Anaerobic Infections Research Articles

Anaerobic bloodstream infections in Italy (ITANAEROBY): A 5-year retrospective nationwide survey

IntroductionA lack of updated data on the burden and profile of anaerobic bloodstream infections (ABIs) exists. We assessed the incidence of ABIs and trends in antimicrobial resistance in anaerobes isolated from blood in Italy. Material and methodsWe conducted a retrospective study on 17 Italian hospitals (2016–2020). Anaerobes isolated from blood culture and their in vitro susceptibility profiles (EUCAST-interpreted) were registered and analyzed. ResultsA total of 1960 ABIs were identified. The mean age of ABIs patients was 68.6 ± 18.5 years, 57.6% were males. The overall incidence rate of ABIs was 1.01 per 10.000 patient-days. Forty-seven% of ABIs occurred in medical wards, 17% in ICUs, 14% in surgical wards, 7% in hemato-oncology, 14% in outpatients. The three most common anti-anaerobic tested drugs were metronidazole (92%), clindamycin (89%) and amoxicillin/clavulanate (83%). The three most common isolated anaerobes were Bacteroides fragilis (n = 529), Cutibacterium acnes (n = 262) and Clostridium perfringens (n = 134). The lowest resistance rate (1.5%) was to carbapenems, whereas the highest rate (51%) was to penicillin. Clindamycin resistance was >20% for Bacteroides spp., Prevotella spp. and Clostridium spp. Metronidazole resistance was 9.2% after excluding C. acnes and Actinomyces spp. Bacteroides spp. showed an increased prevalence of clindamycin resistance through the study period: 19% in 2016, 33% in 2020 (p ≤ 0.001). ConclusionsOur data provide a comprehensive overview of the epidemiology of ABIs in Italy, filling a gap that has existed since 1995. Caution is needed when clindamycin is used as empirical anti-anaerobic drug.

Gentamicin nanogel films based on Carrageenan-Prosopis africana for improved wound healing

<img src=” https://s3.amazonaws.com/production.scholastica/article/35438/large/prnano_722021ga.jpg?1651243768”> Dermal injuries (e. g., trauma, surgical incisions, and burns) are burdensome health care issues in the world. The delayed healing process can be caused by aerobic and anaerobic bacteria infections, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus and Enterococcus species, Pseudomonas aeruginosa, Peptostreptococcus, and Coliforms. Therefore, reduction of bacterial burden to an acceptable level promotes healing. The rationale for this innovative wound dressing relies on good hydration as the single most important external factor responsible for optimal wound healing. Objective: To study the wound healing potential of GEN nanogel films based on natural polymers using carrageenan (CAR) and Prosopis africana (PRO), a local African plant. Method: Nanogel dispersions and films were characterized by their physical and technological properties, microenvironment, Zeta sizer, Differential Scanning Calorimetry, FTIR spectroscopy, in vitro drug release, and efficacy of wound healing in the rat in a full-thickness excisional model. Results: GEN was dispersed in the polymers, with PROGEN films appearing smoother than CARGEN. Wound closure rate was 88, 76, and 72 % with PROGEN, CARGEN, and GENTA cream, respectively, whereas drug release was sustained in the order of PROGEN (~99 % at 8 h), CARGEN (83 % at 10 h), and pure GENTA powder (81% at 1 h). Topical administration of GEN nanogel-films significantly accelerated (< 15 days) wound closure with no scabbing observed compared to the commercial GENTA cream used. Conclusion: GEN-loaded PROGEN and/or CARGEN films have the potential for developing an improved and affordable topical treatment for cutaneous wound healing.

Antimicrobial susceptibility testing of anaerobic bacteria: In routine and research

To date, antimicrobial susceptibility testing (AST) in anaerobes is highly disarrayed. The testing is not routinely performed in many laboratories to direct appropriate antimicrobial therapy, and literature on AST remains scarce. This review aims to present the current methods employed for the AST of anaerobic bacteria both in routine and research. Anaerobes are potential human pathogens and predominate the human microbiota. Despite their significant role in human health and disease, they are not paid enough heed. The educated experience-based treatment has often been instituted with anaerobic infections due to the challenging AST and relatively predictable patterns of antimicrobial resistance (AMR). However, the AMR in anaerobes remains no more predictable, especially in Gram-negative anaerobes like Bacteroides spp., where multi-drug resistance is also emerging. The changing antibiograms and the emergence of resistance determinants in anaerobes necessitate routine AST, periodic monitoring, and literature review of these organisms. The present scenario dictates to limit the empirical management of anaerobic infections and encourage consistent AST practice in routine by employing economical, faster, and more pragmatic approaches, especially in tertiary care setups that cater to a vast patient population and are held accountable for producing accurate clinical and surveillance data. The article may provide a broad perspective on the topic and different AST solutions.

Is there fungal infestation in paediatric chronic otitis media – Mucosal disease?

ObjectivesTo determine the prevalence of fungal infestation in paediatric chronic otitis media (COM)-mucosal disease and to study the various factors that might influence the said infestation. MethodsA cross-sectional study was done on the cited subject in a sample size of 66 paediatric patients [Age group: 1–18 years] suffering from active COM-mucosal disease. In all we had 75 ears from which swab samples were taken as some patients had bilateral disease. Clinical record was documented in each case. Three sample swabs were analysed for aerobic, anaerobic and fungal infection respectively from the discharging ear. Fungal infection was diagnosed by culture. The data was tabulated and statistically analysed for any correlation of fungal infestation with age, sex, background, duration of COM, previous antibiotic/steroid usage and intractable otorrhoea. ResultsWe recorded a prevalence of 32% for fungal colonization of COM-mucosal disease in paediatric population i.e., 24 out of 75 ears. There was no association observed between fungal infestation and specific paediatric age group, sex, background or duration of the disease. However, a distinct statistical correlation was present between fungal infestation and previous antibiotic/steroid usage and intractable otorrhoea. ConclusionFindings of this study lead us to conclude that all cases of paediatric COM-mucosal disease should be analysed for fungal colonization, especially those with intractable otorrhoea and there should be judicious use of antibiotics and steroid ear drops in COM-mucosal disease.

Genomic Insights into the Distribution of Peptidases and Proteolytic Capacity among Prevotella and Paraprevotella Species.

ABSTRACTBacterial peptidases play important roles in health and nutrient digestion in both humans and animals, especially ruminant animals. In this study, we examined and compared the various peptidases (both total and secretory) among species of Prevotella (44 in total) and Paraprevotella (2) revealed in their sequenced genomes that were archived in the MEROPS database. The phylogenetic relationships were also compared among the species based on 16S rRNA gene sequences and the occurrence of peptidases. A rich repertoire of peptidases was found that represents six catalytic types of peptidases (aspartic, cysteine, glutamic, metallo, mixed, and serine), together with some with unknown catalytic mechanisms, and 78 families. Metallopeptidases were the most predominant, followed by serine and cysteine peptidases. Considerable variations in peptidase occurrence and distribution were noted among the species and the different families of peptidases. A total of 48 different families of secretory peptidases were found in the genomes of the Prevotella and Paraprevotella species. Secretory peptidases in the families of S41 and M13 were ubiquitous, and S9, M16, C1, S13, and C69 were found in more than 95% of the species. Multivariate analysis of the peptidases indicated that species were mostly clustered except for a few species. Analysis using a bipartite association network showed that the majority of peptidase families were shared among the species. The relatedness of peptidase distributions among the species did not significantly correlate with their phylogenetic relationship based on the 16S rRNA genes. The genomic overview on the peptidases of Prevotella and Paraprevotella species provided new insights into their potential capacity to degrade proteins.IMPORTANCE Species of Prevotella are prevalent and predominant bacteria residing in animals and humans, and their proteolytic capacity and activity play important roles in nutrient utilization in animals (especially ruminants) and some anaerobic infections of the intestinal, respiratory, and urinary tracts in humans. This study reveals the large repertoire and wide distribution of metallo, serine, and cysteine peptidases, especially secretory peptidases, among the Prevotella species. The information presented here could aid in the identification of the Prevotella species and the peptidases to target to decrease the excessive protein degradation in the rumen and improve dietary nitrogen utilization by ruminant animals.

Candida Parapsilosis As A Cause Of Necrotizing Fasciitis (Fournier’s Gangrene): A Concern For Patient Safety In Low Resource Settings

Fournier's gangrene (FG) is a polymicrobial aerobic and anaerobic bacterial infection with a rare occurrence of primary fungal aetiology. Here we report a case of FG caused by Candida parapsilosis as the primary organism in a 29-year-old man who presented with perineal soft-tissue infection. He had surgical debridement and colostomy for fecal diversion. He was treated with both antifungal and antibacterial drugs. This case highlights the need to consider fungal pathogens as primary aetiologic agents of FG. This further buttresses the need for antimicrobial stewardship with emphasis on targeted therapy to ensure patient safety.

The Role of the Bacteroides spp. in Bacteraemia

Abstract Anaerobic bacteraemia is not frequent (1.2–13.7%), but is associated with a high mortality rate of 16–27%. Anaerobic infections including bacteraemia nearly always arise from contamination by endogenous bacteria into contiguous or other sites like gastrointestinal tracts, genito-urinary tracts, abscesses etc. Factors leading to anaerobic bacteraemia are mainly surgical procedures, crush injuries, the presence of foreign bodies, tissue necrosis, tumours, diabetes. The most common infectious anaerobic agents are Gram-negative bacilli belonging to the Bacteroides/Parabacteroides, which under normal conditions constitute the natural human microflora. An important factor in the virulency of B. fragilis is the ability to form abscesses; a limited infection may progress to bacteraemia and then systemic inflammatory response syndrome and sepsis. Anaerobic bacteraemia mainly affects adults, with elderly patients over 65 years with underlying diseases. The importance of anaerobic bacteria in neonatal bacteraemia and sepsis is a relatively new phenomenon. In contrast the prevalence of anaerobes in bloodstream infections in children is extremely rare with children between 2 and 6 years of age having the least risk ranging 0–0.5% overall. The incidence of recovery of anaerobes in neonatal bacteraemia varies between 1.8% and 12.5%. The majority of cases reported in the literature were due to Bacteroides spp. (41%) other cultured anaerobes belonged to Clostridium spp. (32%), Peptostreptococcus spp. (20%). Blood cultures remain the gold standard for detection of the etiologic both anaerobes and aerobes agent of bloodstream infection. In recent years significant progress has been made in the methods used in the diagnosis of anaerobes, which significantly reduced the time and cost of the examination. Currently, the mass spectometry MALDI-TOF MS and 16S rRNA sequencing are increasingly used for anaerobic bacteria identification. In 2022, EUCAST published standardized disk diffusion method for the drug susceptibility testing of Bacteroides spp. and other 4 clinically significant anaerobes. Determining the sensitivity of anaerobes to antibiotics is important due to the increasing drug resistance in this group. Number of B. fragilis strains resistant to clindamycin and moxifloxacin is increasing. According to the published studies sensitivity to clindamycin and moxifloxacin is significantly rare and occurs in 64% and 68% of isolates and in 50.8% and 58.2% respectively. Carbapenems and metronidazole continue to be the most effective active antibiotics to be used in the empirical therapy of anaerobic bacteraemia. There are case reports of infections caused by multidrug-resistant strains of Bacteroides spp, meaning resistant to at least three antibiotics from different groups. Rapid microbial diagnosis, targeted therapy and surgical treatment of a possible source of infection are crucial in treatment of sepsis caused by anaerobic bacteria.

Gas in the Musculoskeletal System: The Good and the Bad

Gas in the musculoskeletal system is frequently encountered in clinical practice and can indicate either a benign or nefarious process. Anaerobic infection is capable of producing gas in the musculoskeletal system; however, nonaggressive processes accounting for gas are much more common. In some instances, the presence of gas can be used to exclude a diagnosis of infection. Nitrogen gas bubble may be formed with joint manipulation,1–3 in volume loss and degeneration,4 and in traumatic dislocation/diastasis. The presence of gas with fracture is consistent with open fractures, which affect clinical management, and is associated with future complications.5 The referring or treating physician may also introduce gas through arthrocentesis, injection, or surgery. However, infection must always be considered with scrutiny of the radiologic records and correlation with the clinical history and physical examination so that proper treatment is not delayed. Therefore, it is important for the radiologist to recognize the presence of gas on imaging studies and have the expertise to appropriately raise clinical suspicion of infection or assign a typically benign cause to avoid unnecessary diagnostic evaluation and therapeutic procedures.

Design, Synthesis, and Characterization of TNP-2198, a Dual-Targeted Rifamycin-Nitroimidazole Conjugate with Potent Activity against Microaerophilic and Anaerobic Bacterial Pathogens.

TNP-2198, a stable conjugate of a rifamycin pharmacophore and a nitroimidazole pharmacophore, has been designed, synthesized, and evaluated as a novel dual-targeted antibacterial agent for the treatment of microaerophilic and anaerobic bacterial infections. TNP-2198 exhibits greater activity than a 1:1 molar mixture of the parent drugs and exhibits activity against strains resistant to both rifamycins and nitroimidazoles. A crystal structure of TNP-2198 bound to a Mycobacterium tuberculosis RNA polymerase transcription initiation complex reveals that the rifamycin portion of TNP-2198 binds to the rifamycin binding site on RNAP and the nitroimidazole portion of TNP-2198 interacts directly with the DNA template-strand in the RNAP active-center cleft, forming a hydrogen bond with a base of the DNA template strand. TNP-2198 is currently in Phase 2 clinical development for the treatment of Helicobacter pylori infection, Clostridioides difficile infection, and bacterial vaginosis.

Patterns of bacterial culture and antimicrobial susceptibility test results for dogs with retrobulbar abscesses: 133 cases (2002-2019).

To evaluate patterns of bacterial culture and antimicrobial susceptibility test results for dogs with retrobulbar abscesses and generate recommendations for empirical antimicrobial selection. 133 dogs examined between 2002 and 2019. Records were retrospectively reviewed to determine type of bacterial culture, number and type of bacterial isolates, antimicrobial susceptibility test results, concurrent and recent antimicrobial exposure, effect of culture results on antimicrobial regimen, and outcome. Aerobic culture alone was performed in 37 dogs, and aerobic and anaerobic culture was performed in 96 dogs. Isolates were recovered from 96 dogs, with multiple isolates recovered from 54 (56%) of those dogs. Of the 69 dogs for which both aerobic and anaerobic culture was performed and at least 1 isolate was obtained, 34 (49%) had purely aerobic infections, 15 (22%) had mixed aerobic and anaerobic infections, and 20 (29%) had purely anaerobic infections. Pasteurella spp (n = 26), Streptococcus spp (20), and Escherichia coli (12) were the most common aerobic isolates. Bacteroides spp (n = 22), Actinomyces spp (10), and Fusobacterium (10) spp were the most common anaerobic isolates. Susceptibility test results led to changes in the antimicrobial regimen in 37 of 80 (46%) dogs. Of the 76 dogs for which outcome information was available, 78 (97%) recovered. Multipathogen and anaerobic infections were common in dogs with retrobulbar abscesses. Susceptibility data supported the use of amoxicillin-clavulanate or a combination of clindamycin and enrofloxacin as first-line treatments. Additional study is needed to characterize anaerobic antimicrobial susceptibilities and to compare results of susceptibility testing with in vivo responses to antimicrobial administration.

Comparative Contact Angle Analysis of 1% Chlorhexidine and 2% Metronidazole Mouthgel - An In Vitro Study

Introduction: Chlorhexidine is considered to be gold standard drug in treating various oral infections. Metronidazole is effective for treating various anaerobic infections, including dental abscess and periodontal abscess. In current technical applications and material sciences, contact angle is a thermodynamic characteristic that describes the wettability of solid surfaces. The aim of this study is to compare the contact angle of chlorhexidine and metronidazole mouth gel.
 Materials and Methods: Total 10 samples of gel was used, involving 5 samples of 1% chlorhexidine gel and 5 samples of 2 % metronidazole gel. The contact angles were measured with the Ossila Goniometer after each sample was dispensed from a microsyringe on the facial surface of the microtomed tooth. The obtained values from the samples were determined, and the data was statistically analysed using SPSS software version 20.0. The paired t test was used to analyse the data.
 Results: From the data collected it was found that the average contact angle of the mouth gels that is chlorhexidine and metronidazole gel were 51.58 & 67.04 respectively. T test showed p value of 0.157, (p value < 0.05). Hence, it is statistically not significant. Chlorhexidine gel had a lower average mean of contact angle than metronidazole gel, which means chlorhexidine gel had got better wettability when compared to metronidazole gel.
 Conclusion: Thus, it can be concluded that chlorhexidine gel has a better tendency to wet the tooth or oral cavity surfaces when compared to metronidazole gel, thereby it can have better action in treating various oral conditions.

Nano-oxygenated hydrogels for locally and permeably hypoxia relieving to heal chronic wounds

Exposed chronic wounds are usually covered by hypoxic tissues accompanied with necrosis, persistent inflammation and anaerobic infections. Since atmospheric oxygen air can only penetrate about 0.3 mm tissues, meanwhile oxygen from the circulation is difficult to reach chronic wounds through the destroyed blood vessels, a solution to deliver oxygen locally and permeably is urgently needed. Herein we report a technique to reform traditional gel-based wound dressings by adding lyophilized oxygen encapsulated nanoparticles, which can deliver dissolved oxygen locally into wound surface. This delivery technique can potentially evaluate the therapeutic effects on hypoxic epithelial, endothelial and fibroblasts in vitro. Further experiments confirm the effects on both open wounds bearing and flap transplanted diabetic mice models. Considering its biocompatibility, effectiveness and practicality, we believe our hydrogel has significant transformation value to care and accelerate the healing of various clinical wounds, especially chronic wound.

The importance of topical metronidazole in the treatment of acute anal fissure: a double-blind study controlled for prospective randomization.

Acute anal fissure, a disease characterized by severe pain in the anorectal area, reduces quality of life and becomes chronic absent appropriate treatment. More recently, anaerobic infections have been noted as contributive to etiopathogenesis. This study investigates topical metronidazole's effect in the treatment of acute anal fissure. Our prospective randomized controlled double-blind study included 2 groups of 100 patients older than 18 years from our General Surgery Clinic with anal fissure complaints for less than 8 weeks. Topical diltiazem treatment was started in group 1, and topical diltiazem and metronidazole treatment in group 2. Pain levels were evaluated by the visual analogue scale (VAS) score, and recovery status was evaluated by physical examination findings ab initio and at the 1st, 4th, and 6th weeks. VAS score levels, demographic, clinical, and recovery status were then compared. There was no difference between the groups as to age, sex, pain on defecation, bleeding, constipation, and duration of pain, bleeding and constipation (P>0.05). From week 1, fissure epithelialization and healing rates were higher in group 2 (P<0.001); group 2 VAS score levels were lower than in group 1 (P<0.001) and achieved by group 1 only in week 4 (P=0.073). Adding topical metronidazole to treatment reduces the duration and severity of pain, shortens healing time, and increases the healing rate.

Comparative Genomics of Bacteroides fragilis Group Isolates Reveals Species-Dependent Resistance Mechanisms and Validates Clinical Tools for Resistance Prediction.

ABSTRACTBacteroides fragilis group (BFG) are the most frequently recovered anaerobic bacteria from human infections, and resistance to frontline antibiotics is emerging. In the absence of routine antimicrobial susceptibility testing (AST) for BFG in most clinical settings, we assessed the utility of clinical and modern genomics tools to determine BFG species-level identification and resistance patterns. A total of 174 BFG clinical isolates supplemented with 20 archived carbapenem-resistant B. fragilis sensu stricto (BFSS) isolates underwent antimicrobial susceptibility testing, MALDI-ToF mass-spectrometry, and whole-genome sequencing (WGS). Bruker BioTyper and VITEK-MS MALDI-ToF systems demonstrated accurate species-level identifications (91% and 90% agreement, respectively) compared to average nucleotide identity (ANI) analysis of WGS data. Distinct β-lactamase gene profiles were observed between BFSS and non-fragilis Bacteroides species, with significantly higher MICs to piperacillin-tazobactam in B. vulgatus and B. thetaiotaomicron relative to BFSS (P ≤ 0.034). We also uncovered phylogenetic diversity at the genomospecies level between division I and division II BFSS (ANI <0.95) and demonstrate that division II BFSS strains harbor an increased capacity to achieve carbapenem resistance through chromosomal activation of the CfiA carbapenemase. Finally, we report that CfiA detection by the Bruker BioTyper Subtyping Module accurately detected carbapenem resistance in BFSS with positive and negative percent agreement of 94%/90% and 95%/95% compared to ertapenem and meropenem susceptibility, respectively. These comparative analyses indicate that resistance mechanisms are distinct at both the phenotypic and genomic level across species within the BFG and that modern MALDI-ToF identification systems can be used for accurate species-level identification and resistance prediction of the BFG.

Anaerobes in cardiac infections: A decade experience from the tertiary care center

PurposeAnaerobic infections are common yet life-threatening. They are being recovered from all sites of the body, including the cardiovascular system. This study was aimed to determine the retrospective analysis on the isolation of anaerobes in cardiovascular samples received for a decade-long duration. It helps in knowing the frequency of isolation of anaerobic causes of cardiovascular infection. MethodsAll cardiovascular samples from the department of Cardio-thoracic vascular surgery from January 2010 to December 2020 were studied. ResultsOf 601 samples received, predominant samples were vegetations and valvular tissues of 258, followed by 98 samples of pericardial tissues, 92 samples of embolus, 90 samples of blood and post-operative collections, and 63 excised aneurysms and vascular grafts. Of the total, 15 samples grew anaerobes where Clostridium species were the predominant isolates. Clostridioides difficile was isolated in 2 samples. ConclusionsAnaerobes in cardiovascular samples are uncommon yet form a significant cause of morbidity and mortality. Most infections are from the contiguous spread, penetrating trauma, and hematogenous causing endocarditis or valvular infections. These conditions and samples form the seat of infectious focus and clinical suspicion towards the anaerobic cause of these conditions, especially in conventional routine culture-negative samples. Timely diagnosis of anaerobic infections plays a vital role in the good prognostic outcome of patients undergoing cardiothoracic and vascular surgery.

FULMINANT SOFT TISSUE INFECTIONS CAUSED BY ANAEROBIC BACTERIA – THE PARAMEDIC’S PERSPECTIVE

Anaerobic bacterial infections are a broad group of conditions ranging from superficial skin infections to deeply embedded necrotic soft tissue infection. It has been observed that amongst all the microorganisms causing soft tissue infection, as many as 70% of anaerobic bacteria are unquestionably related to mortality in fulminant infections. Such infections are most commonly caused by an open wound that is insufficiently disinfected, or through the wound coming into contact with soil or human or animal faeces. This is particularly the case for wounds resulting from traffic accidents and injuries sustained in agriculture. In 80-95% of cases, the bacteria Clostridium perfringens is responsible for the appearance of gas gangrene manifested by sharp pain and crepitus in the wound area, as well as disseminated intravascular coagulation and septic shock. Due to time-consuming diagnosis and delays in obtaining the results of bacteriological tests, identifying the condition and commencing treatment relies mainly on a paramedic’s knowledge and experience. Improving the body of knowledge on soft tissue infection and awareness of the potential risks can affect the patient’s prognosis.

Pneumoperitoneum, pneumoretroperitoneum, pneumo-mediastinum, pneumothorax, and extensive subcutaneous emphysema caused by wisdom tooth extraction

Pneumoperitoneum is mainly caused by viscus perforation, in most cases by ulcer. However, it may be caused by non-surgical causes in 10 % of the cases, such as patients on respiratory support, renal dialysis, paracentesis, and anaerobic bacterial infections. In addition, there are some sporadic causes of pneumoperitoneum; one of them is wisdom tooth extraction, which is described in our case. In the literature review, there were many documented cases of subcutaneous emphysema, pneumothorax, and pneumomediastinum caused by wisdom tooth extraction. However, there was only one case that represented pneumoperitoneum after that procedure. In this case report, We are reporting a case of a 50-year-old male patient who presented one-week status post wisdom tooth extraction, with significant subcutaneous emphysema involving the face, neck, and upper chest; further workup and assessment showed that the patient has considerable pneumoperitoneum, pneumomediastinum, mild pneumothorax bilaterally, and extensive subcutaneous emphysema on computerized tomography. The patient was managed conservatively, with no need for any surgical intervention, and he was discharged home in a good general condition.

Three cases of Actinomyces isolation from the eye lesions of patients with a chronic and recurrent ophthalmic infection

Actinomycosis is an indolent, slowly progressive infection caused by Gram-positive facultative anaerobic bacteria from the genus Actinomyces. These bacteria have been reported as a cause of ophthalmic infections such as endophthalmitis, keratitis, and canaliculitis. The objective of the present study was to investigate the pattern and antibiotic sensitivity profile of anaerobes isolated from lesions in the medial canthus of the eye. Three pus aspirate samples (from eye) were delivered to a microbiology laboratory in a strict anaerobic condition in Robertson Cooked Meat media (RCM). The samples were inoculated on Blood Agar and incubated anaerobically in a Gas Pack Jar incubator at 5%-10% CO2 and in aerobic condition at 37°C. Pure colonies isolated on anaerobically incubated plates were identified by the VITEK® 2 COMPACT system. Antibiotic sensitivity testing was conducted using an Epsilometer-strip test. Three isolates identified and confirmed with the help of VITEK® 2 were Actinomyces israelii, Actinomyces odontolyticus, and Actinomyces meyeri. All three species of Actinomyces were sensitive to Vancomycin, Moxifloxacin, and Imipenem, but they were resistant to Metronidazole. Since there have been several cases of anaerobic ophthalmic infections reported to date, samples from patients with chronic eye infections should be analyzed for anaerobic culture for correct diagnosis and proper treatment. Moxifloxacin but not Metronidazole is a suitable drug for the treatment of anaerobic eye infection.

Biochemical and structural characterization of Haemophilus influenzae nitroreductase in metabolizing nitroimidazoles.

Nitroheterocycle antibiotics, particularly 5-nitroimidazoles, are frequently used for treating anaerobic infections. The antimicrobial activities of these drugs heavily rely on the in vivo bioactivation, mainly mediated by widely distributed bacterial nitroreductases (NTRs). However, the bioactivation can also lead to severe toxicities and drug resistance. Mechanistic understanding of NTR-mediated 5-nitroimidazole metabolism can potentially aid addressing these issues. Here, we report the metabolism of structurally diverse nitroimidazole drug molecules by a NTR from a human pathogen Haemophilus influenzae (HiNfsB). Our detailed bioinformatic analysis uncovered that HiNfsB represents a group of unexplored oxygen-insensitive NTRs. Biochemical characterization of the recombinant enzyme revealed that HiNfsB effectively metabolizes ten clinically used nitroimidazoles. Furthermore, HiNfsB generated not only canonical nitroreduction metabolites but also stable, novel dimeric products from three nitroimidazoles, whose structures were proposed based on the results of high resolution MS and tandem MS analysis. X-ray structural analysis of the enzyme coupled with site-directed mutagenesis identified four active site residues important to its catalysis and broad substrate scope. Finally, transient expression of HiNfsB sensitized an E. coli mutant strain to 5-nitroimidazoles under anaerobic conditions. Together, these results advance our understanding of the metabolism of nitroimidazole antibiotics mediated by a new NTR group and reinforce the research on the natural antibiotic resistome for addressing the antibiotic resistance crisis.

Fluorescent metabolic labeling-based quick antibiotic susceptibility test for anaerobic bacteria.

Because of the advancements in medicine and science, the numbers of patients surviving complicated diseases are continuously increasing, which in turn leads to elevated chances of anaerobic infections by endogenous bacteria. Traditional growth yield-based antibiotic susceptibility tests (ASTs) against anaerobic bacteria are very time-consuming (≥48 h) and labor intensive, which delays the timely guidance of antibiotic prescription and increases the mortality of patients. Inspired by a fluorescent d-amino acid (FDAA) labeling-based AST (FaAST) that we recently developed for quick determination of aerobic bacteria's susceptibilities, here we report an accurate and fast AST method for anaerobic pathogens. Based on flow cytometry analysis of anaerobes that have been treated with various doses of antibiotics and metabolically labeled with FDAA, the intensities of which can reflect their affected metabolic status by the drugs, the MICs of each drug can then be determined. The whole process can be completed in 5 h. After testing 40 combinations of the representative anaerobic bacteria and antibiotics, our method demonstrates a high susceptibility category accuracy of 95.0%. This FaAST-based protocol is helpful in accurately and quickly guiding antibiotic decisions when treating critical infections caused by anaerobic bacteria.