Unique and specific Proteobacteria diversity in urinary microbiota of tolerant kidney transplanted recipients.

Host-microbiota interactions can modulate the immune system both at local and systemic levels, with potential consequences for organ transplantation outcomes. In this study, we hypothesized that differences in the urinary microbiome following kidney transplantation would be associated with posttransplantation status: stable, minimally immunosuppressed, or tolerant. One hundred thirteen urine samples from stable (n = 51), minimally immunosuppressed (n = 19), and spontaneously tolerant (n = 16) patients, paired with age-matched controls (n = 27) were profiled and compared to each other at a taxonomic level with special interest in the immunosuppressive regimen. All comparisons and correlations were adjusted on sex and time posttransplantation. Our results highlighted a unique and specific urinary microbiota associated with spontaneous tolerance characterized by a high diversity and a clear Proteobacteria profile. Finally, we report that this profile is (1) impacted by gender, (2) inversely correlated with immunosuppressive drugs (calcineurin inhibitors and mammalian target of rapamycin inhibitors), and (3) stable in time.

Mentholation triggers brand-specific shifts in the bacterial microbiota of commercial cigarette products.

Bacterial communities are integral constituents of tobacco products. They originate from tobacco plants and are acquired during manufacturing processes, where they play a role in the production of tobacco-specific nitrosamines. In addition, tobacco bacterial constituents may play an important role in the development of infectious and chronic diseases among users. Nevertheless, tobacco bacterial communities have been largely unexplored, and the influence of tobacco flavor additives such as menthol (a natural antimicrobial) on tobacco bacterial communities is unclear. To bridge this knowledge gap, time series experiments including 5 mentholated and non-mentholated commercially available cigarettes-Marlboro red (non-menthol), Marlboro menthol, Newport menthol box, Newport menthol gold, and Newport non-menthol-were conducted. Each brand was stored under three different temperature and relative humidity conditions. To characterize bacterial communities, total DNA was extracted on days 0 and 14. Resulting DNA was purified and subjected to PCR of the V3V4 region of the 16S rRNA gene, followed by sequencing on the Illumina HiSeq platform and analysis using the QIIME, phyloseq, metagenomeSeq, and DESeq software packages. Ordination analyses showed that the bacterial community composition of Marlboro cigarettes was different from that of Newport cigarettes. Additionally, bacterial profiles significantly differed between mentholated and non-mentholated Newports. Independently of storage conditions, tobacco brands were dominated by Proteobacteria, with the most dominant bacterial genera being Pseudomonas, unclassified Enterobacteriaceae, Bacillus, Erwinia, Sphingomonas, Acinetobacter, Agrobacterium, Staphylococcus, and Terribacillus. These data suggest that the bacterial communities of tobacco products differ across brands and that mentholation of tobacco can alter bacterial community composition of select brands. KEY POINTS: • Bacterial composition differed between the two brands of cigarettes. • Mentholation impacts cigarette microbiota. • Pseudomonas and Bacillus dominated the commercial cigarettes. Graphical abstract.

Temporal instability of the post-surgical maxillary sinus microbiota.

BACKGROUND: Chronic rhinosinusitis is an inflammatory disorder in which the role of bacteria remains uncertain. While sinus outflow obstruction is often an initiating event, mucosal inflammation and dysbiosis may persist or develop in sinuses with widely patent surgical openings. Understanding of the relationship between dysbiosis and chronic sinus inflammation is obfuscated by inter-individual microbiota variability and likely intra-individual temporal variation that has yet to be defined. In this study, long-term microbiota stability is investigated within surgically-opened maxillary sinuses of individuals with and without sinus inflammatory disease. METHODS: Maxillary sinus swabs were performed in 35 subjects with longstanding maxillary antrostomies. Subjects with and without active chronic maxillary sinusitis were included. Repeat swabs were obtained from the same sinuses after a prolonged interval (mean 719 ± 383 days). Patients were categorized based on the inflammatory status of the sinus mucosa at times of sample collection, as assessed by nasal endoscopy. Total DNA from swab eluents was extracted, and the microbiota characterized using 16S rRNA gene sequencing followed by taxonomic classification. Prevalence and abundance of genera were determined by analysis of 16S rRNA gene sequences. Taxa were identified that were stably present between two time points in individual subjects. RESULTS: The overall proportion of stable taxa across time points was 24.5 ± 10.6%. This stability index was consistent across patient groups and not correlated with clinical parameters. Highly prevalent taxa, including Staphylococcus, Corynebacterium, Propionibacterium, and Pseudomonas, were often stably present, but varied in relative abundance. Janthinobacterium, Enterobacter, Lactobacillus, and Acinetobacter were prevalent and moderately abundant taxa in healthy sinuses, but not in inflamed sinuses. Moraxella and Haemophilus were present at low prevalence and proportional abundance in chronically or intermittently inflamed sinuses, but not in healthy sinuses. CONCLUSIONS: A relatively small component of the post-antrostomy maxillary sinus microbiota exhibits long-term stability in individual subjects. Stable bacteria include a limited number of highly prevalent and a larger number of lower prevalence taxa, which vary widely in proportional abundance. The concept of individual-specific core sinus microbiota, durable over time and medical therapy, but fluctuating in proportional abundance, has implications for understanding the role of bacteria in CRS pathogenesis.

Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance.

BACKGROUND: With an increase in the use of colistin methansulfonate (CMS) to treat carbapenem-resistant Acinetobacter baumannii infections, colistin resistance is emerging. METHODS: Patients with infection or colonization due to colistin-resistant A. baumannii were identified at a hospital system in Pennsylvania. Clinical data were collected from electronic medical records. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. To investigate the mechanism of colistin resistance, lipid A was subjected to matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: Twenty patients with colistin-resistant A. baumannii were identified. Ventilator-associated pneumonia was the most common type of infection. Nineteen patients had received intravenous and/or inhaled CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection prior to identification of colistin-resistant isolates. The 30-day all-cause mortality rate was 30%. The treatment regimen for colistin-resistant A. baumannii infection associated with the lowest mortality rate was a combination of CMS, a carbapenem, and ampicillin-sulbactam. The colistin-susceptible and -resistant isolates from the same patients were highly related by PFGE, but isolates from different patients were not, suggesting evolution of resistance during CMS therapy. By MLST, all isolates belonged to the international clone II, the lineage that is epidemic worldwide. Phosphoethanolamine modification of lipid A was present in all colistin-resistant A. baumannii isolates. CONCLUSIONS: Colistin-resistant A. baumannii occurred almost exclusively among patients who had received CMS for treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection. Lipid A modification by the addition of phosphoethanolamine accounted for colistin resistance. Susceptibility testing for colistin should be considered for A. baumannii identified from CMS-experienced patients.

Genotypic and phenotypic analyses of a Pseudomonas aeruginosa chronic bronchiectasis isolate reveal differences from cystic fibrosis and laboratory strains.

BACKGROUND: Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. RESULTS: This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to ß-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. CONCLUSIONS: This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.

A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis-specific lipid A.

Strains of Pseudomonas aeruginosa (PA) isolated from the airways of cystic fibrosis patients constitutively add palmitate to lipid A, the membrane anchor of lipopolysaccharide. The PhoPQ regulated enzyme PagP is responsible for the transfer of palmitate from outer membrane phospholipids to lipid A. This enzyme had previously been identified in many pathogenic Gram-negative bacteria, but in PA had remained elusive, despite abundant evidence that its lipid A contains palmitate. Using a combined genetic and biochemical approach, we identified PA1343 as the PA gene encoding PagP. Although PA1343 lacks obvious primary structural similarity with known PagP enzymes, the ß-barrel tertiary structure with an interior hydrocarbon ruler appears to be conserved. PA PagP transfers palmitate to the 3' position of lipid A, in contrast to the 2 position seen with the enterobacterial PagP. Palmitoylated PA lipid A alters host innate immune responses, including increased resistance to some antimicrobial peptides and an elevated pro-inflammatory response, consistent with the synthesis of a hexa-acylated structure preferentially recognized by the TLR4/MD2 complex. Palmitoylation commonly confers resistance to cationic antimicrobial peptides, however, increased cytokine production resulting in inflammation is not seen with other palmitoylated lipid A, indicating a unique role for this modification in PA pathogenesis.

Enzymatic modification of lipid A by ArnT protects Bordetella bronchiseptica against cationic peptides and is required for transmission.

Pathogen transmission cycles require many steps: initial colonization, growth and persistence, shedding, and transmission to new hosts. Alterations in the membrane components of the bacteria, including lipid A, the membrane anchor of lipopolysaccharide, could affect any of these steps via its structural role protecting bacteria from host innate immune defenses, including antimicrobial peptides and signaling through Toll-like receptor 4 (TLR4). To date, lipid A has been shown to affect only the within-host dynamics of infection, not the between-host dynamics of transmission. Here, we investigate the effects of lipid A modification in a mouse infection and transmission model. Disruption of the Bordetella bronchiseptica locus (BB4268) revealed that ArnT is required for addition of glucosamine (GlcN) to B. bronchiseptica lipid A. ArnT modification of lipid A did not change its TLR4 agonist activity in J774 cells, but deleting arnT decreased resistance to killing by cationic antimicrobial peptides, such as polymyxin B and ß-defensins. In the standard infection model, mutation of arnT did not affect B. bronchiseptica colonization, growth, persistence throughout the respiratory tract, recruitment of neutrophils to the nasal cavity, or shedding of the pathogen. However, the number of bacteria necessary to colonize a host (50% infective dose [ID50]) was 5-fold higher for the arnT mutant. Furthermore, the arnT mutant was defective in transmission between hosts. These results reveal novel functions of the ArnT lipid A modification and highlight the sensitivity of low-dose infections and transmission experiments for illuminating aspects of infectious diseases between hosts. Factors such as ArnT can have important effects on the burden of disease and are potential targets for interventions that can interrupt transmission.

A Bacteriophage Cocktail Targeting Yersinia pestis Provides Strong Post-Exposure Protection in a Rat Pneumonic Plague Model.

Yersinia pestis , one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug resistant strains. The dissemination of such Y. pestis strains could be catastrophic, with public health consequences far more daunting than those caused by the recent COVID-19 pandemic. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines, none FDA approved yet, may not be effective, and those that cannot be controlled by approved antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation with a 100% lytic activity against a panel of 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated ∼88% protection when delivered 18 hours post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies suggest that YPP-401 could provide an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed strains that cannot be managed by vaccines in development and antibiotics.

A bacteriophage cocktail targeting Yersinia pestis provides strong post-exposure protection in a rat pneumonic plague model.

Yersinia pestis, one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug-resistant strains. Indeed, antibiotic-resistant strains (e.g., strains carrying multidrug-resistant or MDR plasmids) have been isolated in various countries and endemic areas. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines (none FDA approved yet) may not be effective and those that cannot be managed by currently available antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation effective against a panel of at least 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated ~88% protection when delivered 18 h post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies provide proof-of-concept that YPP-401 could be an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed multidrug-resistant strains.IMPORTANCECurrently, there are no FDA-approved plague vaccines. Since antibiotic-resistant strains of Y. pestis have emerged or are being intentionally developed to be used as a biothreat agent, new treatment modalities are direly needed. Phage therapy provides a viable option against potentially antibiotic-resistant strains. Additionally, phages are nontoxic and have been approved by the FDA for use in the food industry. Our study provides the first evidence of the protective effect of a cocktail of four phages against pneumonic plague, the most severe form of disease. When treatment was initiated 18 h post infection by either the intranasal or intraperitoneal route in Brown Norway rats, up to 87.5% protection was observed. The phage cocktail had a minimal impact on a representative human microbiome panel, unlike antibiotics. This study provides strong proof-of-concept data for the further development of phage-based therapy to treat plague.

Early Immunomodulatory Program Triggered by Protolerogenic Bifidobacterium pseudolongum Drives Cardiac Transplant Outcomes.

BACKGROUND: Despite ongoing improvements to regimens preventing allograft rejection, most cardiac and other organ grafts eventually succumb to chronic vasculopathy, interstitial fibrosis, or endothelial changes, and eventually graft failure. The events leading to chronic rejection are still poorly understood and the gut microbiota is a known driving force in immune dysfunction. We previously showed that gut microbiota dysbiosis profoundly influences the outcome of vascularized cardiac allografts and subsequently identified biomarker species associated with these differential graft outcomes. METHODS: In this study, we further detailed the multifaceted immunomodulatory properties of protolerogenic and proinflammatory bacterial species over time, using our clinically relevant model of allogenic heart transplantation. RESULTS: In addition to tracing longitudinal changes in the recipient gut microbiome over time, we observed that Bifidobacterium pseudolongum induced an early anti-inflammatory phenotype within 7 d, whereas Desulfovibrio desulfuricans resulted in a proinflammatory phenotype, defined by alterations in leukocyte distribution and lymph node (LN) structure. Indeed, in vitro results showed that B pseudolongum and D desulfuricans acted directly on primary innate immune cells. However, by 40 d after treatment, these 2 bacterial strains were associated with mixed effects in their impact on LN architecture and immune cell composition and loss of colonization within gut microbiota, despite protection of allografts from inflammation with B pseudolongum treatment. CONCLUSIONS: These dynamic effects suggest a critical role for early microbiota-triggered immunologic events such as innate immune cell engagement, T-cell differentiation, and LN architectural changes in the subsequent modulation of protolerant versus proinflammatory immune responses in organ transplant recipients.

Effective Treatment of Staphylococcus aureus Intramammary Infection in a Murine Model Using the Bacteriophage Cocktail StaphLyse™.

Staphylococcus aureus causes intramammary infections (IMIs), which are refractory to antibiotic treatment and frequently result in chronic mastitis. IMIs are the leading cause of conventional antibiotic use in dairy farms. Phage therapy represents an alternative to antibiotics to help better manage mastitis in cows, reducing the global spread of resistance. A mouse mastitis model of S. aureus IMI was used to study the efficacy of a new cocktail of five lytic S. aureus-specific phages (StaphLyse™), administered either via the intramammary (IMAM) route or intravenously (IV). The StaphLyse™ phage cocktail was stable in milk for up to one day at 37 °C and up to one week at 4 °C. The phage cocktail was bactericidal in vitro against S. aureus in a dose-dependent manner. A single IMAM injection of this cocktail given 8 h after infection reduced the bacterial load in the mammary glands of lactating mice infected with S. aureus, and as expected, a two-dose regimen was more effective. Prophylactic use (4 h pre-challenge) of the phage cocktail was also effective, reducing S. aureus levels by 4 log10 CFU per gram of mammary gland. These results suggest that phage therapy may be a viable alternative to traditional antibiotics for the control of S. aureus IMIs.

Strain-specific alterations in gut microbiome and host immune responses elicited by tolerogenic Bifidobacterium pseudolongum.

The beneficial effects attributed to Bifidobacterium are largely attributed to their immunomodulatory capabilities, which are likely to be species- and even strain-specific. However, their strain-specificity in direct and indirect immune modulation remain largely uncharacterized. We have shown that B. pseudolongum UMB-MBP-01, a murine isolate strain, is capable of suppressing inflammation and reducing fibrosis in vivo. To ascertain the mechanism driving this activity and to determine if it is specific to UMB-MBP-01, we compared it to a porcine tropic strain B. pseudolongum ATCC25526 using a combination of cell culture and in vivo experimentation and comparative genomics approaches. Despite many shared features, we demonstrate that these two strains possess distinct genetic repertoires in carbohydrate assimilation, differential activation signatures and cytokine responses signatures in innate immune cells, and differential effects on lymph node morphology with unique local and systemic leukocyte distribution. Importantly, the administration of each B. pseudolongum strain resulted in major divergence in the structure, composition, and function of gut microbiota. This was accompanied by markedly different changes in intestinal transcriptional activities, suggesting strain-specific modulation of the endogenous gut microbiota as a key to immune modulatory host responses. Our study demonstrated a single probiotic strain can influence local, regional, and systemic immunity through both innate and adaptive pathways in a strain-specific manner. It highlights the importance to investigate both the endogenous gut microbiome and the intestinal responses in response to probiotic supplementation, which underpins the mechanisms through which the probiotic strains drive the strain-specific effect to impact health outcomes.

Effect of mupirocin for Staphylococcus aureus decolonization on the microbiome of the nose and throat in community and nursing home dwelling adults.

OBJECTIVE: To characterize the microbial communities of the anterior nares (nose) and posterior pharynx (throat) of adults dwelling in the community and in nursing homes before and after treatment with intranasal mupirocin. METHODS: Staphylococcus aureus-colonized adults were recruited from the community (n = 25) and from nursing homes (n = 7). S. aureus colonization was confirmed using cultures. Participants had specimens taken from nose and throat for S. aureus quantitation using quantitative PCR for the nuc gene and bacterial profiling using 16S rRNA gene sequencing over 12 weeks. After two baseline study visits 4 weeks apart, participants received intranasal mupirocin for 5 days with 3 further visits over a 8 week follow-up period. RESULTS: We found a decrease in the absolute abundance of S. aureus in the nose for 8 weeks after mupirocin (1693 vs 141 fg/ul, p = 0.047). Mupirocin caused a statistically significant disruption in bacterial communities of the nose and throat after 1 week, which was no longer detected after 8 weeks. Bacterial community profiling demonstrated that there was a decrease in the relative abundance of S. aureus (8% vs 0.3%, p<0.01) 8 weeks after mupirocin and a transient decrease in the relative abundance of Staphylococcus epidermidis in the nose (21% vs 5%, p<0.01) 1 week after mupirocin. CONCLUSIONS: Decolonization with mupirocin leads to a sustained effect on absolute and relative abundance of S. aureus but not for other bacteria in the nose. This demonstrates that a short course of mupirocin selectively decreases S. aureus in the nose for up to 8 weeks.

Potential role of the skin and gut microbiota in premenarchal vulvar lichen sclerosus: A pilot case-control study.

The etiology of vulvar lichen sclerosus (LS) remains unclear; however, alterations in cutaneous and gut microbiota may be contributing to the pathogenesis of this inflammatory condition. To explore this hypothesis, we conducted a pilot case-control study, obtaining dermal swab and stool samples from prepubertal girls with vulvar LS (n = 5), girls with nonspecific vulvovaginitis (n = 5), and healthy controls (n = 3). Samples (n = 56) were subjected to total DNA extractions. Resulting DNA was purified, subjected to PCR (targeting the V3V4 region of the 16S rRNA gene), sequenced, and analyzed using QIIME, MetagenomeSeq, and DESeq2 software packages. Our findings showed that there were significant differences in the cutaneous and gut microbiotas of girls with LS compared to controls. On the skin, girls with LS had a statistically significantly higher relative abundance of Porphyromonas spp., Parvimonas spp., Peptoniphilus spp., Prevotella spp., Dialister spp., and Peptostreptococcus spp., but a lower relative abundance of Cornyebacterium compared to the control group. In the gut samples, girls with LS had a significantly higher relative abundance of Dialister spp., Clostridiales spp., Paraprevotella spp., Escherichia coli, Bifidobacterium adolescentis, and Akkermansia muciniphila, and a lower relative abundance of Roseburia faecis and Ruminococcus bromii compared to controls. These results suggest a potential association between cutaneous and gut dysbiosis and pediatric vulvar LS. Future studies involving larger samples sizes are warranted to further evaluate this association.

Low Diversity in Nasal Microbiome Associated With Staphylococcus aureus Colonization and Bloodstream Infections in Hospitalized Neonates.

BACKGROUND: Staphylococcus aureus is a leading cause of infectious morbidity and mortality in neonates. Few data exist on the association of the nasal microbiome and susceptibility to neonatal S. aureus colonization and infection. METHODS: We performed 2 matched case-control studies (colonization cohort-neonates who did and did not acquire S. aureus colonization; bacteremia cohort-neonates who did [colonized neonates] and did not [controls] acquire S. aureus colonization and neonates with S. aureus bacteremia [bacteremic neonantes]). Neonates in 2 intensive care units were enrolled and matched on week of life at time of colonization or infection. Nasal samples were collected weekly until discharge and cultured for S. aureus, and the nasal microbiome was characterized using 16S rRNA gene sequencing. RESULTS: In the colonization cohort, 43 S. aureus-colonized neonates were matched to 82 controls. At 1 week of life, neonates who acquired S. aureus colonization had lower alpha diversity (Wilcoxon rank-sum test P < .05) and differed in beta diversity (omnibus MiRKAT P = .002) even after adjusting for birth weight (P = .01). The bacteremia cohort included 10 neonates, of whom 80% developed bacteremia within 4 weeks of birth and 70% had positive S. aureus cultures within a few days of bacteremia. Neonates with bacteremia had an increased relative abundance of S. aureus sequences and lower alpha diversity measures compared with colonized neonates and controls. CONCLUSIONS: The association of increased S. aureus abundance and decrease of microbiome diversity suggest the need for interventions targeting the nasal microbiome to prevent S. aureus disease in vulnerable neonates.

Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing.

Understanding the complex microbiota of agricultural irrigation water is vital to multiple sectors of sustainable agriculture and public health. To date, microbiome characterization methods have provided comprehensive profiles of aquatic microbiotas, but have not described which taxa are likely metabolically-active. Here, we combined 5­bromo­2'-deoxyuridine (BrdU) labeling with 16S rRNA and shotgun sequencing to identify metabolically-active bacteria in reclaimed and agricultural pond water samples (n = 28) recovered from the Mid-Atlantic United States between March 2017 and January 2018. BrdU-treated samples were significantly less diverse (alpha diversity) compared to non-BrdU-treated samples. The most abundant taxa in the metabolically-active fraction of water samples (BrdU-treated samples) were unclassified Actinobacteria, Flavobacterium spp., Pseudomonas spp. and Aeromonas spp. Interestingly, we also observed that antimicrobial resistance and virulence gene profiles seemed to be more diverse and more abundant in non-BrdU-treated water samples compared to BrdU-treated samples. These findings raise the possibility that these genes may be associated more with relic (inactive) DNA present in the tested water types rather than viable, metabolically-active microorganisms. Our study demonstrates that the coupled use of BrdU labeling and sequencing can enhance understanding of the metabolically-active fraction of bacterial communities in alternative irrigation water sources. Agricultural pond and reclaimed waters are vital to the future of sustainable agriculture, and thus, the full understanding of the pathogenic potential of these waters is important to guide mitigation strategies that ensure appropriate water quality for intended purposes.

The bacterial communities of the small intestine and stool in children with short bowel syndrome.

Short bowel syndrome (SBS) presents an increasing problem in pediatrics. SBS often results from surgical resection of necrotic bowel following necrotizing enterocolitis or treatment of anatomic gastrointestinal defects. SBS is associated with significant morbidity and mortality, and creates substantial burdens for patients, families, and the health system. Recent reports have demonstrated that the fecal microbiome of children with SBS is significantly different from healthy control and severe intestinal microbial imbalances is associated with poor growth. We hypothesized that children with SBS and adverse clinical features such as PN dependent, shorter bowel length and lack of ileocecal valve would demonstrate more gut dysbiosis compare with the SBS non-PN dependent. An improved understanding of SBS pathogenesis would enhance management and potentially suggest new interventions. We studied microbial communities of SBS and control non-SBS patients from the jejunum, obtained endoscopically or by ostomy aspiration, and stool. We enrolled SBS patients who did and did not require parenteral nutrition (PN), as a surrogate marker for the seriousness of their disease. We studied the microbiota using high-throughput DNA sequencing of 16S rRNA genes and statistical analyses. We found that microbial diversity was significantly greater in jejunal aspirate than in stool samples in SBS patients, unlike non-SBS patients; that SBS patients receiving enteral feeds had greater diversity, and that SBS patients on PN and enteral feeds had lower differences in diversity in jejunal vs. stool samples. We found a trend toward increased diversity in patients with an intact ileocecal valve, and found that certain taxa were more abundant in the certain sample types, and in SBS patients vs. non-SBS patients. SBS patients have lower microbial diversity, especially patients with more severe disease, patients requiring PN, and those lacking an ileocecal valve. SBS patients, particularly those with more complex characteristics, exhibit differences in their intestinal microbiota. Particular individual taxa were over- and under-represented in patients with more unfavorable disease. While diminished diversity and alterations in microbiota composition are likely consequences of SBS, future efforts aimed at increasing microbial diversity and interventions targeting specific microbiota characteristics might constitute a testable approach to ameliorate some clinical SBS clinical consequences.

The Association Between the Developing Nasal Microbiota of Hospitalized Neonates and Staphylococcus aureus Colonization.

BACKGROUND: Hospitalized neonates are at high risk for invasive Staphylococcus aureus infections. S. aureus nasal colonization often precedes infection. The nasal microbiota may preclude or support colonization. We aimed to characterize and compare the nasal microbiota of hospitalized neonates who acquire S. aureus colonization (cases) and those who do not acquire S. aureus (controls). METHODS: We obtained residual nares samples from hospitalized neonates who were screened weekly for S. aureus nasal colonization and treated with intranasal mupirocin if colonized. Eight cases were matched based on chronologic age and systemic antibiotic exposure to 7 controls. We extracted DNA, sequenced the V3-V4 region of the 16s rRNA gene, and performed taxonomic assignments. The bacterial species richness, relative abundance, and in silico predicted gene content were compared between cases and controls at 7 days before S. aureus acquisition, at the time of acquisition, and 7 days after acquisition and treatment. RESULTS: Common commensals including nondiphtheriae corynebacteria were more abundant in the nares of controls and Rothia mucilaginosa was more abundant in cases 7 days after intranasal mupirocin treatment than in cases 7 days before S. aureus acquisition. Controls and treated cases had a higher predicted abundance of genes contributing to the synthesis of certain antimicrobial compounds than in cases before S. aureus acquisition. CONCLUSIONS: Neonates without S. aureus nasal colonization had a higher abundance of bacterial species that antagonize S. aureus directly or by selecting for beneficial co-colonizers. These differences may inform novel S. aureus infection prevention strategies in high-risk infants.

Little cigars and cigarillos harbor diverse bacterial communities that differ between the tobacco and the wrapper.

Despite their potential importance with regard to infectious and chronic diseases among tobacco users, microbial constituents of tobacco products lack characterization. Specifically, to our knowledge, there are no data describing the bacterial diversity of little cigars or cigarillos. To address this knowledge gap, we tested four brands of little cigars and cigarillos. Tobacco and wrapper subsamples (n = 132) were separately subjected to DNA extraction, followed by PCR amplification of the V3V4 hypervariable region of the 16S rRNA gene, and sequencing using Illumina HiSeq. Sequences were analyzed using QIIME and Phyloseq implemented in R. We identified 2,681 operational taxonomic units across all products. Significant differences in alpha and beta diversity were observed between Swisher Sweets and Cheyenne products. Alpha and beta diversity was also significantly different between tobacco and wrapper subsamples within the same product. Beta diversity analyses of only tobacco samples identified no significant differences in the bacterial microbiota of different lots of the same products; however, the microbiota in the wrapper differed significantly across lots for all brands. Overall, Firmicutes were found to dominate in the wrapper, whereas Proteobacteria were most abundant in the tobacco. At the genus level, Bacillus and Lactobacillus dominated in the wrappers, and Staphylococcus and Pseudomonas dominated in the tobacco. Our findings suggest that the bacterial microbiota of little cigars and cigarillos is diverse and differs significantly between the tobacco and the wrapper, and across brands. Future work is necessary to evaluate the potential public health implications of these findings.

The Bacterial Communities of Little Cigars and Cigarillos Are Dynamic Over Time and Varying Storage Conditions.

Despite their potential importance with regard to tobacco-related health outcomes, as well as their hypothesized role in the production of tobacco-specific N-nitrosamines, bacterial constituents of tobacco products lack characterization. Specifically, to our knowledge, there has been no comprehensive characterization of the effects of storage conditions on the bacterial communities associated with little cigars and cigarillos. To address this knowledge gap, we characterized the bacterial community composition of the tobacco and wrapper components of the following four products: Swisher Sweets Original; Swisher Sweets, Sweet Cherry; Cheyenne Cigars Full Flavor 100's; and Cheyenne Menthol Box. Each product was stored under three different conditions of temperature and relative humidity to mimic different user storage conditions: room (20°C 50% RH), refrigerator (5°C 18% RH) and pocket (25°C 30% RH). On days 0, 5, 9 and 14, subsamples were collected, the wrapper and tobacco were separated, and their total DNA was extracted separately and purified. Resulting DNA was then used in PCR assays targeting the V3 V4 region of the bacterial 16S rRNA gene, followed by sequencing using Illumina HiSeq 300bp PE. Resulting sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package, followed by analyses in R using the Phyloseq and Vegan packages. A single bacterial phylum, Firmicutes, dominated in the wrapper subsamples whereas the tobacco subsamples were dominated by Proteobacteria. Cheyenne Menthol Box (CMB) samples were characterized by significant differential abundances for 23 bacterial operational taxonomic units (OTUs) in tobacco subsamples and 27 OTUs in the wrapper subsamples between day 0 and day 14 under all conditions. OTUs from the genera Acinetobacter and Bacillus significantly increased in the CMB tobacco subsamples, and OTUs from Bacillus, Streptococcus, Lactobacillus, and Enterococcus significantly increased in the CMB wrapper subsamples over time. These initial results suggest that the bacterial communities of little cigars and cigarillos are dynamic over time and varying storage conditions.