Phenotypical and Genotypical Comparison of Clostridium difficile Isolated from Clinical Samples: Homebrew DNA Fingerprinting versus Antibiotic Susceptibility Testing (AST) and Clostridial Toxin Genes

Background. Clostridium (Clostridioides) difficile is recognized as the major cause of healthcare antibiotic-associated diarrhea. We surveyed a molecular epidemiological correlation between the clinical isolates from two general hospitals in Iran through clustering toxigenic types and antibiotic susceptibility testing (AST) accuracy. Methods. Study population included 460 diarrhoeic specimens from inpatients with a history of antibiotic therapy. All samples underwent enriched anaerobic culture, confirmed by detection of gluD gene with PCR. Toxin status and AST were assessed by the disk diffusion method (DDM) and minimal inhibitory concentrations (MICs) of metronidazole, vancomycin, and rifampin. C. difficile outbreak was analyzed through conventional PCR by tracing toxin genes and Homebrew pulsed-field gel electrophoresis (PFGE) for characterizing isolates within our healthcare systems. Results. A total of 29 C. difficile strains were isolated by enriched anaerobic culture from the clinical samples. Among them, 22 (4.8%) toxigenic profiles yielded toxins A and B (tcdA, tcdB) and binary toxins (cdtA, cdtB). The minimum inhibitory concentration (MIC) was 18.1% and 9% for vancomycin and metronidazole, and all isolates were susceptible to rifampicin and its minimum inhibitory concentration was at <0.003 μg/mL. The most dominant toxigenic and antibiotic-resistant “pulsotype F” was detected through PFGE combined with multiple Clostridial toxigenic pattern and AST. Conclusions. DNA fingerprinting studies represent a powerful tool in surveying hypervirulent C. difficile strains in clinical settings. Resistance to vancomycin and metronidazole, as first-line antibiotics, necessitate accomplishment of proper control strategies and also prescription of tigecycline as a more appropriate option.

Necrotizing enterocolitis

Necrotizing enterocolitis is not a new disease but one that has been reported since special care units began to house preterm infants. It was observed in the foundling hospitals of Paris (Billard 1828) and Vienna (Bednar 1850) and, as it occurred in clusters, was regarded as a nosocomial infection in the infant hospitals of Zurich (Willi 1944) and Berlin (Ylppö 1931). Clinical and pathoanatomical characterization was achieved by Schmidt and Quaiser in 1952. The unproven hypothesis of mesenteric hypoperfusion as a major aetiological factor arose from animal models and analogous perforating disorders in term infants. Despite similarities between necrotizing enterocolitis and clostridial infections, few studies employed anaerobic culture techniques. The pathogenesis remains unclear and its distinction from related disorders uncertain. It is unlikely that strategies to prevent necrotizing enterocolitis will be successful unless the disease is better understood.

Comparative microbiological study of methods of using Metrogyl Dent gel in the treatment of periodontitis

Summary The common drug Metrogyl Denta can be used in different ways in the treatment of periodontitis, in particular, in the form of applications or using high-frequency ultrasound. The article is devoted to the study of the effect of various methods of using Metrogyl Dent gel on the microbiota of periodontal pockets in periodontitis. In 40 patients with a diagnosis of moderate periodontitis, the treatment complex included a combined effect of high-frequency ultrasound and an antibacterial preparation of Metrogyl Dent gel (ultraphonophoresis of Metrogil Dent gel) or Metrogil Dent was used as applications (20 people in the compared groups). The contents of the pathological periodontal pockets were studied twice before treatment (after removing dental deposits), and also after 10 days of treatment. Microbiological examination to identify periodontal pathogenic bacterial microbiota was carried out using the anaerobic culture technique. Ultraphonophoresis of Metrogyl Denta gel has a more pronounced antibacterial effect in comparison with the applications of this drug. The combined effect of high-frequency ultrasound and Metrogyl Denta gel makes it possible to create a drug depot in the periodontal tissues.

Prerequisite Evaluation of Anaerobic Settings for Gut Microbiome Functional Studies

Colon cancer-associated gut bacteria were mostly identified via next-generation sequencing in gut microbiome profiling studies. Anaerobic culture systems can be used to culture colon cells with these gut bacteria to further confirm the tumorigenic properties of these bacteria. Nevertheless, it is unclear how colon cells will grow in an anaerobic environment, as most cells are cultured aerobically. Therefore, we investigated the survival and viability of HT-29, a colon cancer cell line in an anaerobic culture system, and compared it to the usual culture condition in an aerobic setup. Interestingly, we found that HT-29 was able to grow in the anaerobic setup. Its viability was similar for both culture conditions, with only a slower growth rate observed in the anaerobic setup. Furthermore, gene expression studies showed that the cells were not under severe anaerobic stress even when exposed to the oxygen-deprived environment.This study provided results on some baseline parameters of an anaerobic colon cell culture system, and will be useful for journal readers who wish to investigate functional properties of anaerobic bacteria.

The Impact of Norepinephrine on Mono-Species and Dual-Species Staphylococcal Biofilms

The effect of norepinephrine (“NE”) on Gram-negative bacteria is well characterized; however, little is known about the impact of NE on cutaneous Gram-positive skin residents, especially staphylococci. In this study, the impact of NE on monospecies and dual-species biofilms of Staphylococcus epidermidis and S. aureus model strains was investigated for the first time. Biofilms were grown in two different models (on polytetrafluoroethylene (“PTFE”) cubes and glass microfiber filters (“GMFFs”)) and additionally kinetic measurements of bacterial growth was performed. We have shown that NE can affect the biofilm formation of both species with a strong dependence on aerobic or anaerobic culture conditions in different models. It was shown that S. epidermidis suppresses S. aureus growth in dual-species biofilms and that NE can accelerate this process, contributing to the competitive behavior of staphylococci.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 μM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice

Purpose Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an anti-malarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Methods Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. Results In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was 1 day shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs. Conclusion ATV enhanced the effect of ABZ in the treatment of AE in mice.