Antibiotic-treated Patients Research Articles

Genes Contributing to the Unique Biology and Intrinsic Antibiotic Resistance of Enterococcus faecalis.

The enterococci, which are among the leading causes of multidrug-resistant (MDR) hospital infection, are notable for their environmental ruggedness, which extends to intrinsic antibiotic resistance. To identify genes that confer this unique property, we used Tn-seq to comprehensively explore the genome of MDR Enterococcus faecalis strain MMH594 for genes important for growth in nutrient-containing medium and with low-level antibiotic challenge. As expected, a large core of genes for DNA replication, expression, and central metabolism, shared with other bacteria, are intolerant to transposon disruption. However, genes were identified that are important to E. faecalis that are either absent from or unimportant for Staphylococcus aureus and Streptococcus pneumoniae fitness when similarly tested. Further, 217 genes were identified that when challenged by sub-MIC antibiotic levels exhibited reduced tolerance to transposon disruption, including those previously shown to contribute to intrinsic resistance, and others not previously ascribed this role. E. faecalis is one of the few Gram-positive bacteria experimentally shown to possess a functional Entner-Doudoroff pathway for carbon metabolism, a pathway that contributes to stress tolerance in other microbes. Through functional genomics and network analysis we defined the unusual structure of this pathway in E. faecalis and assessed its importance. These approaches also identified toxin-antitoxin and related systems that are unique and active in E. faecalis Finally, we identified genes that are absent in the closest nonenterococcal relatives, the vagococci, and that contribute importantly to fitness with and without antibiotic selection, advancing an understanding of the unique biology of enterococci.IMPORTANCE Enterococci are leading causes of antibiotic-resistant infection transmitted in hospitals. The intrinsic hardiness of these organisms allows them to survive disinfection practices and then proliferate in the gastrointestinal tracts of antibiotic-treated patients. The objective of this study was to identify the underlying genetic basis for its unusual hardiness. Using a functional genomic approach, we identified traits and pathways of general importance for enterococcal survival and growth that distinguish them from closely related pathogens as well as ancestrally related species. We further identified unique traits that enable them to survive antibiotic challenge, revealing a large set of genes that contribute to intrinsic antibiotic resistance and a smaller set of uniquely important genes that are rare outside enterococci.

Immunostimulant as an adjuvant therapy on Toll-like receptor concentration in children with acute otitis media

BackgroundAcute otitis media (AOM) is the commonest pediatric bacterial infection, affecting up to 75% of children at some time before age 5 years. AOM is among the primary reasons for antibiotic prescriptions in pediatric outpatients. This study aimed to detect the value of immunostimulant as an adjuvant therapy with antibiotics for treatment of acute otitis media in children. This study included 60 children suffering from acute otitis media; their age ranged from 3 to 5 years during the period from May 2018 to March 2019. The patients in this study were divided into 2 groups: group A included 30 patients with AOM who received amoxicillin and clavulanic acid antibiotic at attack of AOM. Group B included 30 patients with AOM who received the same antibiotic with immunostimulant (Echinacea extract) for 3 months. Samples of blood were taken from all patients to detect the level of Toll-like receptor by real-time PCR, before and after 3 months of antibiotic and immunostimulant therapy. All cases underwent assessment including complete history taking, otoscopic examination of the ear, and blood sample to detect the level of Toll-like receptor (TLR) before and after the therapy by real-time PCR.ResultsThere was a significant (P < 0.05) decrease in the TLR2 expression in antibiotic-treated patients than its expression before treatment. On the other hand, there was a significant (P < 0.05) increase in the TLR2 expression in immunostimulant plus antibiotic-treated patients than its expression before treatment; there was a high significant (P < 0.001) increase in the expression of TLR2 in the immunostimulant plus antibiotic than the antibiotic-treated patients.ConclusionThe role of antibiotics against bacterial infection causing acute otitis media can be enhanced by immunostimulant which increases the expression of Toll-like receptors which play a major role stimulating immune system to resist bacterial infection.

Changes in the fecal bacterial microbiota associated with disease severity in alcoholic hepatitis patients

ABSTRACT Background and Aims Alcoholic hepatitis is the most severe form of alcohol-related liver disease. While the gut microbiome is known to play a role in disease development and progression, less is known about specific compositional changes of the gut bacterial microbiome associated with disease severity. Therefore, the aim of our study was to correlate gut microbiota features with disease severity in alcoholic hepatitis patients. Methods We used 16S rRNA gene sequencing on fecal samples from 74 alcoholic hepatitis patients, which were enrolled at 9 centers in Europe, the United States, and Mexico in a multi-center observational study. The relative abundance of gut bacterial taxa on genus level, as well as the microbiome diversity, was correlated to various clinical, laboratory, and histologic parameters. Results We observed a negative correlation between the model for end-stage liver disease score and Shannon diversity, independent of potentially confounding factors (Padjust = 0.046). Alcoholic hepatitis patients with more severe disease had significantly decreased relative abundances of Akkermansia while the relative abundance of Veillonella was increased. We observed a reduction in the Bacteroides abundance (Padjust = 0.048) and Shannon diversity (Padjust = 0.018) in antibiotic-treated patients and patients receiving steroids had an increase in Veillonella abundance (Padjust = 0.005), which was both independent of potentially confounding factors. Conclusion We observed distinct changes in the gut bacterial microbiome of alcoholic hepatitis patients with more severe disease. The gut bacterial microbiome might be an attractive target to prevent and treat this deadly disease.

Clinical Impact of an Antibiotic Time Out Initiative at an Academic Medical Center.

Background: Various strategies aimed at limiting inappropriate antimicrobial use including antibiotic time out (ATO) have been proposed to combat the development of antimicrobial resistance, yet there are limited studies that have assessed the impact of ATO on clinical outcomes. Methods: This single-center retrospective study reviewed the effect of a passive ATO strategy by comparing 100 adult patients with an ATO matched by infection type to 100 antibiotic-treated adult patients lacking an ATO note. Results: No difference in clinical outcomes was observed, however, ATO did result in improved optimization of antibiotic selection and duration, and reduction of piperacillin/tazobactam and vancomycin use. Conclusion: Further studies are warranted to evaluate the impact of ATO on clinical outcomes of a larger homogenous population with specified infectious diagnoses and clinical characteristics.

Regulation and Anaerobic Function of the Clostridioides difficile β-Lactamase.

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to β-lactams. These results provide support for the function of a β-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile β-lactamase activity.

2568. Mechanisms of a Specific Probiotic Comprised of Lactobacillus acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 that Interferes with Clostridioides difficile 20291 Toxin Production

Background Clostridioides difficile infections (CDI) result from antibiotic use and cause severe diarrhea (C. difficile-associated diarrhea, CDAD) which is life-threatening and costly. A specific probiotic containing Lactobacillus acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 (Bio-K+) has demonstrated benefits in preventing CDI and has a strong inhibitory effect on the growth of several nosocomial C. difficile strains in vitro. Many Lactobacilli can inhibit CD growth though lactic acidification. Here, we have investigated novel acid-independent mechanisms by which these strains impair C. difficile virulence.MethodsThe hypervirulent strain C. difficile R20291 was co-cultured anaerobically with Bio-K+ probiotic strains in various media and glucose concentrations (5 g/L, 3 g/L, 0 g/L), for 24 hours at 37°C. Parameters such as Log CFU, pH, Toxin A and B, cell cytotoxicity were measured. Statistical comparisons using ANOVA one-way was performed in order to determine whether the groups were significantly different.ResultsAt 5 g/L glucose, no C. difficile toxin was produced and co-culture with these lactobacilli resulted in potent acidification and growth inhibition. At 3 g/L glucose, C. difficile toxin production occurred and acidification by the lactobacilli resulted in growth inhibition as well as >99% reduced Toxin A and B production. In the absence of glucose and a starting pH of 7.0, TY broth, the lactobacilli did not acidify the medium and C. difficile growth was normal yet Toxin A and B production was partially reduced at, 20% and 41% lower. Toxin B from the supernatant of C. difficile grown in TY was cytotoxic to human fibroblast cells, but this was less cytotoxic when co-cultured with the Lactobacilli.ConclusionThese results suggest that the combination of L. acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 interferes with C. difficile pathogenesis through: 1) inhibition of C. difficile growth (via lactic acid secretion), 2) reduced toxin A/B synthesis and (3) toxin neutralization. These results might explain the strain specificity of Bio-K+ probiotic bacteria in potentially preventing C. difficile-associated diarrhea in antibiotic treated patients. Disclosures All authors: No reported disclosures.

517. Treatment Patterns of Hospitalized Adults with Infections Due to Carbapenem Non-Susceptible Gram-Negative Organisms in a Large Electronic Health Record Database in the United States

BackgroundInfections caused by carbapenem non-susceptible (C-NS) Gram-negative (GN) organisms pose a major threat, due in part to limited treatment options. The aim of this study was to assess treatment patterns for these infections in a large US electronic health record database.MethodsA retrospective cohort study of hospitalized adults with complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI), bacterial pneumonia (BP), or bacteremia (BAC) due to C-NS (resistant/intermediate susceptibility to carbapenem) GN organisms from January 2013 to March 2018. Patients with inherently C-NS organisms (e.g., Pseudomonas aeruginosa to ertapenem) were only included if resistance to another carbapenem was identified. The index date was the date of first C-NS culture in a qualifying hospitalization (±3 days from admission/discharge). Clinical characteristics and administered treatments were assessed from admission to discharge with variables summarized descriptively and stratified by infection type.Results7,702 patients met inclusion criteria: 31% cUTI ± BAC, 24% BP ± BAC, 21% cUTI + BP ± BAC, 17% cIAI ± BAC, cUTI, or BP, 7% BAC only. The median age was 66 years, ranging from 60 (BAC) to 69 (cUTI) years; male, 57%. The most common pathogens were Pseudomonas aeruginosa (64%) and Klebsiella pneumoniae (15%). Antibiotics were administered to the majority of patients (87%); of which, 79% received combination therapy (median classes: 3, maximum: 7), the remainder received monotherapy. For antibiotic-treated patients, 93% initiated an antibiotic before the non-susceptibility status of the underlying organism was known. The most common classes given during the index hospitalization were: penicillin (49%), fluoroquinolone (44%), carbapenem (40%), cephalosporin (39%), aminoglycoside (28%) (by infection type, Figure 1). Eleven percent of patients received colistin/polymyxin B.ConclusionVaried antibiotic use was observed in this cohort, with carbapenems frequently detected despite the C-NS nature of the underlying GN organisms. The use of antibiotics to which organisms are non-susceptible could lead to poor health outcomes, supporting the need for new targeted therapies to treat C-NS infections. Disclosures All authors: No reported disclosures.

Predominantly Antibiotic-resistant Intestinal Microbiome Persists in Patients With Pouchitis Who Respond to Antibiotic Therapy

Pouchitis that develops in patients with ulcerative colitis after total proctocolectomy and ileal pouch anal anastomosis is usually treated with antibiotics. Some patients have recurrence of flares, or become antibiotic-dependent, and require repeated courses or prolonged periods of antibiotic therapy. We investigated microbial factors associated with response to antibiotic treatment and development of antibiotic dependence in patients with pouchitis. We performed a prospective study of 49 patients who had undergone pouch surgery at a tertiary center. Disease activity was determined based on clinical, endoscopic, and histologic criteria. Pouch phenotype was defined as recurrent-acute pouchitis (n= 6), chronic pouchitis and Crohn's-like disease of the pouch (n= 27), normal pouch from patient with ulcerative colitis (n= 10), and normal pouch from patient with familial adenomatous polyposis (n= 6). Fecal samples (n= 234) were collected over time during or in the absence of antibiotic treatment (ciprofloxacin and/or metronidazole). Thirty-three patients were treated with antibiotics, for a median of 425 days of cumulative antibiotic therapy, during follow-up. Calprotectin was measured and fecal DNA was sequenced using shotgun metagenomics and analyzed with specifically designed bioinformatic pipelines. Bacterial strains were isolated from fecal samples. We assessed their ciprofloxacin resistance and ability to induce secretion of inflammatory cytokines by HT-29 intestinal epithelial cells. Most antibiotic-treated patients (79%) had a clinical response to each course of antibiotics; however, 89% of those who completed a 4-week course relapsed within 3 months. Median calprotectin levels decreased by 40% in response to antibiotics. Antibiotic treatment reduced disease-associated bacteria such as Clostridium perfringens, Ruminococcus gnavus, and Klebsiella pneumoniae, but also beneficial species, such as Faecalibacterium prausnitzii. The microbiomes of antibiotic-responsive patients were dominated by facultative anaerobic genera (Escherichia, Enterococcus, and Streptococcus), with multiple ciprofloxacin-resistance mutations in drug target genes and confirmed drug resistance. However, these strains had lower potential for virulence and did not induce secretion of inflammatory cytokines by epithelial cells. After antibiotic cessation, patients had an abrupt shift in microbiome composition, with blooms of oral and disease-associated bacteria. In addition, antibiotic treatment enriched for strains that acquired multidrug resistance loci, encoding enzymes that confer resistance to nonrelated antibiotics, including extended-spectrum beta-lactamases. The efficacy of antibiotic treatment of pouchitis might be attributed to the establishment of an antibiotic-resistant microbiome with low inflammatory potential. This microbiome might provide resistance against colonization by bacteria that promote inflammation. To avoid progression to antibiotic-dependent disease and its consequences, strategies such as short-term alternating antibiotics and nutrition- and microbiome-based interventions should be considered.

Systemic antibiotic prophylaxis does not affect infectious complications in pediatric burn injury: A meta-analysis.

In pediatric burns the use of systemic antibiotic prophylaxis is a standard procedure in some burn centers, though its beneficial effect on the infectious complications is debated. The present meta-analysis aimed at determining whether systemic antibiotic prophylaxis prevents infectious complications in pediatric patients with burn injuries. We searched the PubMed, EMBASE, and Cochrane Library databases from inception to August 2019. We included 6 studies, in which event rates of infectious complications were reported in children with burn injuries receiving or not receiving systemic antibiotic prophylaxis. We found that the overall odds ratio (OR) of developing an infection (including local and systemic) was not different between the groups (OR = 1.35; 95% CI, 0.44, 4.18). The chances for systemic infectious complications alone were also not different between antibiotic-treated and non-treated patients (OR = 0.74; 95% CI, 0.38, 1.45). Based on the age, affected total body surface area, and country income level, we did not find any subgroup that benefited from the prophylaxis. Our findings provide quantitative evidence for the inefficacy of systemic antibiotic prophylaxis in preventing infections in pediatric burns. To validate our conclusion, multinational, randomized trials in a diverse population of children with burn injuries are warranted.

Hybrid metagenomic assembly enables high-resolution analysis of resistance determinants and mobile elements in human microbiomes.

Characterization of microbiomes has been enabled by high-throughput metagenomic sequencing. However, existing methods are not designed to combine reads from short- and long-read technologies. We present a hybrid metagenomic assembler named OPERA-MS that integrates assembly-based metagenome clustering with repeat-aware, exact scaffolding to accurately assemble complex communities. Evaluation using defined in vitro and virtual gut microbiomes revealed that OPERA-MS assembles metagenomes with greater base pair accuracy than long-read (>5×; Canu), higher contiguity than short-read (~10× NGA50; MEGAHIT, IDBA-UD, metaSPAdes) and fewer assembly errors than non-metagenomic hybrid assemblers (2×; hybridSPAdes). OPERA-MS provides strain-resolved assembly in the presence of multiple genomes of the same species, high-quality reference genomes for rare species (<1%) with ~9× long-read coverage and near-complete genomes with higher coverage. We used OPERA-MS to assemble 28 gut metagenomes of antibiotic-treated patients, and showed that the inclusion of long nanopore reads produces more contiguous assemblies (200× improvement over short-read assemblies), including more than 80 closed plasmid or phage sequences and a new 263 kbp jumbo phage. High-quality hybrid assemblies enable an exquisitely detailed view of the gut resistome in human patients.

Accuracy of Different Rapid Urease Tests in Comparison with Histopathology in Patients with Endoscopic Signs of Gastritis

Background and Aim: According to several guidelines, both invasive and non-invasive tests can be used to detect Helicobacter pylori (H. pylori). Invasive methods include H. pylori culture, histological staining, rapid urease tests (RUTs) and PCR. Non-invasive methods include urease breath test, stool antigen and serum IgG testing. The aim of our study was to compare all commercially available RUTs and histology in Germany. Material and Methods: One hundred fifty patients were enrolled in our study, irrespective of proton pump inhibitors (PPIs) or antibiotic use. If the results of RUTs and histology were diverging, real-time PCR to detect H. pylori DNA was undertaken. Results: We detected no differences in the sensitivity or specificity between the different RUTs. In PPI and/or antibiotic-treated patients, RUTs seemed to be more sensitive for the detection of H. pylori infection compared to histology. In addition to the cheaper price of RUTs, they are also quicker to process. We show that histological staining in patients with signs of gastritis is expensive and not necessary, if there are no additional histological questions besides H. pylori status. Conclusions: In conclusion, we consider RUTs to be cheap and fast alternatives to histology in patients with endoscopic signs of gastritis, independently of whether PPIs or antibiotic are used. Histological evaluation is expensive, time consuming and may be unnecessary in some cases.

Phenotypic Characterization of Non-toxigenic Clostridioides difficile Strains Isolated From Patients in Mexico.

Clostridioides difficile is a Gram positive, sporulated, rod-shape, anaerobic pathogen responsible for nosocomial diarrhea and colitis, mainly in antibiotic treated patients. C. difficile produce two toxins responsible for disease, toxin A (TcdA) and toxin B (TcdB), although not all strains produce them. Non-toxigenic C. difficile (NTCD) strains are able to colonize the intestinal mucosa and are often isolated from asymptomatic individuals. NTCD are poorly studied, their evolutionary history has not been elucidated, and their relationship with illness remains controversial. The aim of this work was to analyze the phenotype of NTCD strains isolated from clinical cases in hospitals of México, and whether NTCD strains present characteristics that differentiate them from the toxigenic strains. Seventy-four C. difficile strains isolated from patients were tested for cytotoxicity and 14 were identified as NTCD strains. We analyzed phenotypical characteristics that are important for the biology of C. difficile like colony morphology, antibiotic resistance, motility, sporulation, and adherence. Strains were also genotyped to determine the presence of genes coding for TcdA, TcdB and binary toxin and ribotyped for 027 type. When compared with toxigenic strains, NTCD strains presented an enlarged branched colony morphology, higher resistance to metronidazole, and increased sporulation efficiency. This phenotype has been reported associated with mutations that regulates phenotypic characteristics like swimming, sporulation or adhesion. Our results show that phenotype of NTCD strains is heterogeneous but still present characteristics that differentiate them from toxigenic strains.

Persistence of Brucella abortus in the Bone Marrow of Infected Mice.

Brucellosis is a zoonotic bacterial infection that may persist for long periods causing relapses in antibiotic-treated patients. The ability of Brucella to develop chronic infections is linked to their capacity to invade and replicate within the mononuclear phagocyte system, including the bone marrow (BM). Persistence of Brucella in the BM has been associated with hematological complications such as neutropenia, thrombocytopenia, anemia, and pancytopenia in human patients. In the mouse model, we observed that the number of Brucella abortus in the BM remained constant for up to 168 days of postinfection. This persistence was associated with histopathological changes, accompanied by augmented numbers of BM myeloid GMP progenitors, PMNs, and CD4+ lymphocytes during the acute phase (eight days) of the infection in the BM. Monocytes, PMNs, and GMP cells were identified as the cells harboring Brucella in the BM. We propose that the BM is an essential niche for the bacterium to establish long-lasting infections and that infected PMNs may serve as vehicles for dispersion of Brucella organisms, following the Trojan horse hypothesis. Monocytes are solid candidates for Brucella reservoirs in the BM.

858. Preoperative “Dirty” Urinalysis Leads to Antibiotic Use Without Benefit

BackgroundPyuria is often used as a surrogate for bacteriuria and may trigger antibiotic use even in the setting of negative cultures. The impact of preoperative pyuria on empirical antibiotic use and on postoperative outcomes has not been evaluated in large multisite studies. Thus, we investigated rates and outcomes associated with treated versus untreated preoperative pyuria in a national cohort of surgical patients.MethodsAll patients who underwent standardized Surgical Quality review after cardiac, orthopedic implant, or vascular surgery within the national VA health care system from October 1, 2008 to September 30, 2013 and had a urinalysis performed in the 30 days before surgery were eligible. Rates of preoperative pyuria (≥5 WBCs and/or positive leukocyte esterase) and antibiotics were measured. Adjusted rates of 30-day postoperative surgical (SSI) and urinary tract (UTI) infections were determined by a trained nurse reviewer using CDC definitions and compared between pyuria patients who did or did not receive antibiotics before surgery.ResultsAmong 17,749 preoperative urinalyses, 755 were culture-positive and 16,994 were culture-negative. Among culture negative patients, 1,812/16,994 (10.7%) had urinalyses diagnostic of pyuria. Antibiotics were prescribed to 574 (32%) of pyuria-positive, culture-negative patients. After adjusting for diabetes, smoking, age, and ASA score, the rate of post-op SSI was similar among antibiotic-treated pyuria patients (13/574, 2.3%) compared with those not treated (21/1,238, 1.7%), aOR 1.33, 95% (0.66–2.69, P = 0.41). Post-op UTI was also not reduced among pyuria treated (17/351, 4.8%) vs. untreated (39/893, 4.4%), aOR 1.09, 95% (0.60–1.96, P = 0.76).ConclusionIn this large national study cohort, almost 1/3 of pyuria-positive preoperative patients received antibiotics despite negative cultures. Antibiotic treatment was not associated with clinical benefits, including no reduction in post-operative SSI or UTI. There is an opportunity for diagnostic stewardship to reduce preoperative urinalysis testing and unnecessary antibiotic exposure.Disclosures K. Itani, Pfizer: Grant Investigator, Research grant. Sanofi: Grant Investigator, Research grant. K. Gupta, Iterum Therapeutics: Consultant, Consulting fee. Paratek Pharmaceutical: Consultant, Consulting fee. Tetraphase: Consultant, Consulting fee.

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Five-Year Follow-up of Antibiotic Therapy for Uncomplicated Acute Appendicitis in the APPAC Randomized Clinical Trial

Short-term results support antibiotics as an alternative to surgery for treating uncomplicated acute appendicitis, but long-term outcomes are not known. To determine the late recurrence rate of appendicitis after antibiotic therapy for the treatment of uncomplicated acute appendicitis. Five-year observational follow-up of patients in the Appendicitis Acuta (APPAC) multicenter randomized clinical trial comparing appendectomy with antibiotic therapy, in which 530 patients aged 18 to 60 years with computed tomography-confirmed uncomplicated acute appendicitis were randomized to undergo an appendectomy (n = 273) or receive antibiotic therapy (n = 257). The initial trial was conducted from November 2009 to June 2012 in Finland; last follow-up was September 6, 2017. This current analysis focused on assessing the 5-year outcomes for the group of patients treated with antibiotics alone. Open appendectomy vs antibiotic therapy with intravenous ertapenem for 3 days followed by 7 days of oral levofloxacin and metronidazole. In this analysis, prespecified secondary end points reported at 5-year follow-up included late (after 1 year) appendicitis recurrence after antibiotic treatment, complications, length of hospital stay, and sick leave. Of the 530 patients (201 women; 329 men) enrolled in the trial, 273 patients (median age, 35 years [IQR, 27-46]) were randomized to undergo appendectomy, and 257 (median age, 33 years, [IQR, 26-47]) were randomized to receive antibiotic therapy. In addition to 70 patients who initially received antibiotics but underwent appendectomy within the first year (27.3% [95% CI, 22.0%-33.2%]; 70/256), 30 additional antibiotic-treated patients (16.1% [95% CI, 11.2%-22.2%]; 30/186) underwent appendectomy between 1 and 5 years. The cumulative incidence of appendicitis recurrence was 34.0% (95% CI, 28.2%-40.1%; 87/256) at 2 years, 35.2% (95% CI, 29.3%-41.4%; 90/256) at 3 years, 37.1% (95% CI, 31.2%-43.3%; 95/256) at 4 years, and 39.1% (95% CI, 33.1%-45.3%; 100/256) at 5 years. Of the 85 patients in the antibiotic group who subsequently underwent appendectomy for recurrent appendicitis, 76 had uncomplicated appendicitis, 2 had complicated appendicitis, and 7 did not have appendicitis. At 5 years, the overall complication rate (surgical site infections, incisional hernias, abdominal pain, and obstructive symptoms) was 24.4% (95% CI, 19.2%-30.3%) (n = 60/246) in the appendectomy group and 6.5% (95% CI, 3.8%-10.4%) (n = 16/246) in antibiotic group (P < .001), which calculates to 17.9 percentage points (95% CI, 11.7-24.1) higher after surgery. There was no difference between groups for length of hospital stay, but there was a significant difference in sick leave (11 days more for the appendectomy group). Among patients who were initially treated with antibiotics for uncomplicated acute appendicitis, the likelihood of late recurrence within 5 years was 39.1%. This long-term follow-up supports the feasibility of antibiotic treatment alone as an alternative to surgery for uncomplicated acute appendicitis. ClinicalTrials.gov Identifier: NCT01022567.

Use of broad-spectrum antibiotics impacts outcome in patients treated with immune checkpoint inhibitors

ABSTRACT We carried out a retrospective cohort study on patients with advanced cancer treated with immune checkpoint inhibitors (ICIs) to determine whether antibiotics affect treatment outcome. Sixty consecutive patients were identified, and 17 received systemic antibiotics within 2 weeks before and/or after first dose of ICI. Antibiotic-treated patients were significantly younger (p = 0.0008) and less likely to receive nivolumab (p = 0.08) or had neutrophil:lymphocyte ratio < 5 (p = 0.08). They had a lower response rate (RR) (29.4% vs 62.8%) (p = 0.024) and more inferior progression-free survival (PFS) (p = 0.048). Narrow-spectrum antibiotics did not affect the RR. However, broad-spectrum antibiotics were associated with a lower RR (25% vs 61%) (p = 0.02) and a trend towards longer time to response (median: 14 weeks vs 12 weeks) (p = 0.1). They also had shorter PFS (p = 0.012). Multivariate analysis identified antibiotics as the only factor affecting RR (p = 0.0038) and PFS (p = 0.01). We next examined the 21 patients whose PFS lasted for 12 weeks or more. Five of the 21 patients received broad-spectrum antibiotics within 10 weeks before disease progression. There was a trend towards shorter PFS in these patients (p = 0.1). Finally, antibiotic-treated patients experienced shorter overall survival (OS) (median: 24 months vs 89 months) (p = 0.003). Multivariate analysis found age (p = 0.035) and antibiotics (p = 0.038) to be the only factors affecting OS. Our results point to a detrimental effect of broad-spectrum antibiotics on treatment outcome to ICI therapy.

Incidence of Antibiotic-Related Rash in Children with Epstein-Barr Virus Infection and Evaluation of the Frequency of Confirmed Antibiotic Hypersensitivity

Background: The incidence of rash after aminopenicillin treatment in children with Epstein-Barr Virus (EBV) infection was reported to be 80–100%. A few recent studies suggested that the incidence may be much lower during EBV infection. There are no clear data on the incidence of true drug hypersensitivity among these patients. The aim of this study is to determine the incidence of rash and antibiotic allergy after antibiotic treatment in children with EBV infection. Methods: Drug hypersensitivity was investigated in antibiotic-treated patients with a positive EBV IgM who developed a rash between 2013 and 2016. Results: During the study period, 221 children were diagnosed with EBV infection, and 120 (54.3%) patients were treated with antibiotics during disease. Rash developed in 41 (41/221, 18.6%) patients, and 20 of them (total 120; 16.6%) were treated with antibiotics (most frequently aminopenicillins: 72.5%), and 21 of them (total 101, 20.8%) were not treated with antibiotics (p = 0.43). For 10 of the 20 antibiotic-treated patients with a rash, parents did not consent to an allergy workup. Three of the 10 patients who were tested for drug allergy were proven to have amoxicillin-clavulanate hypersensitivity (30%). Five of the patients without workup reacted after reuse of the suspected drug for infection treatment. Conclusion: In this study, the incidence of drug hyper­sensitivity was much lower than previously reported. Some of the reactions that occur during infectious mononucleosis are transient, and some are true drug hypersensitivity reactions. Thus, these patients should be evaluated with allergy tests before these drugs are used again after EBV infection.

The long‐term effects of faecal microbiota transplantation for gastrointestinal symptoms and general health in patients with recurrent Clostridium difficile infection

Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection. In short-term the treatment has been shown to be safe, however, there are no large, long-term follow-up studies looking into the potential adverse effects. To analyse the long-term effect of FMT treatment in patients with recurrent C. difficile infection and to compare the outcome to antibiotic treated patients. Altogether 84 patients of which 45 received a FMT treatment and 39 served as controls receiving antibiotics for the infection were followed on average for 3.8years. Their recovery and medical status was evaluated using a retrospective questionnaire, determining their quality of life, gastrointestinal symptoms and new diseases potentially related to the FMT. There was no difference in the incidence of severe diseases (inflammatory bowel disease, cancer, autoimmune disease, allergy, neurological diseases) between the patient groups. In addition, weight gain did not differ between treatment groups. The FMT treated patients reported that their bowel habits improved significantly faster, they had less irregular bowel function and less symptoms of upper GI-tract when compared to the patients treated with antibiotics. Significantly more patients in FMT-group reported that their mental health improved after the treatment. The willingness to receive FMT treatment for potential new C. difficile infection was significantly higher in both treatment groups compared to other treatment options. Our study highlights that FMT is a durable, safe and acceptable treatment option for patients with recurrent C. difficile infection also in long term, and it shows potential benefits over antimicrobial treatment.