The gut metagenome harbors metabolic and antibiotic resistance signatures of moderate-to-severe asthma.

Asthma is a common allergic airway disease that has been associated with the development of the human microbiome early in life. Both the composition and function of the infant gut microbiota have been linked to asthma risk, but functional alterations in the gut microbiota of older patients with established asthma remain an important knowledge gap. Here, we performed whole metagenomic shotgun sequencing of 95 stool samples from a cross-sectional cohort of 59 healthy and 36 subjects with moderate-to-severe asthma to characterize the metagenomes of gut microbiota in adults and children 6 years and older. Mapping of functional orthologs revealed that asthma contributes to 2.9% of the variation in metagenomic content even when accounting for other important clinical demographics. Differential abundance analysis showed an enrichment of long-chain fatty acid (LCFA) metabolism pathways, which have been previously implicated in airway smooth muscle and immune responses in asthma. We also observed increased richness of antibiotic resistance genes (ARGs) in people with asthma. Several differentially abundant ARGs in the asthma cohort encode resistance to macrolide antibiotics, which are often prescribed to patients with asthma. Lastly, we found that ARG and virulence factor (VF) richness in the microbiome were correlated in both cohorts. ARG and VF pairs co-occurred in both cohorts suggesting that virulence and antibiotic resistance traits are coselected and maintained in the fecal microbiota of people with asthma. Overall, our results show functional alterations via LCFA biosynthetic genes and increases in antibiotic resistance genes in the gut microbiota of subjects with moderate-to-severe asthma and could have implications for asthma management and treatment.

Everything but the Kitchen Sink: An Analysis of Bacterial and Chemical Contaminants Found in Syringe Residue From People Who Inject Drugs.

Background: People who inject drugs (PWID) are at high risk of severe wounds, invasive infections, and overdoses. To date, there are few data on the bacterial and chemical contaminants PWID are exposed to when using illicitly manufactured fentanyls and stimulants. Methods: Previously used injection drug use equipment was recovered in St Louis, Missouri, by harm reduction organizations over a 12-month period. Syringe residue was analyzed for bacterial contaminants by routine culturing followed by whole genome sequencing of single bacterial isolates. Chemical adulterants in syringe residue were identified by liquid chromatography-mass spectrometry. Results: Bacteria were cultured from 58.75% of 160 syringes analyzed. Polymicrobial growth was common and was observed in 23.75% of samples. Bacillus cereus was the most common pathogen present and was observed in 20.6% of syringe residues, followed closely by Staphylococcus aureus at 18.8%. One hundred syringes underwent mass spectrometry, which demonstrated that chemical adulterants were common and included caffeine, diphenhydramine, lidocaine, quinine, and xylazine. Conclusions: Analysis of syringe residue from discarded drug use equipment demonstrates both chemical and biological contaminants, including medically important pathogens and adulterants.

Gut microbiome and antibiotic resistance effects during travelers' diarrhea treatment and prevention.

IMPORTANCE: The travelers' gut microbiome is potentially assaulted by acute and chronic perturbations (e.g., diarrhea, antibiotic use, and different environments). Prior studies of the impact of travel and travelers' diarrhea (TD) on the microbiome have not directly compared antibiotic regimens, and studies of different antibiotic regimens have not considered travelers' microbiomes. This gap is important to be addressed as the use of antibiotics to treat or prevent TD-even in moderate to severe cases or in regions with high infectious disease burden-is controversial based on the concerns for unintended consequences to the gut microbiome and antimicrobial resistance (AMR) emergence. Our study addresses this by evaluating the impact of defined antibiotic regimens (single-dose treatment or daily prophylaxis) on the gut microbiome and resistomes of deployed servicemembers, using samples collected during clinical trials. Our findings indicate that the antibiotic treatment regimens that were studied generally do not lead to adverse effects on the gut microbiome and resistome and identify the relative risks associated with prophylaxis. These results can be used to inform therapeutic guidelines for the prevention and treatment of TD and make progress toward using microbiome information in personalized medical care.

Effect of amoxicillin on the gut microbiome of children with severe acute malnutrition in Madarounfa, Niger: a retrospective metagenomic analysis of a placebo-controlled trial.

BACKGROUND: Children with severe acute malnutrition are treated with antibiotics as outpatients. We aimed to determine the effect of 7 days of amoxicillin on acute and long-term changes to the gut microbiome and antibiotic resistome in children treated for severe acute malnutrition. METHODS: We conducted a secondary analysis of a randomised, double-blinded, placebo-controlled trial (NCT01613547) of amoxicillin in children (aged 6-59 months) with severe acute malnutrition treated as outpatients in Madarounfa, Niger. We randomly selected 161 children from the overall cohort (n=2399) for initial 12-week follow-up from Sept 23, 2013 to Feb 3, 2014. We selected a convenience sample of those 161 children, on the basis of anthropometric measures, for follow-up 2 years later (Sept 28 to Oct 27, 2015). Children provided faecal samples at baseline, week 1, week 4, week 8, week 12, and, for those in the 2-year follow-up cohort, week 104. We conducted metagenomic sequencing followed by microbiome and resistome profiling of faecal samples. 38 children without severe acute malnutrition and six children with severe acute malnutrition matching the baseline ages of the original cohort were used as reference controls. FINDINGS: In the 12-week follow-up group, amoxicillin led to an immediate decrease in gut microbiome richness from 37·6 species (95% CI 32·6-42·7) and Shannon diversity index (SDI) 2·18 (95% CI 1·97-2·39) at baseline to 27·7 species (95% CI 22·9-32·6) species and SDI 1·55 (95% CI 1·35-1·75) at week 1. Amoxicillin increased gut antibiotic resistance gene abundance to 6044 reads per kilobase million (95% CI 4704-7384) at week 1, up from 4800 (3391-6208) at baseline, which returned to baseline 3 weeks later. 35 children were included in the 2-year follow-up; the amoxicillin-treated children (n=22) had increased number of species in the gut microbiome compared with placebo-treated children (n=13; 60·7 [95% CI 54·7-66·6] vs 36·9 [29·4-44·3]). Amoxicillin-treated children had increased Prevotella spp and decreased Bifidobacterium spp relative to age-matched placebo-treated children, indicating a more mature, adult-like microbiome. INTERPRETATION: Amoxicillin treatment led to acute but not sustained increases in antimicrobial resistance genes and improved gut microbiome maturation 2 years after severe acute malnutrition treatment. FUNDING: Bill & Melinda Gates Foundation; Médecins sans Frontières Operational Center Paris; National Institute of Allergy and Infectious Diseases; National Institute of General Medical Sciences; Eunice Kennedy Shriver National Institute of Child Health and Human Development; Edward Mallinckrodt Jr Foundation; Doris Duke Foundation.

Gut pathogen colonization precedes bloodstream infection in the neonatal intensive care unit.

Bacterial bloodstream infections (BSIs) resulting in late-onset sepsis affect up to half of extremely preterm infants and have substantial morbidity and mortality. Bacterial species associated with BSIs in neonatal intensive care units (NICUs) commonly colonize the preterm infant gut microbiome. Accordingly, we hypothesized that the gut microbiome is a reservoir of BSI-causing pathogenic strains that increase in abundance before BSI onset. We analyzed 550 previously published fecal metagenomes from 115 hospitalized neonates and found that recent ampicillin, gentamicin, or vancomycin exposure was associated with increased abundance of Enterobacteriaceae and Enterococcaceae in infant guts. We then performed shotgun metagenomic sequencing on 462 longitudinal fecal samples from 19 preterm infants (cases) with BSI and 37 non-BSI controls, along with whole-genome sequencing of the BSI isolates. Infants with BSI caused by Enterobacteriaceae were more likely than infants with BSI caused by other organisms to have had ampicillin, gentamicin, or vancomycin exposure in the 10 days before BSI. Relative to controls, gut microbiomes of cases had increased relative abundance of the BSI-causing species and clustered by Bray-Curtis dissimilarity according to BSI pathogen. We demonstrated that 11 of 19 (58%) of gut microbiomes before BSI, and 15 of 19 (79%) of gut microbiomes at any time, harbored the BSI isolate with fewer than 20 genomic substitutions. Last, BSI strains from the Enterobacteriaceae and Enterococcaceae families were detected in multiple infants, indicating BSI-strain transmission. Our findings support future studies to evaluate BSI risk prediction strategies based on gut microbiome abundance in hospitalized preterm infants.

The gut metagenome harbors metabolic and antibiotic resistance signatures of moderate-to-severe asthma.

Asthma is a common allergic airway disease that develops in association with the human microbiome early in life. Both the composition and function of the infant gut microbiota have been linked to asthma risk, but functional alterations in the gut microbiota of older patients with established asthma remain an important knowledge gap. Here, we performed whole metagenomic shotgun sequencing of 95 stool samples from 59 healthy and 36 subjects with moderate-to-severe asthma to characterize the metagenomes of gut microbiota in children and adults 6 years and older. Mapping of functional orthologs revealed that asthma contributes to 2.9% of the variation in metagenomic content even when accounting for other important clinical demographics. Differential abundance analysis showed an enrichment of long-chain fatty acid (LCFA) metabolism pathways which have been previously implicated in airway smooth muscle and immune responses in asthma. We also observed increased richness of antibiotic resistance genes (ARGs) in people with asthma. One differentially abundant ARG was a macrolide resistance marker, ermF, which significantly co-occurred with the Bacteroides fragilis toxin, suggesting a possible relationship between enterotoxigenic B. fragilis, antibiotic resistance, and asthma. Lastly, we found multiple virulence factor (VF) and ARG pairs that co-occurred in both cohorts suggesting that virulence and antibiotic resistance traits are co-selected and maintained in the fecal microbiota of people with asthma. Overall, our results show functional alterations via LCFA biosynthetic genes and increases in antibiotic resistance genes in the gut microbiota of subjects with moderate-to-severe asthma and could have implications for asthma management and treatment.

ENDOGENOUS ENDOPHTHALMITIS CAUSED BY GROUP B STREPTOCOCCUS IN A HEALTHY, TERM NEONATE.

PURPOSE: The purpose of this study was to describe an unusual case of unilateral, endogenous endophthalmitis in an otherwise healthy, term neonate. METHODS: A 3-week-old otherwise healthy, term male infant was referred to St. Louis Children's Hospital for a second opinion of presumed panuveitis of the right eye. RESULTS: Diffusion-weighted magnetic resonance imaging demonstrating purulent intraocular contents facilitated the diagnosis of endophthalmitis. Examination of surgical vitreous samples by staining and cytology demonstrated gram-positive bacterial cocci in short chains, thereby confirming endophthalmitis. Polymerase chain reaction testing of vitreous fluid identified Streptococcus agalactiae , despite an unremarkable systemic workup and a negative prepartum maternal Group B streptococcal screen. CONCLUSION: Endogenous endophthalmitis is a rare but devastating cause of vision loss in otherwise healthy, term neonates. Prompt diagnosis may be facilitated by magnetic resonance imaging and diagnostic vitreous biopsy.

Impact of international travel and diarrhea on gut microbiome and resistome dynamics.

International travel contributes to the global spread of antimicrobial resistance. Travelers' diarrhea exacerbates the risk of acquiring multidrug-resistant organisms and can lead to persistent gastrointestinal disturbance post-travel. However, little is known about the impact of diarrhea on travelers' gut microbiomes, and the dynamics of these changes throughout travel. Here, we assembled a cohort of 159 international students visiting the Andean city of Cusco, Peru and applied next-generation sequencing techniques to 718 longitudinally-collected stool samples. We find that gut microbiome composition changed significantly throughout travel, but taxonomic diversity remained stable. However, diarrhea disrupted this stability and resulted in an increased abundance of antimicrobial resistance genes that can remain high for weeks. We also identified taxa differentially abundant between diarrheal and non-diarrheal samples, which were used to develop a classification model that distinguishes between these disease states. Additionally, we sequenced the genomes of 212 diarrheagenic Escherichia coli isolates and found those from travelers who experienced diarrhea encoded more antimicrobial resistance genes than those who did not. In this work, we find the gut microbiomes of international travelers' are resilient to dysbiosis; however, they are also susceptible to colonization by multidrug-resistant bacteria, a risk that is more pronounced in travelers with diarrhea.

The resistance within: Antibiotic disruption of the gut microbiome and resistome dynamics in infancy.

Intestinal host-microbiota interactions during the first year of life are critical for infant development. Early-life antibiotic exposures disrupt stereotypical gut microbiota maturation and adversely affect childhood health. Furthermore, antibiotics increase the abundance of resistant bacteria and enrich the resistome-the compendium of antibiotic resistance genes-within the gut microbiota. Here, we discuss acute and persistent impacts of antibiotic exposure during infancy on pediatric health, the gut microbiome, and, particularly, the resistome. Reviewing our current understanding of antibiotic resistance acquisition and dissemination within and between microbiomes, we highlight open questions, which are imperative to resolve in the face of rising bacterial resistance.

Taxonomic changes in the gut microbiota are associated with cartilage damage independent of adiposity, high fat diet, and joint injury.

Lipodystrophic mice are protected from cartilage damage following joint injury. This protection can be reversed by the implantation of a small adipose tissue graft. The purpose of this study was to evaluate the relationship between the gut microbiota and knee cartilage damage while controlling for adiposity, high fat diet, and joint injury using lipodystrophic (LD) mice. LD and littermate control (WT) mice were fed a high fat diet, chow diet, or were rescued with fat implantation, then challenged with destabilization of the medial meniscus surgery to induce osteoarthritis (OA). 16S rRNA sequencing was conducted on feces. MaAslin2 was used to determine associations between taxonomic relative abundance and OA severity. While serum LPS levels between groups were similar, synovial fluid LPS levels were increased in both limbs of HFD WT mice compared to all groups, except for fat transplanted animals. The Bacteroidetes:Firmicutes ratio of the gut microbiota was significantly reduced in HFD and OA-rescued animals when compared to chow. Nine novel significant associations were found between gut microbiota taxa and OA severity. These findings suggest the presence of causal relationships the gut microbiome and cartilage health, independent of diet or adiposity, providing potential therapeutic targets through manipulation of the microbiome.

Microbiota restoration reduces antibiotic-resistant bacteria gut colonization in patients with recurrent Clostridioides difficile infection from the open-label PUNCH CD study.

BACKGROUND: Once antibiotic-resistant bacteria become established within the gut microbiota, they can cause infections in the host and be transmitted to other people and the environment. Currently, there are no effective modalities for decreasing or preventing colonization by antibiotic-resistant bacteria. Intestinal microbiota restoration can prevent Clostridioides difficile infection (CDI) recurrences. Another potential application of microbiota restoration is suppression of non-C. difficile multidrug-resistant bacteria and overall decrease in the abundance of antibiotic resistance genes (the resistome) within the gut microbiota. This study characterizes the effects of RBX2660, a microbiota-based investigational therapeutic, on the composition and abundance of the gut microbiota and resistome, as well as multidrug-resistant organism carriage, after delivery to patients suffering from recurrent CDI. METHODS: An open-label, multi-center clinical trial in 11 centers in the USA for the safety and efficacy of RBX2660 on recurrent CDI was conducted. Fecal specimens from 29 of these subjects with recurrent CDI who received either one (N = 16) or two doses of RBX2660 (N = 13) were analyzed secondarily. Stool samples were collected prior to and at intervals up to 6 months post-therapy and analyzed in three ways: (1) 16S rRNA gene sequencing for microbiota taxonomic composition, (2) whole metagenome shotgun sequencing for functional pathways and antibiotic resistome content, and (3) selective and differential bacterial culturing followed by isolate genome sequencing to longitudinally track multidrug-resistant organisms. RESULTS: Successful prevention of CDI recurrence with RBX2660 correlated with taxonomic convergence of patient microbiota to the donor microbiota as measured by weighted UniFrac distance. RBX2660 dramatically reduced the abundance of antibiotic-resistant Enterobacteriaceae in the 2 months after administration. Fecal antibiotic resistance gene carriage decreased in direct relationship to the degree to which donor microbiota engrafted. CONCLUSIONS: Microbiota-based therapeutics reduce resistance gene abundance and resistant organisms in the recipient gut microbiome. This approach could potentially reduce the risk of infections caused by resistant organisms within the patient and the transfer of resistance genes or pathogens to others. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01925417 ; registered on August 19, 2013.

Improving ICI outcomes with a little help from my microbial friends.

Gut microbiome composition correlates with responsiveness to immune checkpoint inhibitor therapy. In a recent study in Science, Baruch et al. manipulated gut microbiome composition in patients with refractory metastatic melanoma using fecal microbiota transplants. Fecal microbiota transplant was safe and partially effective in inducing remission in refractory patients.

Understanding the impact of antibiotic perturbation on the human microbiome.

The human gut microbiome is a dynamic collection of bacteria, archaea, fungi, and viruses that performs essential functions for immune development, pathogen colonization resistance, and food metabolism. Perturbation of the gut microbiome's ecological balance, commonly by antibiotics, can cause and exacerbate diseases. To predict and successfully rescue such perturbations, first, we must understand the underlying taxonomic and functional dynamics of the microbiome as it changes throughout infancy, childhood, and adulthood. We offer an overview of the healthy gut bacterial architecture over these life stages and comment on vulnerability to short and long courses of antibiotics. Second, the resilience of the microbiome after antibiotic perturbation depends on key characteristics, such as the nature, timing, duration, and spectrum of a course of antibiotics, as well as microbiome modulatory factors such as age, travel, underlying illness, antibiotic resistance pattern, and diet. In this review, we discuss acute and chronic antibiotic perturbations to the microbiome and resistome in the context of microbiome stability and dynamics. We specifically discuss key taxonomic and resistance gene changes that accompany antibiotic treatment of neonates, children, and adults. Restoration of a healthy gut microbial ecosystem after routine antibiotics will require rationally managed exposure to specific antibiotics and microbes. To that end, we review the use of fecal microbiota transplantation and probiotics to direct recolonization of the gut ecosystem. We conclude with our perspectives on how best to assess, predict, and aid recovery of the microbiome after antibiotic perturbation.

Complex interactions between the microbiome and cancer immune therapy.

Immuno-oncology has rapidly grown in the last thirty years, and immunotherapeutic agents are now approved to treat many disparate cancers. Immune checkpoint inhibitors (ICIs) are employed to augment cytotoxic anti-cancer activity by inhibiting negative regulatory elements of the immune system. Modulating the immune system to target neoplasms has improved survivability of numerous cancers in many individuals, but forecasting outcomes post therapy is difficult due to insufficient predictive biomarkers. Recently, the tumor and gastrointestinal microbiome and immune milieu have been investigated as predictors and influencers of cancer immune therapy. In this review, we discuss: (1) ways to measure the microbiome including relevant bioinformatic analyses, (2) recent developments in animal studies and human clinical trials utilizing gut microbial composition and function as biomarkers of cancer immune therapy response and toxicity, and (3) using prebiotics, probiotics, postbiotics, antibiotics, and fecal microbiota transplant (FMT) to modulate immune therapy. We discuss the respective benefits of 16S ribosomal RNA (rRNA) gene and shotgun metagenomic sequencing including important considerations in obtaining samples and in designing and interpreting human and animal microbiome studies. We then focus on studies discussing the differences in response to ICIs in relation to the microbiome and inflammatory mediators. ICIs cause colitis in up to 25% of individuals, and colitis is often refractory to common immunosuppressive medications. Researchers have measured microbiota composition prior to ICI therapy and correlated baseline microbiota composition with efficacy and colitis. Certain bacterial taxa that appear to enhance therapeutic benefit are also implicated in increased susceptibility to colitis, alluding to a delicate balance between pro-inflammatory tumor killing and anti-inflammatory protection from colitis. Pre-clinical and clinical models have trialed probiotic administration, e.g. Bifidobacterium spp. or FMT, to treat colitis when immune suppressive agents fail. We are excited about the future of modulating the microbiome to predict and influence cancer outcomes. Furthermore, novel therapies employed for other illnesses including bacteriophage and genetically-engineered microbes can be adapted in the future to promote increased advancements in cancer treatment and side effect management.

Comparative Genomics of Antibiotic-Resistant Uropathogens Implicates Three Routes for Recurrence of Urinary Tract Infections.

The rise of antimicrobial resistance in uropathogens has complicated the management of urinary tract infections (UTIs), particularly in patients who are afflicted by recurrent episodes of UTIs. Antimicrobial-resistant (AR) uropathogens persistently colonizing individuals at asymptomatic time points have been implicated in the pathophysiology of UTIs. The dynamics of uropathogen persistence following the resolution of symptomatic disease are, however, mostly unclear. To further our understanding, we determined longitudinal AR uropathogen carriage and clonal persistence of uropathogenic Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae isolates in the intestinal and urinary tracts of patients affected by recurrent and nonrecurrent UTIs. Clonal tracking of isolates in consecutively collected urine and fecal specimens indicated repeated transmission of uropathogens between the urinary tract and their intestinal reservoir. Our results further implicate three independent routes of recurrence of UTIs: (i) following an intestinal bloom of uropathogenic bacteria and subsequent bladder colonization, (ii) reinfection of the urinary tract from an external source, and (iii) bacterial persistence within the urinary tract. Taken together, our observation of clonal persistence following UTIs and uropathogen transmission between the intestinal and urinary tracts warrants further investigations into the connection between the intestinal microbiome and recurrent UTIs.IMPORTANCE The increasing antimicrobial resistance of uropathogens is challenging the continued efficacy of empiric antibiotic therapy for UTIs, which are among the most frequent bacterial infections worldwide. It has been suggested that drug-resistant uropathogens could persist in the intestine after the resolution of UTI and cause recurrences following periurethral contamination. A better understanding of the transmission dynamics between the intestinal and urinary tracts, combined with phenotypic characterization of the uropathogen populations in both habitats, could inform prudent therapies designed to overcome the rising resistance of uropathogens. Here, we integrate genomic surveillance with clinical microbiology to show that drug-resistant clones persist within and are readily transmitted between the intestinal and urinary tracts of patients affected by recurrent and nonrecurrent UTIs. Thus, our results advocate for understanding persistent intestinal uropathogen colonization as part of the pathophysiology of UTIs, particularly in patients affected by recurrent episodes of symptomatic disease.

Ureaplasma urealyticum pyelonephritis presenting with progressive dysuria, renal failure, and neurologic symptoms in an immunocompromised patient.

Ureaplasma urealyticum is a bacterial species correlated with urethritis in healthy individuals and invasive infections in immunocompromised patients. We describe a 20-year-old female with a history of remote heart transplant on everolimus, mycophenolate, and rituximab presenting with progressive urinary tract symptoms, renal failure, and neurologic symptoms. An extensive workup ultimately identified U urealyticum infection, and the patient successfully recovered after a course of azithromycin and doxycycline.

A mucosal imprint left by prior Escherichia coli bladder infection sensitizes to recurrent disease.

Recurrent bacterial infections are a significant burden worldwide, and prior history of infection is often a significant risk factor for developing new infections. For urinary tract infection (UTI), a history of two or more episodes is an independent risk factor for acute infection. However, mechanistic knowledge of UTI pathogenesis has come almost exclusively from studies in naive mice. Here we show that, in mice, an initial Escherichia coli UTI, whether chronic or self-limiting, leaves a long-lasting molecular imprint on the bladder tissue that alters the pathophysiology of subsequent infections, affecting host susceptibility and disease outcome. In bladders of previously infected versus non-infected, antibiotic-treated mice, we found (1) an altered transcriptome and defects in cell maturation, (2) a remodelled epithelium that confers resistance to intracellular bacterial colonization, and (3) changes to cyclooxygenase-2-dependent inflammation. Furthermore, in mice with a history of chronic UTI, cyclooxygenase-2-dependent inflammation allowed a variety of clinical E. coli isolates to circumvent intracellular colonization resistance and cause severe recurrent UTI, which could be prevented by cyclooxygenase-2 inhibition or vaccination. This work provides mechanistic insight into how a history of infection can impact the risk for developing recurrent infection and has implications for the development of therapeutics for recurrent UTI.

Subinhibitory antibiotic therapy alters recurrent urinary tract infection pathogenesis through modulation of bacterial virulence and host immunity.

UNLABELLED: The capacity of subinhibitory levels of antibiotics to modulate bacterial virulence in vitro has recently been brought to light, raising concerns over the appropriateness of low-dose therapies, including antibiotic prophylaxis for recurrent urinary tract infection management. However, the mechanisms involved and their relevance in influencing pathogenesis have not been investigated. We characterized the ability of antibiotics to modulate virulence in the uropathogens Staphylococcus saprophyticus and Escherichia coli. Several antibiotics were able to induce the expression of adhesins critical to urothelial colonization, resulting in increased biofilm formation, colonization of murine bladders and kidneys, and promotion of intracellular niche formation. Mice receiving subinhibitory ciprofloxacin treatment were also more susceptible to severe infections and frequent recurrences. A ciprofloxacin prophylaxis model revealed this strategy to be ineffective in reducing recurrences and worsened infection by creating larger intracellular reservoirs at higher frequencies. Our study indicates that certain agents used for antibiotic prophylaxis have the potential to complicate infections. IMPORTANCE: Antibiotics are the mainstay treatment for bacterial infections; however, evidence is emerging that argues these agents may have off-target effects if sublethal concentrations are present. Most studies have focused on changes occurring in vitro, leaving questions regarding the clinical relevance in vivo. We utilized a murine urinary tract infection model to explore the potential impact of low-dose antibiotics on pathogenesis. Using this model, we showed that subinhibitory antibiotics prime uropathogens for adherence and invasion of murine urothelial tissues. These changes in initial colonization promoted the establishment of chronic infection. Furthermore, treatment of chronically infected mice with subtherapeutic ciprofloxacin served to exacerbate infection. A part of these changes was thought to be due to suppression of mucosal immunity, as demonstrated through reductions in cytokine secretion and migration of leukocytes into the urinary tract. This work identifies novel risk factors associated with antibiotic therapy when dosing strategies fall below subtherapeutic levels.

Uropathogenic Escherichia coli superinfection enhances the severity of mouse bladder infection.

Urinary tract infections (UTIs) afflict over 9 million women in America every year, often necessitating long-term prophylactic antibiotics. One risk factor for UTI is frequent sexual intercourse, which dramatically increases the risk of UTI. The mechanism behind this increased risk is unknown; however, bacteriuria increases immediately after sexual intercourse episodes, suggesting that physical manipulation introduces periurethral flora into the urinary tract. In this paper, we investigated whether superinfection (repeat introduction of bacteria) resulted in increased risk of severe UTI, manifesting as persistent bacteriuria, high titer bladder bacterial burdens and chronic inflammation, an outcome referred to as chronic cystitis. Chronic cystitis represents unchecked luminal bacterial replication and is defined histologically by urothelial hyperplasia and submucosal lymphoid aggregates, a histological pattern similar to that seen in humans suffering chronic UTI. C57BL/6J mice are resistant to chronic cystitis after a single infection; however, they developed persistent bacteriuria and chronic cystitis when superinfected 24 hours apart. Elevated levels of interleukin-6 (IL-6), keratinocyte cytokine (KC/CXCL1), and granulocyte colony-stimulating factor (G-CSF) in the serum of C57BL/6J mice prior to the second infection predicted the development of chronic cystitis. These same cytokines have been found to precede chronic cystitis in singly infected C3H/HeN mice. Furthermore, inoculating C3H/HeN mice twice within a six-hour period doubled the proportion of mice that developed chronic cystitis. Intracellular bacterial replication, regulated hemolysin (HlyA) expression, and caspase 1/11 activation were essential for this increase. Microarrays conducted at four weeks post inoculation in both mouse strains revealed upregulation of IL-1 and antimicrobial peptides during chronic cystitis. These data suggest a mechanism by which caspase-1/11 activation and IL-1 secretion could predispose certain women to recurrent UTI after frequent intercourse, a predisposition predictable by several serum biomarkers in two murine models.