Whole-Genome Sequencing-Based Characterization of 100 Listeria monocytogenes Isolates Collected from Food Processing Environments over a Four-Year Period

Listeria monocytogenes is frequently found in foods and processing facilities, where it can persist, creating concerns for the food industry. Its ability to survive under a wide range of environmental conditions enhances the potential for cross-contamination of the final food products, leading to possible outbreaks of listeriosis. In this study, whole-genome sequencing (WGS) was applied as a tool to characterize and track 100 L. monocytogenes isolates collected from three food processing environments. These WGS data from environmental and food isolates were analyzed to (i) assess the genomic diversity of L. monocytogenes, (ii) identify possible source(s) of contamination, cross-contamination routes, and persistence, (iii) detect absence/presence of antimicrobial resistance-encoding genes, (iv) assess virulence genotypes, and (v) explore in vivo pathogenicity of selected L. monocytogenes isolates carrying different virulence genotypes. The predominant L. monocytogenes sublineages (SLs) identified were SL101 (21%), SL9 (17%), SL121 (12%), and SL5 (12%). Benzalkonium chloride (BC) tolerance-encoding genes were found in 62% of these isolates, a value that increased to 73% among putative persistent subgroups. The most prevalent gene was emrC followed by bcrABC, qacH-Tn6188, and qacC. The L. monocytogenes major virulence factor inlA was truncated in 31% of the isolates, and only one environmental isolate (L. monocytogenes CFS086) harbored all major virulence factors, including Listeria pathogenicity island 4 (LIPI-4), which has been shown to confer hypervirulence. A zebrafish embryo infection model showed a low (3%) embryo survival rate for all putatively hypervirulent L. monocytogenes isolates assayed. Higher embryo survival rates were observed following infection with unknown virulence potential (20%) and putatively hypovirulent (53 to 83%) L. monocytogenes isolates showing predicted pathogenic phenotypes inferred from virulence genotypes

Conversion of RpoS− Attenuated Salmonella enterica Serovar Typhi Vaccine Strains to RpoS+ Improves Their Resistance to Host Defense Barriers

ABSTRACT The vast majority of live attenuated typhoid vaccines are constructed from the Salmonella enterica serovar Typhi strain Ty2, which is devoid of a functioning alternative sigma factor, RpoS, due to the presence of a frameshift mutation. RpoS is a specialized sigma factor that plays an important role in the general stress response of a number of Gram-negative organisms, including Salmonella. Previous studies have demonstrated that this sigma factor is necessary for survival following exposure to acid, hydrogen peroxide, nutrient-limiting conditions, and starvation. In addition, studies with Salmonella enterica serovar Typhimurium and the mouse model of typhoid fever have shown that RpoS is important in colonization and survival within the infected murine host. We converted 4 clinically studied candidate typhoid vaccine strains derived from Ty2 [CVD908-htrA, Ty800, and χ9639(pYA3493)] and the licensed live typhoid vaccine Ty21a (also derived from Ty2) to RpoS+ and compared their abilities to withstand environmental stresses that may be encountered within the host to those of the RpoS− parent strains. The results of our study indicate that strains that contain a functional RpoS were better able to survive following stress and that they would be ideal for further development as safe, effective vaccines to prevent S. Typhi infections or as vectors in recombinant attenuated Salmonella vaccines (RASVs) designed to protect against other infectious disease agents in humans. The S. Typhi strains constructed and described here will be made freely available upon request, as will the suicide vector used to convert rpoS mutants to RpoS+. IMPORTANCE Recombinant attenuated Salmonella vaccines (RASVs) represent a unique prevention strategy to combating infectious disease because they utilize the ability of Salmonella to invade and colonize deep effector lymphoid tissues and deliver hetero- and homologous derived antigens at the lowest immunizing dose. Our recent clinical trial in human volunteers indicated that an RpoS+ derivative of Ty2 was better at inducing immune responses than its RpoS− counterpart. In this study, we demonstrate that a functional RpoS allele is beneficial for developing effective live attenuated vaccines against S. Typhi or in using S. Typhi as a recombinant attenuated vaccine vector to deliver other protective antigens

Genomewide Identification of Essential Genes and Fitness Determinants of Streptococcus mutans UA159

ABSTRACT Transposon mutagenesis coupled with next-generation DNA sequencing (Tn-seq) is a powerful tool for discovering regions of the genome that are required for the survival of bacteria in different environments. We adapted this technique to the dental caries pathogen Streptococcus mutans UA159 and identified 11% of the genome as essential, with many genes encoding products required for replication, translation, lipid metabolism, and cell wall biogenesis. Comparison of the essential genome of S. mutans UA159 with those of selected other streptococci for which such information is available revealed several metabolic pathways and genes that are required in S. mutans, but not in some Streptococcus spp. We further identified genes that are essential for sustained growth in rich or defined medium, as well as for persistence in vivo in a rodent model of oral infection. Collectively, our results provide a novel and comprehensive view of the genes required for essential processes of S. mutans, many of which could represent potential targets for therapeutics. IMPORTANCE Tooth decay (dental caries) is a common cause of pain, impaired quality of life, and tooth loss in children and adults. It begins because of a compositional change in the microorganisms that colonize the tooth surface driven by repeated and sustained carbohydrate intake. Although several bacterial species are associated with tooth decay, Streptococcus mutans is the most common cause. Therefore, it is important to identify biological processes that contribute to the survival of S. mutans in the human mouth, with the aim of disrupting the processes with antimicrobial agents. We successfully applied Tn-seq to S. mutans, discovering genes that are required for survival, growth, and persistence, both in laboratory environments and in a mouse model of tooth decay. This work highlights new avenues for the control of an important human pathogen

Comparison between Listeria sensu stricto and Listeria sensu lato strains identifies novel determinants involved in infection

Abstract The human pathogen L. monocytogenes and the animal pathogen L. ivanovii, together with four other species isolated from symptom-free animals, form the “Listeria sensu stricto” clade. The members of the second clade, “Listeria sensu lato”, are believed to be solely environmental bacteria without the ability to colonize mammalian hosts. To identify novel determinants that contribute to infection by L. monocytogenes, the causative agent of the foodborne disease listeriosis, we performed a genome comparison of the two clades and found 151 candidate genes that are conserved in the Listeria sensu stricto species. Two factors were investigated further in vitro and in vivo. A mutant lacking an ATP-binding cassette transporter exhibited defective adhesion and invasion of human Caco-2 cells. Using a mouse model of foodborne L. monocytogenes infection, a reduced number of the mutant strain compared to the parental strain was observed in the small intestine and the liver. Another mutant with a defective 1,2-propanediol degradation pathway showed reduced persistence in the stool of infected mice, suggesting a role of 1,2-propanediol as a carbon and energy source of listeriae during infection. These findings reveal the relevance of novel factors for the colonization process of L. monocytogenes

The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade both staphylococcal and human proteins