The epidemiology and impact of persistent Campylobacter infections on childhood growth among children 0-24 months of age in resource-limited settings.

Background: Campylobacter is the leading cause of bacterial gastroenteritis worldwide. It is generally associated with an acute gastrointestinal infection causing a self-limiting diarrheal episode. However, there is evidence that persistent/recurrent carriage of Campylobacter also occurs. In hyperendemic settings the epidemiology and consequences of persistent Campylobacter enteric infections is poorly studied. Methods: Risk factors for and growth consequences of persistent Campylobacter infections detected by polymerase chain reaction (qPCR) were evaluated with data from the MAL-ED birth cohort study in children 0-24 months of age between November 2009 and February 2012. A persistent Campylobacter infection was defined as three or more consecutive Campylobacter positive monthly stools. Findings: Across all study sites, 45.5% (781/1715) of children experienced at least one persistent Campylobacter episode. The average cumulative duration of days in which children with persistent Campylobacter were positive for Campylobacter spp. was 150 days (inter-quartile range: 28-236 days). Children who experienced a persistent Campylobacter episode had an attained 24-month length-for-age (LAZ) score that was 0.23 (95% (CI): -0.31, -0.15) less than children without a persistent Campylobacter episode. Among children who had at least one episode of Campylobacter over a 3-month or 9-month window, persistent episodes were not significantly associated with poorer 3-month weight gain (-28.7 g, 95% CI: -63.4 g, 6.0 g) but were associated with poorer 9-month linear growth (-0.134 cm 95% CI: -0.246, -0.022) compared to children with an episode that resolved within 31 days. Interpretation: Persistent/recurrent Campylobacter infection is common among children and has a measurable negative impact on linear growth in early childhood. Funding: Funding for this study was provided by the Bill and Melinda Gates Foundation (OPP1066146 and OPP1152146), the National Institutes of Health United States (R01AI158576 and R21AI163801 to MNK and CTP; K43TW012298 to FS; K01AI168493 to JMC; GOL was supported by K01AI145080. This research was also supported in part by USDA-ARS CRIS project 2030-42000-055-00D. The funders had no role in study design, study implementation, data analysis, or interpretation of the results.

Machine learning to attribute the source of Campylobacter infections in the United States: A retrospective analysis of national surveillance data.

OBJECTIVES: Integrating pathogen genomic surveillance with bioinformatics can enhance public health responses by identifying risk and guiding interventions. This study focusses on the two predominant Campylobacter species, which are commonly found in the gut of birds and mammals and often infect humans via contaminated food. Rising incidence and antimicrobial resistance (AMR) are a global concern, and there is an urgent need to quantify the main routes to human infection. METHODS: During routine US national surveillance (2009-2019), 8856 Campylobacter genomes from human infections and 16,703 from possible sources were sequenced. Using machine learning and probabilistic models, we target genetic variation associated with host adaptation to attribute the source of human infections and estimate the importance of different disease reservoirs. RESULTS: Poultry was identified as the primary source of human infections, responsible for an estimated 68% of cases, followed by cattle (28%), and only a small contribution from wild birds (3%) and pork sources (1%). There was also evidence of an increase in multidrug resistance, particularly among isolates attributed to chickens. CONCLUSIONS: National surveillance and source attribution can guide policy, and our study suggests that interventions targeting poultry will yield the greatest reductions in campylobacteriosis and spread of AMR in the US. DATA AVAILABILITY: All sequence reads were uploaded and shared on NCBI's Sequence Read Archive (SRA) associated with BioProjects; PRJNA239251 (CDC / PulseNet surveillance), PRJNA287430 (FSIS surveillance), PRJNA292668 & PRJNA292664 (NARMS) and PRJNA258022 (FDA surveillance). Publicly available genomes, including reference genomes and isolates sampled worldwide from wild birds are associated with BioProject accessions: PRJNA176480, PRJNA177352, PRJNA342755, PRJNA345429, PRJNA312235, PRJNA415188, PRJNA524300, PRJNA528879, PRJNA529798, PRJNA575343, PRJNA524315 and PRJNA689604. Contiguous assemblies of all genome sequences compared are available at Mendeley data (assembled C. coli genomes doi: 10.17632/gxswjvxyh3.1; assembled C. jejuni genomes doi: 10.17632/6ngsz3dtbd.1) and individual project and accession numbers can be found in Supplementary tables S1 and S2, which also includes pubMLST identifiers for assembled genomes. Figshare (10.6084/m9.figshare.20279928). Interactive phylogenies are hosted on microreact separately for C. jejuni (https://microreact.org/project/pascoe-us-cjejuni) and C. coli (https://microreact.org/project/pascoe-us-ccoli).

Bacterial diversity of cantaloupes and soil from Arizona and California commercial fields at the point of harvest.

Across the United States, melons are a high demand crop reaching a net production of 2.7 million tons in 2020 with an economic value of $915 million dollars. The goal of this study was to characterize the bacterial diversity of cantaloupe rinds and soil from commercial melon fields at the point of harvest from two major production regions, Arizona, and California. Cantaloupes and composite soil samples were collected from three different commercial production fields, including Imperial Valley, CA, Central Valley, CA, and Yuma Valley, AZ, at the point of harvest over a three-month period, and 16S rRNA gene amplicon sequencing was used to assess bacterial diversity and community structure. The Shannon Diversity Index showed higher diversity among soil compared to the cantaloupe rind regardless of the sampling location. Regional diversity of soil differed significantly, whereas there was no difference in diversity on cantaloupe surfaces. Bray-Curtis Principal Coordinate Analysis (PCoA) dissimilarity distance matrix found the samples clustered by soil and melon individually, and then clustered tighter by region for the soil samples compared to the cantaloupe samples. Taxonomic analysis found total families among the regions to be 52 for the soil samples and 12 among cantaloupes from all three locations, but composition and abundance did vary between the three locations. Core microbiome analysis identified two taxa shared among soil and cantaloupe which were Bacillaceae and Micrococcaceae. This study lays the foundation for characterizing the cantaloupe microbiome at the point of harvest that provides the cantaloupe industry with those bacterial families that are potentially present entering post-harvest processing, which could assist in improving cantaloupe safety, shelf-life, cantaloupe quality and other critical aspects of cantaloupe post-harvest practices.

Proximity to humans is associated with antimicrobial-resistant enteric pathogens in wild bird microbiomes.

Humans are radically altering global ecology, and one of the most apparent human-induced effects is urbanization, where high-density human habitats disrupt long-established ecotones. Changes to these transitional areas between organisms, especially enhanced contact among humans and wild animals, provide new opportunities for the spread of zoonotic pathogens. This poses a serious threat to global public health, but little is known about how habitat disruption impacts cross-species pathogen spread. Here, we investigated variation in the zoonotic enteric pathogen Campylobacter jejuni. The ubiquity of C. jejuni in wild bird gut microbiomes makes it an ideal organism for understanding how host behavior and ecology influence pathogen transition and spread. We analyzed 700 C. jejuni isolate genomes from 30 bird species in eight countries using a scalable generalized linear model approach. Comparing multiple behavioral and ecological traits showed that proximity to human habitation promotes lineage diversity and is associated with antimicrobial-resistant (AMR) strains in natural populations. Specifically, wild birds from urban areas harbored up to three times more C. jejuni genotypes and AMR genes. This study provides novel methodology and much-needed quantitative evidence linking urbanization to gene pool spread and zoonoses.

Bacterial diversity and composition on the rinds of specific melon cultivars and hybrids from across different growing regions in the United States.

The goal of this study was to characterize the bacterial diversity on different melon varieties grown in different regions of the US, and determine the influence that region, rind netting, and variety of melon has on the composition of the melon microbiome. Assessing the bacterial diversity of the microbiome on the melon rind can identify antagonistic and protagonistic bacteria for foodborne pathogens and spoilage organisms to improve melon safety, prolong shelf-life, and/or improve overall plant health. Bacterial community composition of melons (n = 603) grown in seven locations over a four-year period were used for 16S rRNA gene amplicon sequencing and analysis to identify bacterial diversity and constituents. Statistically significant differences in alpha diversity based on the rind netting and growing region (p < 0.01) were found among the melon samples. Principal Coordinate Analysis based on the Bray-Curtis dissimilarity distance matrix found that the melon bacterial communities clustered more by region rather than melon variety (R2 value: 0.09 & R2 value: 0.02 respectively). Taxonomic profiling among the growing regions found Enterobacteriaceae, Bacillaceae, Microbacteriaceae, and Pseudomonadaceae present on the different melon rinds at an abundance of ≥ 0.1%, but no specific core microbiome was found for netted melons. However, a core of Pseudomonadaceae, Bacillaceae, and Exiguobacteraceae were found for non-netted melons. The results of this study indicate that bacterial diversity is driven more by the region that the melons were grown in compared to rind netting or melon type. Establishing the foundation for regional differences could improve melon safety, shelf-life, and quality as well as the consumers' health.

Bacterial community shifts of commercial apples, oranges, and peaches at different harvest points across multiple growing seasons.

Assessing the microbes present on tree fruit carpospheres as the fruit enters postharvest processing could have useful applications, as these microbes could have a major influence on spoilage, food safety, verification of packing process controls, or other aspects of processing. The goal of this study was to establish a baseline profile of bacterial communities associated with apple (pome fruit), peach (stone fruit), and Navel orange (citrus fruit) at harvest. We found that commercial peaches had the greatest bacterial richness followed by oranges then apples. Time of harvest significantly changed bacterial diversity in oranges and peaches, but not apples. Shifts in diversity varied by fruit type, where 70% of the variability in beta diversity on the apple carposphere was driven by the gain and loss of species (i.e., nestedness). The peach and orange carposphere bacterial community shifts were driven by nearly an even split between turnover (species replacement) and nestedness. We identified a small core microbiome for apples across and between growing seasons that included only Methylobacteriaceae and Sphingomonadaceae among the samples, while peaches had a larger core microbiome composed of five bacterial families: Bacillaceae, Geodermtophilaceae, Nocardioidaceae, Micrococcaeceae, and Trueperaceae. There was a relatively diverse core microbiome for oranges that shared all the families present on apples and peaches, except for Trueperaceae, but also included an additional nine bacterial families not shared including Oxalobacteraceae, Cytophagaceae, and Comamonadaceae. Overall, our findings illustrate the important temporal dynamics of bacterial communities found on major commercial tree fruit, but also the core bacterial families that constantly remain with both implications being important entering postharvest packing and processing.

Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool.

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.

Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru.

OBJECTIVES: Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. METHODS: Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. RESULTS: The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. CONCLUSIONS: Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.

Etiology of Acute Febrile Illness in the Peruvian Amazon as determined by modular formatted quantitative PCR: A Protocol for RIVERA, a Health Facility-Based Case-Control Study.

Background: The study of the etiology of acute febrile illness (AFI) has historically been designed as a prevalence of pathogens detected from a case series. This strategy has an inherent unrealistic assumption that all pathogen detection allows for causal attribution, despite known asymptomatic carriage of the principal causes of acute febrile illness in most low- and middle-income countries (LMICs). We designed a semi-quantitative PCR in a modular format to detect bloodborne agents of acute febrile illness that encompassed common etiologies of AFI in the region, etiologies of recent epidemics, etiologies that require an immediate public health response and additional pathogens of unknown endemicity. We then designed a study that would delineate background levels of transmission in the community in the absence of symptoms to provide corrected estimates of attribution for the principal determinants of AFI. Methods: A case-control study of acute febrile illness in patients ten years or older seeking health care in Iquitos, Loreto, Peru, was planned. Upon enrollment, we will obtain blood, saliva, and mid-turbinate nasal swabs at enrollment with a follow-up visit on day 21-28 following enrollment to attain vital status and convalescent saliva and blood samples, as well as a questionnaire including clinical, socio-demographic, occupational, travel, and animal contact information for each participant. Whole blood samples are to be simultaneously tested for 32 pathogens using TaqMan array cards. Mid-turbinate samples will be tested for SARS-CoV-2, Influenza A and Influenza B. Conditional logistic regression models will be fitted treating case/control status as the outcome and with pathogen-specific sample positivity as predictors to attain estimates of attributable pathogen fractions for AFI. Discussion: The modular PCR platforms will allow for reporting of all primary results of respiratory samples within 72 hours and blood samples within one week, allowing for results to influence local medical practice and enable timely public health responses. The inclusion of controls will allow for a more accurate estimate of the importance of specific, prevalent pathogens as a cause of acute illness. Study Registration: Project 1791, Registro de Proyectos de Investigación en Salud Pública (PRISA), Instituto Nacional de Salud, Perú.

Etiology of acute febrile illness in the peruvian amazon as determined by modular formatted quantitative PCR: a protocol for RIVERA, a health facility-based case-control study.

BACKGROUND: The study of the etiology of acute febrile illness (AFI) has historically been designed as a prevalence of pathogens detected from a case series. This strategy has an inherent unrealistic assumption that all pathogen detection allows for causal attribution, despite known asymptomatic carriage of the principal causes of acute febrile illness in most low- and middle-income countries (LMICs). We designed a semi-quantitative PCR in a modular format to detect bloodborne agents of acute febrile illness that encompassed common etiologies of AFI in the region, etiologies of recent epidemics, etiologies that require an immediate public health response and additional pathogens of unknown endemicity. We then designed a study that would delineate background levels of transmission in the community in the absence of symptoms to provide corrected estimates of attribution for the principal determinants of AFI. METHODS: A case-control study of acute febrile illness in patients ten years or older seeking health care in Iquitos, Loreto, Peru, was planned. Upon enrollment, we will obtain blood, saliva, and mid-turbinate nasal swabs at enrollment with a follow-up visit on day 21-28 following enrollment to attain vital status and convalescent saliva and blood samples, as well as a questionnaire including clinical, socio-demographic, occupational, travel, and animal contact information for each participant. Whole blood samples are to be simultaneously tested for 32 pathogens using TaqMan array cards. Mid-turbinate samples will be tested for SARS-CoV-2, Influenza A and Influenza B. Conditional logistic regression models will be fitted treating case/control status as the outcome and with pathogen-specific sample positivity as predictors to attain estimates of attributable pathogen fractions for AFI. DISCUSSION: The modular PCR platforms will allow for reporting of all primary results of respiratory samples within 72 h and blood samples within one week, allowing for results to influence local medical practice and enable timely public health responses. The inclusion of controls will allow for a more accurate estimate of the importance of specific prevalent pathogens as a cause of acute illness. STUDY REGISTRATION: Project 1791, Registro de Proyectos de Investigación en Salud Pública (PRISA), Instituto Nacional de Salud, Perú.

The Missing Pieces: The Role of Secretion Systems in Campylobacter jejuni Virulence.

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

Deletion of Constitutive Androstane Receptor Led to Intestinal Alterations and Increased Imidacloprid in Murine Liver.

Imidacloprid (IMI) is the most frequently detected neonicotinoid pesticide in the environment. Despite typically low toxicity in vertebrates, IMI exposure is associated with liver and gastrointestinal toxicity. The mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, such as the constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. This study examined the role of CAR in mediating IMI off-target toxicity. Female Car-/- and wild-type (WT) mice were orally administered imidacloprid (50 mg/kg, twice daily) for 21 days, following which serum, liver, and intestinal tissues were collected. Liver tissue analysis indicated mild inflammation and induction of detoxification gene Cyp2b10 in IMI-exposed WT mice. The absence of CAR increased hepatic IMI accumulation. Microbiome analysis of ileal samples revealed IMI altered microbial diversity in a genotype-specific manner, with increased α-diversity in Car-/- mice while decreased α-diversity in WT mice. We observed Car-/- mice exhibit intestinal alterations with decreased CYP-P450 expression, blunted villi height, and increased small intestine length and weight independent of IMI exposure. Our results suggest that IMI is not overtly toxic. However, the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the villi structure and Cyp gene expression in the intestine.

"Candidatus Campylobacter infans" detection is not associated with diarrhea in children under the age of 2 in Peru.

A working hypothesis is that less common species of Campylobacter (other than C. jejuni and C. coli) play a role in enteric disease among children in low resource settings and explain the gap between the detection of Campylobacter using culture and culture independent methods. "Candidatus Campylobacter infans" (C. infans), was recently detected in stool samples from children and hypothesized to play a role in Campylobacter epidemiology in low- and middle-income countries (LMIC). This study determined the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru. Stool samples from 215 children with diarrhea and 50 stool samples from children without diarrhea under the age of two were evaluated using a multiplex qPCR assay to detect Campylobacter spp. (16S rRNA), Campylobacter jejuni / Campylobacter coli (cadF gene), C. infans (lpxA), and Shigella spp. (ipaH). C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The association between diarrhea and the presence of these targets was evaluated using univariate logistic regressions. C. infans was not associated with diarrhea. Fifty-one percent (75/146) of Campylobacter positive fecal samples were negative for C. jejuni, C. coli, and C. infans via qPCR. Shotgun metagenomics confirmed the presence of C. infans among 13 out of 14 positive C. infans positive stool samples. C infans explained only 20.7% of the diagnostic gap in stools from children with diarrhea and 16.7% of the gap in children without diarrhea. We posit that poor cadF primer performance better explains the observed gap than the prevalence of atypical non-C. jejuni/coli species.

Shotgun metagenomics of fecal samples from children in Peru reveals frequent complex co-infections with multiple Campylobacter species.

Campylobacter spp. are a major cause of bacterial diarrhea worldwide and are associated with high rates of mortality and linear growth faltering in children living in low- to middle-income countries (LMICs). Campylobacter jejuni and Campylobacter coli are most often the causative agents of enteric disease among children in LMICs. However, previous work on a collection of stool samples from children under 2 years of age, living in a low resource community in Peru with either acute diarrheal disease or asymptomatic, were found to be qPCR positive for Campylobacter species but qPCR negative for C. jejuni and C. coli. The goal of this study was to determine if whole-genome shotgun metagenomic sequencing (WSMS) could identify the Campylobacter species within these samples. The Campylobacter species identified in these stool samples included C. jejuni, C. coli, C. upsaliensis, C. concisus, and the potential new species of Campylobacter, "Candidatus Campylobacter infans". Moreover, WSMS results demonstrate that over 65% of the samples represented co-infections with multiple Campylobacter species present in a single stool sample, a novel finding in human populations.

Genomic population structure associated with repeated escape of Salmonella enterica ATCC14028s from the laboratory into nature.

Salmonella enterica serovar Typhimurium strain ATCC14028s is commercially available from multiple national type culture collections, and has been widely used since 1960 for quality control of growth media and experiments on fitness ("laboratory evolution"). ATCC14028s has been implicated in multiple cross-contaminations in the laboratory, and has also caused multiple laboratory infections and one known attempt at bioterrorism. According to hierarchical clustering of 3002 core gene sequences, ATCC14028s belongs to HierCC cluster HC20_373 in which most internal branch lengths are only one to three SNPs long. Many natural Typhimurium isolates from humans, domesticated animals and the environment also belong to HC20_373, and their core genomes are almost indistinguishable from those of laboratory strains. These natural isolates have infected humans in Ireland and Taiwan for decades, and are common in the British Isles as well as the Americas. The isolation history of some of the natural isolates confirms the conclusion that they do not represent recent contamination by the laboratory strain, and 10% carry plasmids or bacteriophages which have been acquired in nature by HGT from unrelated bacteria. We propose that ATCC14028s has repeatedly escaped from the laboratory environment into nature via laboratory accidents or infections, but the escaped micro-lineages have only a limited life span. As a result, there is a genetic gap separating HC20_373 from its closest natural relatives due to a divergence between them in the late 19th century followed by repeated extinction events of escaped HC20_373.

RNA and Sugars, Unique Properties of Bacteriophages Infecting Multidrug Resistant Acinetobacter radioresistens Strain LH6.

Bacteriophages (phages) are predicted to be the most ubiquitous biological entity on earth, and yet, there are still vast knowledge gaps in our understanding of phage diversity and phage-host interactions. Approximately one hundred Acinetobacter-infecting DNA viruses have been identified, and in this report, we describe eight more. We isolated two typical dsDNA lytic podoviruses (CAP1-2), five unique dsRNA lytic cystoviruses (CAP3-7), and one dsDNA lysogenic siphovirus (SLAP1), all capable of infecting the multidrug resistant isolate Acinetobacter radioresistens LH6. Using transmission electron microscopy, bacterial mutagenesis, phage infectivity assays, carbohydrate staining, mass-spectrometry, genomic sequencing, and comparative studies, we further characterized these phages. Mutation of the LH6 initiating glycosyltransferase homolog, PglC, necessary for both O-linked glycoprotein and capsular polysaccharide (CPS) biosynthesis, prevented infection by the lytic podovirus CAP1, while mutation of the pilin protein, PilA, prevented infection by CAP3, representing the lytic cystoviruses. Genome sequencing of the three dsRNA segments of the isolated cystoviruses revealed low levels of homology, but conserved synteny with the only other reported cystoviruses that infect Pseudomonas species. In Pseudomonas, the cystoviruses are known to be enveloped phages surrounding their capsids with the inner membrane from the infected host. To characterize any membrane-associated glycoconjugates in the CAP3 cystovirus, carbohydrate staining was used to identify a low molecular weight lipid-linked glycoconjugate subsequently identified by mutagenesis and mass-spectrometry as bacterial lipooligosaccharide. Together, this study demonstrates the isolation of new Acinetobacter-infecting phages and the determination of their cell receptors. Further, we describe the genomes of a new genus of Cystoviruses and perform an initial characterization of membrane-associated glycoconjugates.

Genomic Characterization of Salmonella typhimurium DT104 Strains Associated with Cattle and Beef Products.

Salmonella enterica subsp. enterica serovar Typhimurium DT104, a multidrug-resistant phage type, has emerged globally as a major cause of foodborne outbreaks particularly associated with contaminated beef products. In this study, we sequenced three S. Typhimurium DT104 strains associated with a 2009 outbreak caused by ground beef, including the outbreak source strain and two clinical strains. The goal of the study was to gain a stronger understanding of the genomics and genomic epidemiology of highly clonal S. typhimurium DT104 strains associated with bovine sources. Our study found no single nucleotide polymorphisms (SNPs) between the ground beef source strain and the clinical isolates from the 2009 outbreak. SNP analysis including twelve other S. typhimurium strains from bovine and clinical sources, including both DT104 and non-DT104, determined DT104 strains averaged 55.0 SNPs between strains compared to 474.5 SNPs among non-DT104 strains. Phylogenetic analysis separated the DT104 strains from the non-DT104 strains, but strains did not cluster together based on source of isolation even within the DT104 phage type. Pangenome analysis of the strains confirmed previous studies showing that DT104 strains are missing the genes for the allantoin utilization pathway, but this study confirmed that the genes were part of a deletion event and not substituted or disrupted by the insertion of another genomic element. Additionally, cgMLST analysis revealed that DT104 strains with cattle as the source of isolation were quite diverse as a group and did not cluster together, even among strains from the same country. Expansion of the analysis to 775 S. typhimurium ST19 strains associated with cattle from North America revealed diversity between strains, not limited to just among DT104 strains, which suggests that the cattle environment is favorable for a diverse group of S. typhimurium strains and not just DT104 strains.

Genomic Characterization of Campylobacter jejuni Adapted to the Guinea Pig (Cavia porcellus) Host.

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide with excessive incidence in low-and middle-income countries (LMIC). During a survey for C. jejuni from putative animal hosts in a town in the Peruvian Amazon, we were able to isolate and whole genome sequence two C. jejuni strains from domesticated guinea pigs (Cavia porcellus). The C. jejuni isolated from guinea pigs had a novel multilocus sequence type that shared some alleles with other C. jejuni collected from guinea pigs. Average nucleotide identity and phylogenetic analysis with a collection of C. jejuni subsp. jejuni and C. jejuni subsp. doylei suggest that the guinea pig isolates are distinct. Genomic comparisons demonstrated gene gain and loss that could be associated with guinea pig host specialization related to guinea pig diet, anatomy, and physiology including the deletion of genes involved with selenium metabolism, including genes encoding the selenocysteine insertion machinery and selenocysteine-containing proteins.

Transcriptomic analysis of cyanobacterial alkane overproduction reveals stress-related genes and inhibitors of lipid droplet formation.

The cyanobacterium Nostoc punctiforme can form lipid droplets (LDs), internal inclusions containing triacylglycerols, carotenoids and alkanes. LDs are enriched for a 17 carbon-long alkane in N. punctiforme, and it has been shown that the overexpression of the aar and ado genes results in increased LD and alkane production. To identify transcriptional adaptations associated with increased alkane production, we performed comparative transcriptomic analysis of an alkane overproduction strain. RNA-seq data identified a large number of highly upregulated genes in the overproduction strain, including genes potentially involved in rRNA processing, mycosporine-glycine production and synthesis of non-ribosomal peptides, including nostopeptolide A. Other genes encoding helical carotenoid proteins, stress-induced proteins and those for microviridin synthesis were also upregulated. Construction of N. punctiforme strains with several upregulated genes or operons on multi-copy plasmids resulted in reduced alkane accumulation, indicating possible negative regulators of alkane production. A strain containing four genes for microviridin biosynthesis completely lost the ability to synthesize LDs. This strain exhibited wild-type growth and lag phase recovery under standard conditions, and slightly faster growth under high light. The transcriptional changes associated with increased alkane production identified in this work will provide the basis for future experiments designed to use cyanobacteria as a production platform for biofuel or high-value hydrophobic products.