An in vivo atlas of host-pathogen transcriptomes during Streptococcus pneumoniae colonization and disease.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo Spn and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three Spn strains. We identified Spn genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct Spn and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby Spn adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by Spn during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of Spn and identify therapeutic targets.

Inhibition of Necroptosis to Prevent Long-term Cardiac Damage During Pneumococcal Pneumonia and Invasive Disease.

BACKGROUND: Streptococcus pneumoniae infection can result in bacteremia with devastating consequences including heart damage. Necroptosis is a proinflammatory form of cell death instigated by pore-forming toxins such as S. pneumoniae pneumolysin. Necroptosis-inhibiting drugs may lessen organ damage during invasive pneumococcal disease (IPD). METHODS: In vitro experiments were carried out with human and mouse cardiomyocytes. Long-term cardiac damage was assessed using high-resolution echocardiography in ampicillin-rescued mice 3 months after challenge with S. pneumoniae. Ponatinib, a necroptosis-inhibiting and Food and Drug Administration-approved drug for lymphocytic leukemia treatment, was administered intraperitoneally alongside ampicillin to test its therapeutic efficacy. Histology of heart sections included hematoxylin-eosin staining for overt damage, immunofluorescence for necroptosis, and Sirius red/fast green staining for collagen deposition. RESULTS: Cardiomyocyte death and heart damage was due to pneumolysin-mediated necroptosis. IPD leads to long-term cardiac damage, as evidenced by de novo collagen deposition in mouse hearts and a decrease in fractional shortening. Adjunct necroptosis inhibition reduced the number of S. pneumoniae foci observed in hearts of acutely infected mice and serum levels of troponin I. Ponatinib reduced collagen deposition and protected heart function in convalescence. CONCLUSIONS: Acute and long-term cardiac damage incurred during IPD is due in part to cardiomyocyte necroptosis. Necroptosis inhibitors may be a viable adjunct therapy.

Intraclade Variability in Toxin Production and Cytotoxicity of Bacillus cereus Group Type Strains and Dairy-Associated Isolates.

While some species in the Bacillus cereus group are well-characterized human pathogens (e.g., B. anthracis and B. cereus sensu stricto), the pathogenicity of other species (e.g., B. pseudomycoides) either has not been characterized or is presently not well understood. To provide an updated characterization of the pathogenic potential of species in the B. cereus group, we classified a set of 52 isolates, including 8 type strains and 44 isolates from dairy-associated sources, into 7 phylogenetic clades and characterized them for (i) the presence of toxin genes, (ii) phenotypic characteristics used for identification, and (iii) cytotoxicity to human epithelial cells. Overall, we found that B. cereus toxin genes are broadly distributed but are not consistently present within individual species and/or clades. After growth at 37°C, isolates within a clade did not typically show a consistent cytotoxicity phenotype, except for isolates in clade VI (B. weihenstephanensis/B. mycoides), where none of the isolates were cytotoxic, and isolates in clade I (B. pseudomycoides), which consistently displayed cytotoxic activity. Importantly, our study highlights that B. pseudomycoides is cytotoxic toward human cells. Our results indicate that the detection of toxin genes does not provide a reliable approach to predict the pathogenic potential of B. cereus group isolates, as the presence of toxin genes is not always consistent with cytotoxicity phenotype. Overall, our results suggest that isolates from multiple B. cereus group clades have the potential to cause foodborne illness, although cytotoxicity is not always consistently found among isolates within each clade.IMPORTANCE Despite the importance of the Bacillus cereus group as a foodborne pathogen, characterizations of the pathogenic potential of all B. cereus group species were lacking. We show here that B. pseudomycoides (clade I), which has been considered a harmless environmental microorganism, produces toxins and exhibits a phenotype consistent with the production of pore-forming toxins. Furthermore, B. mycoides/B. weihenstephanensis isolates (clade VI) did not show cytotoxicity when grown at 37°C, despite carrying multiple toxin genes. Overall, we show that the current standard methods to characterize B. cereus group isolates and to detect the presence of toxin genes are not reliable indicators of species, phylogenetic clades, or an isolate's cytotoxic capacity, suggesting that novel methods are still needed for differentiating pathogenic from nonpathogenic species within the B. cereus group. Our results also contribute data that are necessary to facilitate risk assessments and a better understanding as to which B. cereus group species are likely to cause foodborne illness.

Production of hemolysin BL by Bacillus cereus group isolates of dairy origin is associated with whole-genome phylogenetic clade.

BACKGROUND: Bacillus cereus group isolates that produce diarrheal or emetic toxins are frequently isolated from raw milk and, in spore form, can survive pasteurization. Several species within the B. cereus group are closely related and cannot be reliably differentiated by established taxonomical criteria. While B. cereus is traditionally recognized as the principal causative agent of foodborne disease in this group, there is a need to better understand the distribution and expression of different toxin and virulence genes among B. cereus group food isolates to facilitate reliable characterization that allows for assessment of the likelihood of a given isolate to cause a foodborne disease. RESULTS: We performed whole genome sequencing of 22 B. cereus group dairy isolates, which represented considerable genetic diversity not covered by other isolates characterized to date. Maximum likelihood analysis of these genomes along with 47 reference genomes representing eight validly published species revealed nine phylogenetic clades. Three of these clades were represented by a single species (B. toyonensis -clade V, B. weihenstephanensis - clade VI, B. cytotoxicus - VII), one by two dairy-associated isolates (clade II; representing a putative new species), one by two species (B. mycoides, B. pseudomycoides - clade I) and four by three species (B. cereus, B. thuringiensis, B. anthracis - clades III-a, b, c and IV). Homologues of genes encoding a principal diarrheal enterotoxin (hemolysin BL) were distributed across all, except the B. cytotoxicus clade. Using a lateral flow immunoassay, hemolysin BL was detected in 13 out of 18 isolates that carried hblACD genes. Isolates from clade III-c (which included B. cereus and B. thuringiensis) consistently did not carry hblACD and did not produce hemolysin BL. Isolates from clade IV (B. cereus, B. thuringiensis) consistently carried hblACD and produced hemolysin BL. Compared to others, clade IV was significantly (p = 0.0001) more likely to produce this toxin. Isolates from clade VI (B. weihenstephanensis) carried hblACD homologues, but did not produce hemolysin BL, possibly due to amino acid substitutions in different toxin-encoding genes. CONCLUSIONS: Our results demonstrate that production of diarrheal enterotoxin hemolysin BL is neither inclusive nor exclusive to B. cereus sensu stricto, and that phylogenetic classification of isolates may be better than taxonomic identification for assessment of B. cereus group isolates risk for causing a diarrheal foodborne disease.

Bacillus wiedmannii sp. nov., a psychrotolerant and cytotoxic Bacillus cereus group species isolated from dairy foods and dairy environments.

A facultatively anaerobic, spore-forming Bacillus strain, FSL W8-0169T, collected from raw milk stored in a silo at a dairy powder processing plant in the north-eastern USA was initially identified as a Bacillus cereus group species based on a partial sequence of the rpoB gene and 16S rRNA gene sequence. Analysis of core genome single nucleotide polymorphisms clustered this strain separately from known B. cereus group species. Pairwise average nucleotide identity blast values obtained for FSL W8-0169T compared to the type strains of existing B. cereus group species were <95 % and predicted DNA-DNA hybridization values were <70 %, suggesting that this strain represents a novel B. cereus group species. We characterized 10 additional strains with the same or closely related rpoB allelic type, by whole genome sequencing and phenotypic analyses. Phenotypic characterization identified a higher content of iso-C16 : 0 fatty acid and the combined inability to ferment sucrose or to hydrolyse arginine as the key characteristics differentiating FSL W8-0169T from other B. cereus group species. FSL W8-0169T is psychrotolerant, produces haemolysin BL and non-haemolytic enterotoxin, and is cytotoxic in a HeLa cell model. The name Bacillus wiedmannii sp. nov. is proposed for the novel species represented by the type strain FSL W8-0169T (=DSM 102050T=LMG 29269T).

Paenibacillus odorifer, the Predominant Paenibacillus Species Isolated from Milk in the United States, Demonstrates Genetic and Phenotypic Conservation of Psychrotolerance but Clade-Associated Differences in Nitrogen Metabolic Pathways.

Paenibacillus is a spore-forming bacterial genus that is frequently isolated from fluid milk and is proposed to play a role in spoilage. To characterize the genetic and phenotypic diversity of Paenibacillus spp., we first used rpoB allelic typing data for a preexisting collection of 1,228 Paenibacillus species isolates collected from raw and processed milk, milk products, and dairy environmental sources. Whole-genome sequencing (WGS) and average nucleotide identity by BLAST (ANIb) analyses performed for a subset of 58 isolates representing unique and overrepresented rpoB allelic types in the collection revealed that these isolates represent 21 different Paenibacillus spp., with P. odorifer being the predominant species. Further genomic characterization of P. odorifer isolates identified two distinct phylogenetic clades, clades A and B, which showed significant overrepresentation of 172 and 164 ortholog clusters and 94 and 52 gene ontology (GO) terms, respectively. While nitrogen fixation genes were found in both clades, multiple genes associated with nitrate and nitrite reduction were overrepresented in clade A isolates; additional phenotypic testing demonstrated that nitrate reduction is specific to isolates in clade A. Hidden Markov models detected 9 to 10 different classes of cold shock-associated genetic elements in all P. odorifer isolates. Phenotypic testing revealed that all isolates tested here can grow in skim milk broth at 6°C, suggesting that psychrotolerance is conserved in P. odorifer Overall, our data suggest that Paenibacillus spp. isolated from milk in the United States represent broad genetic diversity, which may provide challenges for targeted-control strategies aimed at reducing fluid milk spoilage.IMPORTANCE Although Paenibacillus species isolates are frequently isolated from pasteurized fluid milk, the link between the genetic diversity and phenotypic characteristics of these isolates was not well understood, especially as some Bacillales isolated from milk are unable to grow at refrigeration temperatures. Our data demonstrate that Paenibacillus spp. isolated from fluid milk represent tremendous interspecies diversity, with P. odorifer being the predominant Paenibacillus sp. isolated. Furthermore, genetic and phenotypic data support that P. odorifer is well suited to transition from a soil-dwelling environment, where nitrogen fixation (and other nitrate/nitrite reduction pathways present only in clade A) may facilitate growth, to fluid milk, where its multiple cold shock-associated adaptations enable it to grow at refrigeration temperatures throughout the storage of milk. Therefore, efforts to reduce bacterial contamination of milk will require a systematic approach to reduce P. odorifer contamination of raw milk.

Genes Associated With Psychrotolerant Bacillus cereus Group Isolates.

The Bacillus cereus group comprises 18 different species, including human pathogens as well as psychrotolerant strains that are an important cause of fluid milk spoilage. To enhance our understanding of the genetic markers associated with psychrotolerance (defined here as > 1 log10 increase in cfu/mL after 21 days incubation at 6°C) among dairy-associated B. cereus group isolates, we used genetic (whole genome sequencing) and phenotypic methods [growth in Skim Milk Broth (SMB) and Brain Heart Infusion (BHI) broth] to characterize 23 genetically-distinct representative isolates from a collection of 503 dairy-associated isolates. Quality threshold clustering identified three categories of psychrotolerance: (i) 14 isolates that were not psychrotolerant in BHI or SMB, (ii) 6 isolates that were psychrotolerant in BHI but not in SMB, and (iii) 2 isolates that were psychrotolerant in BHI and SMB. One isolate, which was psychrotolerant in BHI broth but was just below the cut-off of >1 log10 cfu/mL increase in SMB was not assigned to a cluster. A maximum likelihood phylogeny constructed with core genome single nucleotide polymorphisms classified all psychrotolerant isolates (i.e., psychrotolerant in BHI) into clade VI (representing B. mycoides/weihenstephanensis). Analysis of correlations between gene ortholog presence or absence patterns and psychrotolerance identified 206 orthologous gene clusters that were significantly overrepresented among psychrotolerant strains, including two clusters of cold shock proteins, which were identified in 8/9 and 7/9 psychrotolerant isolates. Gene ontology analyses revealed 36 gene ontology terms that were overrepresented in psychrotolerant isolates, including putrescine catabolic processes and putrescine transmembrane transporter activity. Lastly, Hidden Markov Model searches identified three protein family motifs, including cold shock domain proteins and fatty acid hydroxylases that were significantly associated with psychrotolerance in BHI broth. Analyses of CspA sequences revealed a positive association between psychrotolerant strains and a previously identified "psychrotolerant" CspA sequence. Overall, our data highlight genetic and phenotypic differences in psychrotolerance among B. cereus group dairy-associated isolates and show that psychrotolerance is dependent on the growth medium. We also identified a number of gene targets that could be used for specific detection or control of psychrotolerant B. cereus group isolates.

Corrigendum: Genes Associated With Psychrotolerant Bacillus cereus Group Isolates.

[This corrects the article DOI: 10.3389/fmicb.2019.00662.].

Severity and properties of cardiac damage caused by Streptococcus pneumoniae are strain dependent.

Streptococcus pneumoniae is an opportunistic Gram-positive pathogen that can cause invasive disease. Recent studies have shown that S. pneumoniae is able to invade the myocardium and kill cardiomyocytes, with one-in-five adults hospitalized for pneumococcal pneumonia having a pneumonia-associated adverse cardiac event. Furthermore, clinical reports have shown up to a 10-year increased risk of adverse cardiac events in patients formerly hospitalized for pneumococcal bacteremia. In this study, we investigated the ability of nine S. pneumoniae clinical isolates, representing eight unique serotypes, to cause cardiac damage in a mouse model of invasive disease. Following intraperitoneal challenge of C57BL/6 mice, four of these strains (D39, WU2, TIGR4, and 6A-10) caused high-grade bacteremia, while CDC7F:2617-97 and AMQ16 caused mid- and low-grade bacteremia, respectively. Three strains did not cause any discernible disease. Of note, only the strains capable of high-grade bacteremia caused cardiac damage, as inferred by serum levels of cardiac troponin-I. This link between bacteremia and heart damage was further corroborated by Hematoxylin & Eosin and Trichrome staining which showed cardiac cytotoxicity only in D39, WU2, TIGR4, and 6A-10 infected mice. Finally, hearts infected with these strains showed varying histopathological characteristics, such as differential lesion formation and myocytolysis, suggesting that the mechanism of heart damage varied between strains.

Development and Validation of Pathogen Environmental Monitoring Programs for Small Cheese Processing Facilities.

Pathogen environmental monitoring programs (EMPs) are essential for food processing facilities of all sizes that produce ready-to-eat food products exposed to the processing environment. We developed, implemented, and evaluated EMPs targeting Listeria spp. and Salmonella in nine small cheese processing facilities, including seven farmstead facilities. Individual EMPs with monthly sample collection protocols were designed specifically for each facility. Salmonella was detected in only one facility, with likely introduction from the adjacent farm indicated by pulsed-field gel electrophoresis data. Listeria spp. were isolated from all nine facilities during routine sampling. The overall Listeria spp. (other than Listeria monocytogenes ) and L. monocytogenes prevalences in the 4,430 environmental samples collected were 6.03 and 1.35%, respectively. Molecular characterization and subtyping data suggested persistence of a given Listeria spp. strain in seven facilities and persistence of L. monocytogenes in four facilities. To assess routine sampling plans, validation sampling for Listeria spp. was performed in seven facilities after at least 6 months of routine sampling. This validation sampling was performed by independent individuals and included collection of 50 to 150 samples per facility, based on statistical sample size calculations. Two of the facilities had a significantly higher frequency of detection of Listeria spp. during the validation sampling than during routine sampling, whereas two other facilities had significantly lower frequencies of detection. This study provides a model for a science- and statistics-based approach to developing and validating pathogen EMPs.