A new therapeutic strategy for infectious diseases against intracellular multidrug-resistant bacteria.

Bacterial infections result in 7,700,000 deaths per year globally, with intracellular bacteria causing repeated and resistant infection. No drug is currently licenced for the treatment of intracellular bacteria. A new screening platform mimicking the host milieu has been established to explore phytochemical antibiotic adjuvants. Previously neglected isoprenylated flavonoids were found to be effective against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Specifically, the synergistic effect between glabrol and streptomycin against intracellular bacteria was observed for the first time. The glabrol-streptomycin combination targets bacterial inner membrane phospholipids, disrupts arginine biosynthesis, inhibits cell wall proteins and biofilm formation genes (agrA/B/C/D), and promotes ROS production, causing subsequent membrane and wall damage. To enhance the selective uptake of combination drug into infected cells, hyaluronic acid-streptomycin-lipoic acid-glabrol nanoparticles (HSLGS-S) were designed and synthesized to trigger the intracellular delivery of the glabrol-streptomycin combination. Thus, the treatment can be transported into the infected intracellular region and selectively release the glabrol-streptomycin combination to the bacterial at site. The bioactivity of HSLGS-S in clearing intracellular bacteria was 20-fold higher than that of the glabrol-streptomycin combination alone in vitro and 2- to 10-fold higher in vivo.

Medicated Scaffolds Prepared with Hydroxyapatite/Streptomycin Nanoparticles Encapsulated into Polylactide Microfibers.

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanoparticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaffolds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles' crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concentrations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties.

Mobility of ß-lactam resistance under ampicillin treatment in gut microbiota suffering from pre-disturbance.

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to dissemination of antibiotic resistance genes (ARGs) in the gut microbiota. The gut microbiota often suffers from various disturbances. It is not clear whether and how disturbed microbiota may affect ARG mobility under antibiotic treatments. For proof of concept, in the presence or absence of streptomycin pre-treatment, mice were inoculated orally with a ß-lactam-susceptible Salmonella enterica serovar Heidelberg clinical isolate (recipient) and a ß-lactam resistant Escherichia coli O80:H26 isolate (donor) carrying a blaCMY-2 gene on an IncI2 plasmid. Immediately following inoculation, mice were treated with or without ampicillin in drinking water for 7 days. Faeces were sampled, donor, recipient and transconjugant were enumerated, blaCMY-2 abundance was determined by quantitative PCR, faecal microbial community composition was determined by 16S rRNA amplicon sequencing and cecal samples were observed histologically for evidence of inflammation. In faeces of mice that received streptomycin pre-treatment, the donor abundance remained high, and the abundance of S. Heidelberg transconjugant and the relative abundance of Enterobacteriaceae increased significantly during the ampicillin treatment. Co-blooming of the donor, transconjugant and commensal Enterobacteriaceae in the inflamed intestine promoted significantly (P<0.05) higher and possibly wider dissemination of the blaCMY-2 gene in the gut microbiota of mice that received the combination of streptomycin pre-treatment and ampicillin treatment (Str-Amp) compared to the other mice. Following cessation of the ampicillin treatment, faecal shedding of S. Heidelberg transconjugant persisted much longer from mice in the Str-Amp group compared to the other mice. In addition, only mice in the Str-Amp group shed a commensal E. coli O2:H6 transconjugant, which carries three copies of the blaCMY-2 gene, one on the IncI2 plasmid and two on the chromosome. The findings highlight the significance of pre-existing gut microbiota for ARG dissemination and persistence during and following antibiotic treatments of infectious diseases.

Polyurethane-functionalized starch nanocrystals as anti-tuberculosis drug carrier.

Studies related to loading ability and delivery of clinically used first-line anti-tuberculosis drugs (ATDs) such as isoniazid, rifampicin, pyrazinamide and streptomycin on the surface of starch-derived bulk and nanopolyurethanes (SBPUs and SNPUs) as drug delivery systems (DDS) have been focused to minimise or remove the drug-associated adverse effects. The efficiencies of nanopolyurethanes obtained from the differently substituted cyclic aliphatic and aromatic isocyanates have been studied for drug loading and release purposes. Different advanced instrumental techniques analysed the structural and morphological properties, thermal stability and crystallinity of the starch nanopolyurethans. Average particle sizes ranging from 27.35-42.38 nm to 126.89-218.60 nm for starch nanopolyurethans, SNPU3i and SNPU4i, respectively, were determined by high-resolution transmission electron microscopy. Similarly, the loading efficiency of ATDs to the surfaces of SNPUs and SBPUs was observed in the range of 60-97% while ATDs-loaded SNPUs showed a sustainable release profile for all ATDs except for streptomycin. However, most SBPUs provided burst-release for all the above-mentioned ATDs in pH-dependent studies. The anti-tuberculosis assay against the Mycobacterium tuberculosis H37Rv strain revealed that streptomycin-loaded SNPU4i and isoniazid-loaded SNPU7i are approximately 42 and 7 times more active than the native streptomycin and isoniazid, respectively.

Streptomycin sulphate loaded solid lipid nanoparticles show enhanced uptake in macrophage, lower MIC in Mycobacterium and improved oral bioavailability.

Present study addresses the challenge of incorporating hydrophilic streptomycin sulphate (STRS; log P -6.4) with high dose (1 g/day) into a lipid matrix of SLNs. Cold high-pressure homogenization technique used for SLN preparation achieved 30% drug loading and 51.17 ± 0.95% entrapment efficiency. Polyethylene glycol 600 as a supporting-surfactant assigned small size (218.1 ± 15.46 nm) and mucus-penetrating property. It was conceived to administer STRS-SLNs orally rather than intramuscularly. STRS-SLNs remained stable on incubation for varying times in SGF or SIF. STRS-SLNs were extensively characterised for microscopic (TEM and AFM), thermal (DSC), diffraction (XRD) and spectroscopic (NMR and FTIR) properties and showed zero-order controlled release. Enhanced (60 times) intracellular uptake was observed in THP-1 and Pgp expressing LoVo and DLD-1 cell lines, using fluorescein-SLNs. Presence of SLNs in LoVo cells was also revealed by TEM studies. STRS-SLNs showed 3 times reduction in MIC against Mycobacterium tuberculosis H37RV (256182) in comparison to free STRS. It also showed better activity against both M. bovis BCG and Mycobacterium tuberculosis H37RV (272994) in comparison to free STRS. Cytotoxicity and acute toxicity studies (OECD 425 guidelines) confirmed in vitro and in vivo safety of STRS-SLNs. Single-dose oral pharmacokinetic studies in rat plasma using validated LCMS/MS technique or the microbioassay showed significant oral absorption and bioavailability (160% - 710% increase than free drug).

Tuberculosis in the intensive care unit: alternative treatment regimens and association with mortality.

OBJECTIVES: Adequate anti-tuberculosis (TB) treatment is an important factor that can affect the patient's outcome. Higher mortality is found in patients who do not receive optimal treatment that includes isoniazid and rifampicin. The objective of this study is to evaluate the association of use of alternative TB treatment regimens (without rifampicin and isoniazid) and mortality among patients requiring intensive care. METHODS: Retrospective cohort study, from January 2010 to December 2018. Patients aged > 18 years with a TB diagnosis, admitted to the ICU of a general, tertiary care, university-affiliated hospital (Hospital de Clínicas de Porto Alegre - HCPA) were included. Data on TB treatment used and outcomes of treatment were collected. RESULTS: 462 patients met the inclusion criteria and were included in the analysis; 284 used the usual treatment regimen (rifampicin, isoniazid, pyrazinamide and ethambutol - all orally), and 178 used alternative treatment regimens (IV levofloxacin plus oral ethambutol plus IM streptomycin or IV amikacin, without rifampicin and isoniazid). The mortality was higher among users of alternative treatment regimens (63.5%) than among usual treatment regimen users (51.4%) (P = 0.011). In a multivariate analysis, age, albumin and death were independently associated with alternative treatment regimens use. CONCLUSIONS: TB programmes in which IV rifampicin is not widely available should consider including it, especially for critically ill TB patients, for whom there may be improved survival.

OBJECTIFS: Un traitement antituberculeux (TB) adéquat est un facteur important pouvant influencer les résultats du patient. Une mortalité plus élevée est observée chez les patients qui ne reçoivent pas un traitement optimal comprenant de l'isoniazide et de la rifampicine. L'objectif de cette étude est d'évaluer l'association entre l'utilisation d'autres schémas thérapeutiques anti-TB (sans rifampicine ni isoniazide) et la mortalité chez les patients nécessitant des soins intensifs. MÉTHODES: Etude de cohorte rétrospective, de janvier 2010 à décembre 2018. Les patients âgés de >18 ans avec un diagnostic de TB, admis à l'unité de soins intensifs d'un hôpital général, avec des soins tertiaires, affilié à l'Université (Hôpital de Clínicas de Porto Alegre-HCPA) ont été inclus. Des données sur le traitement anti-TB utilisé et les résultats du traitement ont été collectés. RÉSULTATS: 462 patients répondaient aux critères d'inclusion et ont été inclus dans l'analyse; 284 ont utilisé le schéma thérapeutique habituel (rifampicine, isoniazide, pyrazinamide et éthambutol - tous par voie orale) et 178 ont utilisé des schémas thérapeutiques alternatifs (lévofloxacine IV plus éthambutol oral plus streptomycine IM ou amikacine IV, sans rifampicine ni isoniazide). La mortalité était plus élevée chez les utilisateurs de schémas thérapeutiques alternatifs (63,5%) que chez les utilisateurs de schémas thérapeutiques habituels (51,4%) (P = 0,011). Dans l'analyse multivariée, l'âge, l'albumine et le décès ont été indépendamment associés à l'utilisation de schémas thérapeutiques alternatifs. CONCLUSIONS: Les programmes de lutte contre la TB dans lesquels la rifampicine IV n'est pas largement disponible devraient envisager de l'inclure, en particulier pour les patients atteints de TB et sévèrement malades, pour lesquels la survie peut être améliorée.

Enhancing mucosal immunity by transient microbiota depletion.

Tissue resident memory CD8+ T cells (Trm) are poised for immediate reactivation at sites of pathogen entry and provide optimal protection of mucosal surfaces. The intestinal tract represents a portal of entry for many infectious agents; however, to date specific strategies to enhance Trm responses at this site are lacking. Here, we present TMDI (Transient Microbiota Depletion-boosted Immunization), an approach that leverages antibiotic treatment to temporarily restrain microbiota-mediated colonization resistance, and favor intestinal expansion to high densities of an orally-delivered Listeria monocytogenes strain carrying an antigen of choice. By augmenting the local chemotactic gradient as well as the antigenic load, this procedure generates a highly expanded pool of functional, antigen-specific intestinal Trm, ultimately enhancing protection against infectious re-challenge in mice. We propose that TMDI is a useful model to dissect the requirements for optimal Trm responses in the intestine, and also a potential platform to devise novel mucosal vaccination approaches.

Transient Intestinal Colonization by a Live-Attenuated Oral Cholera Vaccine Induces Protective Immune Responses in Streptomycin-Treated Mice.

Current mouse models for evaluating the efficacy of live oral cholera vaccines (OCVs) have important limitations. Conventionally raised adult mice are resistant to intestinal colonization by Vibrio cholerae, but germfree mice can be colonized and have been used to study OCV immunogenicity. However, germfree animals have impaired immune systems and intestinal physiology; also, live OCVs colonize germfree mice for many months, which does not mimic the clearance kinetics of live OCVs in humans. In this study, we leveraged antibiotic-treated, conventionally raised adult mice to study the effects of transient intestinal colonization by a live OCV V. cholerae strain. In a single-dose vaccination regimen, we found that HaitiV, a live-attenuated OCV candidate, was cleared by streptomycin-treated adult mice within 2 weeks after oral inoculation. This transient colonization elicited far stronger adaptive immune correlates of protection against cholera than did inactivated whole-cell HaitiV. Infant mice from HaitiV-vaccinated dams were also significantly more protected from choleric disease than pups from inactivated-HaitiV-vaccinated dams. Our findings establish the benefits of antibiotic-treated mice for live-OCV studies as well as their limitations and underscore the immunogenicity of HaitiV.IMPORTANCE Oral cholera vaccines (OCVs) are being deployed to combat cholera, but current killed OCVs require multiple doses and show little efficacy in young children. Live OCVs have the potential to overcome these limitations, but small-animal models for testing OCVs have shortcomings. We used an antibiotic treatment protocol for conventional adult mice to study the effects of short-term colonization by a single dose of HaitiV, a live-OCV candidate. Vaccinated mice developed vibriocidal antibodies against V. cholerae and delivered pups that were resistant to cholera, whereas mice vaccinated with inactivated HaitiV did not. These findings demonstrate HaitiV's immunogenicity and suggest that this antibiotic treatment protocol will be useful for evaluating the efficacy of live OCVs.

Rifampicin and clarithromycin (extended release) versus rifampicin and streptomycin for limited Buruli ulcer lesions: a randomised, open-label, non-inferiority phase 3 trial.

BACKGROUND: Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans infection that damages the skin and subcutis. It is most prevalent in western and central Africa and Australia. Standard antimicrobial treatment with oral rifampicin 10 mg/kg plus intramuscular streptomycin 15 mg/kg once daily for 8 weeks (RS8) is highly effective, but streptomycin injections are painful and potentially harmful. We aimed to compare the efficacy and tolerability of fully oral rifampicin 10 mg/kg plus clarithromycin 15 mg/kg extended release once daily for 8 weeks (RC8) with that of RS8 for treatment of early Buruli ulcer lesions. METHODS: We did an open-label, non-inferiority, randomised (1:1 with blocks of six), multicentre, phase 3 clinical trial comparing fully oral RC8 with RS8 in patients with early, limited Buruli ulcer lesions. There were four trial sites in hospitals in Ghana (Agogo, Tepa, Nkawie, Dunkwa) and one in Benin (Pobè). Participants were included if they were aged 5 years or older and had typical Buruli ulcer with no more than one lesion (caterories I and II) no larger than 10 cm in diameter. The trial was open label, and neither the investigators who took measurements of the lesions nor the attending doctors were masked to treatment assignment. The primary clinical endpoint was lesion healing (ie, full epithelialisation or stable scar) without recurrence at 52 weeks after start of antimicrobial therapy. The primary endpoint and safety were assessed in the intention-to-treat population. A sample size of 332 participants was calculated to detect inferiority of RC8 by a margin of 12%. This study was registered with ClinicalTrials.gov, NCT01659437. FINDINGS: Between Jan 1, 2013, and Dec 31, 2017, participants were recruited to the trial. We stopped recruitment after 310 participants. Median age of participants was 14 years (IQR 10-29) and 153 (52%) were female. 297 patients had PCR-confirmed Buruli ulcer; 151 (51%) were assigned to RS8 treatment, and 146 (49%) received oral RC8 treatment. In the RS8 group, lesions healed in 144 (95%, 95% CI 91 to 98) of 151 patients, whereas lesions healed in 140 (96%, 91 to 99) of 146 patients in the RC8 group. The difference in proportion, -0·5% (-5·2 to 4·2), was not significantly greater than zero (p=0·59), showing that RC8 treatment is non-inferior to RS8 treatment for lesion healing at 52 weeks. Treatment-related adverse events were recorded in 20 (13%) patients receiving RS8 and in nine (7%) patients receiving RC8. Most adverse events were grade 1-2, but one (1%) patient receiving RS8 developed serious ototoxicity and ended treatment after 6 weeks. No patients needed surgical resection. Four patients (two in each study group) had skin grafts. INTERPRETATION: Fully oral RC8 regimen was non-inferior to RS8 for treatment of early, limited Buruli ulcer and was associated with fewer adverse events. Therefore, we propose that fully oral RC8 should be the preferred therapy for early, limited lesions of Buruli ulcer. FUNDING: WHO with additional support from MAP International, American Leprosy Missions, Fondation Raoul Follereau France, Buruli ulcer Groningen Foundation, Sanofi-Pasteur, and BuruliVac.

Role of local streptomycin in prevention of surgical site infection in TB spine.

PURPOSE: Surgical site infection (SSI) continues to be one of the most common post-operative complications in most spine surgeries. Patients with tuberculosis (TB) of spine are more at risk of developing this complication due to a number of reasons. This adds to significant morbidity and economic burden on patients adversely affecting the mental status and quality of life of patients. The aim of this study was to investigate the role of local streptomycin in preventing SSI in patients undergoing surgical management of spinal TB. METHODS: In total, 56 patients who underwent surgical management for radiologically proven TB spine divided into two groups were included in the study. Group A included 30 patients with no local streptomycin administered intraoperatively, while group B included 26 patients operated in the later part of study with the use of local streptomycin intraoperatively. The two groups were compared and the outcome criteria analysed were SSI rate, length of hospital stay, duration of post-operative antibiotics and need for debridement. RESULTS: Length of hospital stay (group A: 18.4 ± 6.9 days; group B: 9.7 ± 3.9 days) and duration of post-operative antibiotics (group A: 8.1 ± 1.6 days; group B: 6.2 ± 2.1 days) were significantly higher in group A when compared with group B. SSI rate (group A: 13.34%; group B: 3.84%) and need for debridement (group A: 10%; group B: 3.84%) were higher in group A, but the difference was not statistically significant. CONCLUSION: Intraoperative administration of local streptomycin significantly reduces the length of hospital stay and duration of antibiotic administration in post-operative period in patients undergoing surgery for TB spine.

Chronotoxicity of Streptomycin-Induced Renal Injury in Mice.

The aim of the present study was to investigate the "chronotoxicity" of streptomycin (SM) in relation to its circadian periodicity. Male ICR mice were injected intraperitoneally with SM (780 mg/kg, one shot) one of six time points throughout the day. Mortality was monitored until 14 d after the injection and clearly differed depending on the timing of the injection (i.e., mice were more sensitive to injection during the dark phase). Moreover, when mice were administered with non-lethal doses of SM (550 mg/kg, every 24 h for 3 d, in the light phase or dark phase), the levels of nephrotoxicity indicators (blood urea nitrogen and renal levels of malondialdehyde and cyclooxygenase-2) were significantly increased by the injection in the dark phase, but not in the light phase. These results suggested that SM showed clear chronotoxicity. Our current data indicated that chronotoxicology may provide valuable information on the importance of injection timings for evaluations of toxicity and undesirable side effects.

Antibacterial mechanism of the synergistic combination between streptomycin and alcohol extracts from the Chimonanthus salicifolius S. Y. Hu. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Chimonanthus salicifolius S. Y. Hu. Is a unique traditional medicinal plant in ancient China, and it can eliminate turbid pathogens with aromatics, clear heat, detoxify, prevent colds and influenza, Xinhua Compendium of Materia Medica records that. AIM OF THE STUDY: In previous study, we investigated the regulation of ethanol extracts (EEs) from C. salicifolius S. Y. Hu. leaves on three common antibiotics (chloramphenicol, streptomycin, imipenem) by the checkerboard method. The combination exhibited the best synergy among all combinations, which were composed of streptomycin and 50% EE (SE) from the C. salicifolius S. Y. Hu. leaves. The aim of this study was to investigate the antibacterial mechanism of the SE against Escherichia coli (E. coli, G-) and Staphylococcus aureus (S. aureus, G+). MATERIALS AND METHODS: The antibacterial mechanism of the SE was explored by the time-kill test, the phosphorus metabolism, cell membrane integrity assays, the SDS-PAGE, the SEM and TEM observation. RESULTS: The time-kill test illustrated that the SE was bacteriostatic with a time-dependent relationship, not sterilization. The phosphorus metabolism indicated that the SE lowered phosphorus consumption. The cell membrane integrity assays demonstrated that the cell membrane was damaged, with the nucleic acid flowing out. The SDS-PAGE analysis found that the SE inhibited the synthesis of the total protein. The SEM and TEM results revealed that the surface and internal ultrastructure of bacteria were damaged. The surface of the bacteria was shriveled and deformed, and the internal structure of the cells was also mutilated. CONCLUSIONS: The SE damaged the cell membrane, with the cytoplasm flowing out, disturbed the synthesis of total protein and phosphorus metabolism, and ultimately killed the bacteria.

Delivery of long-term-injectable agents for TB by lay carers: pragmatic randomised trial.

BACKGROUND: People with recurrent or drug-resistant TB require long courses of intramuscular injections. We evaluate a novel system in which patient-nominated lay carers were trained to deliver intramuscular injections to patients in their own homes. METHODS: A pragmatic, individually randomised non-inferiority trial was conducted at two hospitals in Malawi. Adults starting TB retreatment were recruited. Patients randomised to the intervention received home-based care from patient-nominated lay people trained to deliver intramuscular streptomycin. Patients receiving standard care were admitted to hospital for 2 months of streptomycin. The primary outcome was successful treatment (alive and on treatment) at the end of the intervention. RESULTS: Of 456 patients screened, 204 participants were randomised. The trial was terminated early due to futility. At the end of the intervention, 97/101 (96.0%) in the hospital arm were still alive and on treatment compared with 96/103 (93.2%) in the home-based arm (risk difference -0.03 (95% CI -0.09 to 0.03); p value 0.538). There were no differences in the proportion completing 8 months of anti-TB treatment; or the proportion experiencing 2-month sputum culture conversion. The mean cost of hospital-based management was US$1546.3 per person, compared to US$729.2 for home-based management. Home-based care reduced risk of catastrophic household costs by 84%. CONCLUSIONS: Although this trial failed to meet target recruitment, the available data demonstrate that training patient-nominated lay people has potential to provide a feasible solution to the operational challenges associated with delivering long-term-injectable drugs to people with recurrent or drug-resistant TB in resource-limited settings, and substantially reduce costs. Further data under operational conditions are required. TRIAL REGISTRATION NUMBER: ISRCTN05815615.

Chronic Brucellosis in Japan.

Human brucellosis, one of the most common zoonoses worldwide, rarely occurs in Japan, and only a few chronic cases have been reported. We herein report the case of a 39-year-old Japanese woman with chronic human brucellosis, considered a Brucella canis infection, that persisted for 19 years. Her medical history and fever pattern suggested chronic brucellosis, and the diagnosis was made based on the results of a serum tube agglutination test (SAT). After undergoing combination therapy with streptomycin and doxycycline, she achieved symptomatic relief and showed negative SAT results. Even in non-endemic areas, chronic brucellosis is an important differential diagnosis in patients with long-term persistent fatigue or a fever.

Perianal Tuberculosis: Lessons Learned in 57 Patients From 743 Samples of Histopathology and Polymerase Chain Reaction and a Systematic Review of Literature.

BACKGROUND: Few data are published on perianal tuberculosis. OBJECTIVE: This study aimed to determine the best method to diagnose tuberculosis in patients with fistula-in-ano and to conduct a systematic review to determine the incidence and characteristics of tuberculosis fistula-in-ano. DATA SOURCES: The prospective study data and existing literature were derived from PubMed, Google scholar, and Scopus STUDY SELECTION:: Prospective analysis of patients with tuberculous fistula-in-ano treated between 2014 and 2018 was conducted, and a systematic review of studies describing ≥3 patients with tuberculosis fistula-in-ano was completed. INTERVENTION: Testing of tuberculosis was performed by histopathology or polymerase chain reaction of tissue or pus from the fistula tract. MAIN OUTCOME MEASURES: The primary outcomes measured were the detection rate of various tests to detect tuberculosis in fistula-in-ano and the prevalence rate of tuberculosis in simple versus complex fistulas. RESULTS: In 637 samples (410 patients) tested, tuberculosis was detected in 49 samples (43 patients). Additional samples (n = 106) sent in patients with a high index of suspicion tested positive in 14 more patients. Thus, overall, 63 samples tested positive in 57 patients (total: 743 samples in 410 patients were tested). Tuberculosis was detected in 2 of 181 patients (1.1%) in tissue (histopathology), in 28 of 341 patients (8.2%) in tissue (polymerase chain reaction), and in 19 of 115 patients (16.5%) in pus (polymerase chain reaction) samples. To detect tuberculosis, tissue (polymerase chain reaction) was significantly better than tissue (histopathology) (28/341 vs 2/181, p < 0.00001) and pus (polymerase chain reaction) was significantly better than tissue (polymerase chain reaction) (19/115 vs 28/341, p < 0.0009). Tuberculosis was significantly more common in complex fistulas than in simple fistulas (20.3% vs 7.2%, p = 0.0002). The systematic review (n = 199) highlighted that tubercular fistulas are more common in recurrent and complex fistulas and in tuberculosis endemic regions. LIMITATIONS: The true sensitivity and specificity of each testing modality could not be determined because not all patients with tuberculosis fistula-in-ano were tested by every diagnostic modality studied. CONCLUSIONS: The tuberculosis detection rate of polymerase chain reaction was significantly higher than histopathology. Among polymerase chain reaction, pus had higher detection rate than tissue. Tuberculosis was associated with more complex and recurrent fistulas.

Foodborne infection of mice with Salmonella Typhimurium.

The bacterial pathogen Salmonella enterica serovar Typhimurium is one of the most common causes of foodborne disease in humans and is also an important model system for bacterial pathogenesis. Oral inoculation of C57Bl/6 mice, which are genetically susceptible to Salmonella, results in systemic infection but the murine intestine is not efficiently colonized unless the intestinal microbiota is disrupted. Pretreatment of C57Bl/6 mice with streptomycin, followed by oral inoculation with Salmonella Typhimurium results in colitis resembling human intestinal Salmonellosis. The predominant method of delivery of bacteria is oral gavage, during which organisms are deposited directly into the stomach via a feeding needle. Although convenient, this method can be stressful for mice, and may lead to unwanted tracheal or systemic introduction of bacteria. Here, we developed a method for oral infection of mice by voluntary consumption of regular mouse chow inoculated with bacteria. Mice readily ate chow fragments containing up to 108 CFU Salmonella, allowing for a wide range of infectious doses. In mice pretreated with streptomycin, infection with inoculated chow resulted in reproducible infections with doses as low as 103 CFU. Mice not treated with streptomycin, as well as resistant Nramp1 reconstituted C57Bl/6J mice, were also readily infected using this method. In summary, voluntary consumption of chow inoculated with Salmonella represents a natural route of infection for foodborne salmonellosis and a viable alternative to oral gavage.

Co-administration of a Rhododendron tomentosum extract does not affect mercury tissue concentrations and excretion rate in methylmercury-treated adult male rats.

OBJECTIVES: Consumption of fish/seafood is clearly linked to higher mercury levels in human tissue samples. However, correlations between methylmercury (MeHg) intakes calculated from dietary surveys and mercury body burdens are usually weak and can vary across populations. Different factors may affect MeHg absorption, distribution, metabolism and excretion, including co-exposures to phytochemicals and antibiotics, which were shown to affect mercury body burdens in rodents. Based on the observation that rat pups developmentally exposed to MeHg and a Rhododendron tomentosum extract (Labrador Tea) presented significantly higher blood mercury levels at weaning compared to pups exposed to MeHg alone, the modulation of MeHg toxicokinetics by Labrador Tea was further investigated in adult rats. RESULTS: Total mercury levels were quantified in the blood, liver, kidney and feces of adult male rats exposed to MeHg (1.2 mg/kg bodyweight/day, for 3 weeks) administered either alone or in combination with Labrador Tea (100 mg/kg bodyweight/day) or with an antibiotics cocktail (to inhibit MeHg demethylation by gut bacteria). While the reduced fecal excretion and higher blood mercury levels expected from antibiotics-treated rats were observed, mercury levels in samples from Labrador Tea-treated rats were not significantly different from those measured in samples from rats exposed to MeHg alone.

Pollen reverses decreased lifespan, altered nutritional metabolism and suppressed immunity in honey bees (Apis mellifera) treated with antibiotics.

Nutrition is involved in regulating multiple aspects of honey bee biology such as caste, immunity, lifespan, growth and behavioral development. Deformed wing virus (DWV) is a major pathogenic factor which threatens honey bee populations, and its replication is regulated by the nutrition status and immune response of honey bees. The alimentary canal of the honey bee is home to a diverse microbial community that provides essential nutrients and serves to bolster immune responses. However, to what extent gut bacteria affect honey bee nutrition metabolism and immunity with respect to DWV has not been investigated fully. In this study, newly emerged worker bees were subjected to four diets that contained (1) pollen, (2) pollen and antibiotics, (3) neither pollen nor antibiotics or (4) antibiotics alone. The expression level of two nutrition genes target of rapamycin (tor) and insulin like peptide (ilp1), one nutritional marker gene vitellogenin (vg), five major royal jellyprotein genes (mrjp1-5), one antimicrobial peptide regulating gene relish (rel), and DWV virus titer and its replication intermediate, negative RNA strand, were determined by qRT-PCR from the honey bees at 7 days post-antibiotic treatment. Additionally, honey bee head mass and survival rate were measured. We observed that antibiotics decreased the expression of tor and rel, and increased DWV titer and its replication activity. Expression of ilp1, mrjp1-5 and vg, and honey bee head mass were also reduced compared with bees on a pollen diet. Antibiotics also caused a significant drop in survivorship, which could be rescued by addition of pollen to the diet. Of importance, pollen could partially rescue the loss of vg and mrjp2 while also increasing the head mass of antibiotic-treated bees. Our results illuminate the roles of bacteria in honey bee nutrition, metabolism and immunity, which confer the ability to inhibit virus replication, extend honey bee lifespan and improve overall health.

Rapid direct drug susceptibility testing of Mycobacterium tuberculosis based on culture droplet digital polymerase chain reaction.

SETTING: Early diagnosis and drug susceptibility testing are important for anti-tuberculosis treatment. OBJECTIVE: To develop a rapid method for detecting Mycobacterium tuberculosis and drug susceptibility based on culture and droplet digital polymerase chain reaction (ddPCR). DESIGN: M. tuberculosis in 102 sputum samples was detected using ddPCR, Xpert, quantitative PCR (qPCR) and MGIT™ 960™. The susceptibility of ddPCR-positive samples to rifampicin (RMP), isoniazid (INH) and streptomycin (SM) was tested by measuring changes in DNA quantity over 4 days of culture. For comparison, susceptibility of MGIT 960-positive samples was tested using the standard agar proportion method. RESULTS: The sensitivity and specificity of M. tuberculosis detection using ddPCR and MGIT 960 were respectively 95.7% (95%CI 80.0-99.2) and 88.9% (95%CI 76.7-95.4). Compared with agar proportion, the susceptibility of 44 specimens positive on culture-ddPCR showed sensitivity and specificity for RMP, INH and SM of respectively 83.3% (95%CI 50.9-97.1) and 90.6% (95%CI 73.8-97.6); 79.0% (95%CI 53.9-93.0) and 92% (95%CI 72.5-98.6); 94.1% (95%CI 69.2-99.7) and 92.6% (95%CI 74.3-98.7). CONCLUSION: Culture ddPCR could detect M. tuberculosis within 5 h and drug susceptibility within 4 days directly from sputum, which would greatly reduce the laboratory time needed for tuberculosis diagnosis.