Genome sequencing of an historic Staphylococcus aureus collection reveals new enterotoxin genes and sheds light on the evolution and genomic organisation of this key virulence gene family

We take advantage of an historic collection of 133 Staphylococcus aureus strains accessioned between 1924 and 2016, whose genomes have been long-read sequenced as part of a major National Collection of Type Cultures (NCTC) initiative, to conduct a gene family-wide computational analysis of enterotoxin genes. We identify two novel Staphylococcal enterotoxin (pseudo)genes (sel29p and sel30), the former of which has not been observed in any contemporary strain to date. We provide further information on five additional enterotoxin genes or gene variants that have either recently entered the literature or for which the nomenclature or description is currently unclear (selz, sel26, sel27, sel28 and ses-2p). An examination of over 11,000 RefSeq genomes in search of wider support for these seven (pseudo)genes led to the identification of an additional three novel enterotoxin gene family members (sel31, sel32 and sel33) plus two new variants (seh-2p and ses-3p). We cast light on the genomic distribution of the enterotoxin genes, further defining their arrangement in gene clusters. Finally, we show that co-occurrence of enterotoxin genes is prevalent, with individual NCTC strains possessing as many as eighteen enterotoxin genes and pseudogenes, and that Clonal Complex membership rather than time of isolation is the key factor in determining enterotoxin load. Importance: Staphylococcus aureus strains pose a significant health risk to both human and animal populations. Key amongst this species’ virulence factors are the Staphylococcal enterotoxin gene family. Certain enterotoxin forms can induce a potentially life-threatening immune response, while others are implicated in less fatal though often severe conditions such as food poisoning. Genetic characterisation of Staphylococcal enterotoxin gene family members has steadily accumulated over recent decades, with over 20 genes now established in the literature. Despite the current wealth of knowledge on this important gene family, questions remain about the presence of additional enterotoxin genes and the genomic composition of family members. This study further expands knowledge of the Staphylococcal enterotoxins while shedding light on their evolution over the last century.

Enterotoxin Gene Distribution and Genotypes of Bacillus cereus sensu lato Isolated from Cassava Starch

Bacillus cereus is a human pathogenic bacterium found in foods with the potential to cause emesis and diarrhea. This study estimated the presence, toxigenic and genomic diversity of B. cereus s.l. obtained from cassava starch samples collected in bakeries and powdered food companies in Medellín (Colombia). Bacillus cereuss.l. was found in 43 of 75 (57%) cassava starch samples and 98 isolates were obtained. The nheABC, hblCDAB, cytK2, entFM and cesB toxin genes were detected by multiplex PCR and the most frequent operon was nheABC, whereas cesB gene was not found. Twelve toxigenic profiles were determined by the detection of toxin genes, and the most frequent profiles harbored all enterotoxin genes. A broad genomic diversity was detected according to GTG5-PCR fingerprinting results with 76 B. cereus s.l. grouped in sixteen clusters and the 22 isolates clustering separately. No relationship was observed between genomic background and toxigenic profiles. In general, the results showed a high genomic and enterotoxigenic diversity in B. cereus s.l. found in cassava starch. These results should incentive future studies to understand the distribution of B. cereus s.l. isolated on raw materials in comparison with finished products.

Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSaβ typing

Currently only five (SEA-SEE) out of 27 known staphylococcal enterotoxins can be analyzed using commercially available kits.Six genes (seg, sei, sem, sen, seo, and seu), encoding putative and undetectable enterotoxins, are located on the enterotoxin gene cluster (egc) which is part of the Staphylococcus aureus genomic island vSaβ. These enterotoxins have been described as likely being involved in staphylococcal food poisoning outbreaks.The aim of the present study was to determine if whole genome data can be used for the prediction of staphylococcal egc enterotoxin production, particularly enterotoxin G (SEG) and enterotoxin I (SEI). For this purpose whole genome sequences of 75 Staphylococcus aureus (S. aureus) strains from different origins (food poisoning outbreaks, human, and animal) were investigated applying bioinformatics methods (phylogenetic analysis using the core genome and different alignments). SEG and SEI expression was tested in vitro using a sandwich ELISA method.Strains could be allocated to 14 different vSaβ types, each type being associated with a single clonal complex (CC). In addition the vSaβ type and CC were associated with the origin of the strain (human or cattle derived). The amount of SEG and SEI produced also correlated with the vSaβ type and the CC of a strain. The present results show promising indications that the in vitro production of SEG and SEI can be predicted based on the vSaβ type or CC of a strain.IMPORTANCE Beside the infection properties in human and animals, S. aureus can produce different enterotoxins in food. The enterotoxins can cause vomiting and diarrhea, often involving many people. Most of these outbreaks remain undiscovered as detection methods for enterotoxins are only available for a few enterotoxins, but not for the more recently discovered enterotoxin G (SEG) and I (SEI).In this study we show promising results that in vitro production of SEG and SEI can be predicted based on the whole genome sequencing data of a strain. In addition, this data could be used to find the source (human- or cattle-derived) of an outbreak strain, which is the key for a better understanding of the role SEG and SEI play in foodborne outbreaks caused by S. aureus.

Occurrence and enterotoxin gene profiles of Staphylococcus aureus isolated from retail chicken meat

The present study was conducted to evaluate the prevalence and enterotoxin gene profiles of Staphylococcus aureus isolated from 120 chicken meat marketed in retail outlets of Chennai, India. It was observed that total of 120 meat samples collected from different retail outlets, 66.67% (80/120) of the samples were positive for the presence of S. aureus based on biochemical characterization and species specific PCR based on thermonuclease gene ( nuc). Enterotoxin gene profiling of the isolates for 9 genes ( sea- sej) revealed that 52.50% (42/80) of the isolates in the present study were enterotoxigenic harboring either one or more gene. It was evident that majority of the isolates harbored seb, followed by seg, sei, sec, sed and sej either alone or in combination. None of the isolates harbored sea, see and seh either alone or in combination. The results of the study clearly indicated higher prevalence of enterotoxigenic S. aureus in retail meat marketed in Chennai, India indicating the potential of retail chicken meat to act as vehicle for food borne intoxication and a major public health threat.

Microbiological and molecular aspects of Staphylococcus aureus isolated from bovine mastitis in West-Central Brazil

Bovine mastitis is a major disease affecting dairy cattle, and Staphylococcus aureus is one of the most important agent involved in this condition due to its capacity to produce enterotoxins and develop resistance to antimicrobial agents. This study aimed to detect S. aureus strains in milk samples from cows with subclinical mastitis employing microbiological and molecular analysis. Eleven farms were visited and from 187 lactating cows sampled, 33 S. aureus strains were isolated. Only one of the 33 strains was positive for mecA resistance gene, 23 were positive for sea enterotoxin gene, and none was positive for seb or sec enterotoxin gene. S. aureus strains were submitted to in vitro antimicrobial susceptibility test and 63.6% (21/33) were susceptible to all antimicrobials tested, while 36.3% (12/33) were resistant to one or more antimicrobial agents. Identification of mecA and the sea genes highlighted the need to elaborate strategies to reduce problems related to animal. Furthermore, the identification of bovine mastitis caused by S. aureus is very important to manage herd and to public health, since milk contaminated by this pathogen can lead to serious health problems.

Non-structural Enterotoxin (NSP4) Gene based Molecular Characterization of Caprine and Ovine Rotavirus A, India

Rotavirus A (RVA) causes viral gastroenteritis in humans and animals, including calves, piglets, and foals. The current study reports the genetic characterization of the full-length enterotoxin gene, NSP4, from caprine and ovine species. Upon characterizing eight full-length NSP4 genes by sequencing, it was found that the four caprine and three ovine RVAs NSP4 genes are of E2 genotype and the sole ovine RVA isolate was found to be of E1 genotype. In the sequence and phyloanalysis of the NSP4 gene the seven E2 genotypes clustered with bovine, human, and caprine isolates from India and Bangladesh, respectively. The E1 genotype of ovine RVA was closer to human RVA isolate from India. The nucleotide per cent identity analysis revealed that all E2 genotype strains of caprine and ovine species ranged from 88.4% to 90.4% and it was found common to both the reference human RVA isolates DS-1 and AU-1. Whereas, the E1 genotype ovine strain clustered with human RVA isolates with 93.1% nucleotide per cent identity. The RVA strains circulating in caprine and ovine populations may share a common origin which is usually found in artiodactyl species because humans share a common dwelling with animals. Future studies are needed to confirm these findings of their relationship with humans and large animals.

Prevalence of enterotoxins and other virulence genes of Staphylococcus aureus caused subclinical mastitis in dairy cows

Background and Aim: Milk production is one of the main props for the national economy. One of the crucial problems in this industry is subclinical mastitis, which harms this industry that considered the backbone of the economy. It is an infectious and zoonotic disease; the infection can spread between dairy animals through milkers' hands, and milking machines, while the human infection occurs due to the consumption of apparently hygienic milk. Staphylococcus aureus is one of the main causative agents of clinical and subclinical mastitis. It is also considered one of the bacteria incriminated in food intoxication of humans due to its virulence factors as enterotoxins and toxic shock syndrome. The current study was designed to assess the prevalence of S. aureus and its enterotoxins, as well as, its other virulence factors in milk collected from cows that suffer from subclinical mastitis. Materials and Methods: Sixty cows were collected from different dairy farms located in Assiut Governorate, Egypt. These cows were subjected to the clinical examination of the udder and its lymph nodes before sampling. Milk samples were collected from clinically healthy udders. All the milk samples were examined by California mastitis test (CMT), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) for confirmation subclinical mastitis, presence of S. aureus and its enterotoxins genes and other virulence factors in the examined milk samples. Results: The cows included in the current study had healthy udders. The sixty collected milk samples were tested by CMT. 48/60 (80.0%) were positive samples; from the 48 positive samples, 46 (95.83%) samples were confirmed positive by S. aureus 16s rRNA PCR assay. Multiplex PCRs confirmed the presence of staphylococcus enterotoxin gene C (sec) in one sample, staphylococcus enterotoxin gene D (sed) in 23 samples, while ELISA assay confirmed the presence of the same enterotoxin in only two samples. On the other hand, other groups of genes responsible for some other virulence factors of S. aureus like the extracellular thermostable nuclease (nuc) gene were found in 33 samples, while toxic shock syndrome (tsst) gene and methicillin restraint S. aureus (mecA) gene were not detected in this study. Conclusion: Subclinical mastitis is one of the hidden factors that adversely affect the health of both animals and humans. The milk is usually appeared good and may be consumed by humans especially children; however, it causes severe public health problems. In addition, the infected animals with this form of mastitis can spread the infection to other dairy animals and may be turned to a clinical case of contagious mastitis that may be ended by animal culling or death. S. aureus is one of the main causes of subclinical mastitis in cattle. In addition to extracellular thermostable nuclease (nuc) gene, staphylococcus enterotoxin gene C (sec) and staphylococcus enterotoxin gene D (sed) are the most common virulence genes confirmed in subclinical mastitis milk. These results highlighted the need to apply more hygienic measures in the dairy farms to avoid spreading the infection between animals to ensure the production of safe and healthy food to humans.