 8 -10 -10 9 9 9 8 9 9 10 9 8 8 9 8 9 9 8 8 10 gerKC (ABG86274.1) 6 6 6 7 6 -10 -10 -10 9 9 9 6 6 6 9 6 6 7 6 6 6 6 10 10 6 gerAA (ABG86934.1) 0 3 3 5 4 3 -10 -10 4 4 4 3 3 3 8 4 4 2 3 4 3 3 2 2 3 spo0A (ABG85493.1) 4 4 4 4 4 4 -10 57 4 4 4 4 4 4 5 4 3 4 4 4 4 4 4 4 4 Table 7: Nucleotide sequence alignments of Saudi Arabian isolates showing the recorded similarities and differences   S 001 S 002 S 003 S 004 S 005 S 006 S 007 S 008 S 009 S 010 S 011 S 012 S 013 S 014 S 015 S 016 S 017 S 018 S 019 S 020 S 021 S 022 S 023 S 024 S 025 sleC (ABG87393.1) 121 118 120 121 122 NH 153 NH 95 95 95 119 95 119 112 118 95 118 95 97 95 95 117 117 95 virS (ABG86783.1) 74 64 68 67 68 NH NH NH 71 71 71 68 62 44 122 70 68 67 62 65 62 62 68 68 62 sigF (ABG87692.1) 10 10 11 8 11 11 NH NH 11 11 11 10 11 11 27 11 11 9 11 11 11 11 18 18 11 sigG (ABG86124.1) 6 6 8 7 6 NH NH NH 5 5 5 6 7 6 13 6 8 6 6 6 6 6 12 12 6 sigE (ABG85707.1) 10 11 11 10 12 11 NH NH 12 12 12 12 12 12 28 12 10 12 12 12 12 12 10 10 12 plc (ABG86694.1) 26 23 27 28 27 NH 0 NH 24 24 24 22 25 27 45 28 26 29 25 27 25 25 28 28 25 cpe (ABG85760.1) NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH NH cspB (ABG86463.1) NH 137 137 167 136 NH NH NH 132 132 132 134 135 132 164 137 134 134 135 135 135 135 151 151 135 gerKB (ABG85755.1) 38 30 30 42 30 33 NH NH 60 60 60 42 38 38 62 38 29 42 38 38 38 38 66 66 38 gerKA (ABG86956.1) 51 57 57 49 52 51 NH NH 75 75 75 52 50 50 77 50 54 51 50 49 50 50 55 55 51 gerKC (ABG86274.1) 24 23 23 27 25 NH NH NH 51 51 51 25 23 24 49 23 23 28 23 24 23 23 56 56 23 gerAA (ABG86934.1) NH 39 37 29 25 32 NH NH 28 28 28 37 24 26 72 42 35 21 26 32 26 26 41 41 24 spo0A (ABG85493.1) 9 11 10 10 9 11 NH 53 10 10 10 8 10 10 17 9 8 10 10 9 10 10 16 16 10 To determine the degree of protein conservation between the reference and KSA strains, we ran multiple alignments of a single region for some of the genes in C. perfringens that regulate toxin production and sporulation. Sequence logos were used for the multiple alignments of the protein sequences identified in each isolate for the following genes: gerKC (ABG86274.1) [39,40], spo0A (ABG85493.1) [41], plc (ABG86694.1) [42], and virS (ABG86783.1) [43]. Sequence logos showed a high sequence similarity between gene versions (Figure 2–5). We found that most Saudi Arabian samples had highly conservative proteins with the C. perfringens reference strain SM101, as shown in gerKC (ABG86274.1) (Figure 2), spo0A (ABG85493.1) (Figure 3), plc (ABG86694.1) (Figure 4), and virS (ABG86783.1) (Figure 5). Figure 2: Protein sequence motif represented as a motif logo for gerKC (ABG86274) Figure 3: Protein sequence motif represented as a motif logo for spo0A (ABG85493) Figure 4: Protein sequence motif represented as a motif logo for plc (ABG86694) Figure 5: Protein sequence motif represented as a motif logo for virS (ABG86783)

4. Conclusion

Our study successfully surveyed soil and hospital environmental samples from the Hail and Qassim areas and isolated 25 C. perfringens isolates. All these isolates were plc and \(\beta\)-toxin positive. However, none of the isolates were cpe-positive. Furthermore, genome-sequencing analyses showed that Saudi C. perfringens isolates are genotypically similar to the American and European isolates. However, some proteins between isolates might be functionally different, which should be investigated. Interestingly, among six toxinotyping gene alignments, plc aligned to all 25, and etx to only 3, Saudi C. perfringens genomes. However, cpe, cpb, iap, ibp and netB did not align to any of the 25 Saudi isolates. Collectively, our findings suggest that C. perfringens are highly prevalent in the Hail and Qassim environment, with 88% isolates are type A (plc-positive) and 12% are type D (plc-and etx-positive). The absence of C. perfringens type F (cpe-positive) in Hail and Qassim area might be a reason for limited reports on C. perfringens type F FP outbreaks in KSA compared to USA. Future surveys using samples from more KSA states are needed to confirm this hypothesis.

5. Abbrevations

CDI, Clostridioides difficile infection; CPE, Clostridium perfringens enterotoxin; NetB, Clostridium perfringens necrotic enteritis B-like toxin; NFB, non-foodborne; PFO, perfringolysin O; SNPs, single nucleotide polymorphisms; TGY, trypticase-glucose-yeast extract; TSC, tryptose-sulfite-cycloserine

Data Availability

C. perfringens sequences were deposited into GenBank under BioProject PRJNA954388.

Acknowledgment

This research was supported by grant from the Deanship of Postgraduate Studies and Scientific Research at Majmaah University grant number (R-2024-1080) (to S.B); by grants from the N.L. Tartar foundation (FS009N-AMMS) and the Agricultural Research Foundation (VMD512-ALX9) of Oregon State University (to M.R.S).

Authors Contribution

SB,MJ,AQ,BK and MS: Conceptualization, Methodology, Software SB,MJ,AQ,BK and MS: Data curation, Writing- Original draft preparation. SB,MJ: Visualization, Investigation. MS: Supervision.: BK: Software, Validation.: SB,MJ,BK nad MS: Writing- Reviewing and Editing.

Conflict of Interest

The authors declare no conflict of interests. All authors read and approved final version of the paper.

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