Anaerobic Pathogens Research Articles

A-280 Impact of 2022 CLSI Revised Piperacillin/Tazobactam Clinical Breakpoints on Enterobacterales Isolates Identified at a Tertiary Medical Center in Southern Taiwan

Abstract Background Piperacillin-tazobactam (TZP) is one of the most common antibiotics administered to hospitalized patients. Its broad activity against gram-negative, gram-positive, and anaerobic pathogens. The Clinical and Laboratory Standards Institute (CLSI) publishes revised TZP breakpoints in 2022. The purpose of this study was to evaluate the impact of changing the TZP breakpoint in a regional teaching hospital in southern Taiwan. Methods We retrospectively evaluated TZP for 1347 Enterobacterales isolates identified by MALDI-TOF (Bruker Daltonik, Bremen, Germany) between September 1, 2022, through December 31, 2022, at Chang Gung Memorial Hospital in Chiayi. Blood antimicrobial susceptibility testing was performed using an NMIC-411 panel on the BD Phoenix™ M50. The antibiotic susceptibility test of Enterobacterales from other sources of infection used the Disk diffusion method (Kirby-Bauer Method). Susceptibilities were assessed based on historic CLSI breakpoints in 2021 as well as the current CLSI breakpoints from the most recent CLSI MIC breakpoints, the 32th edition of document M100, published in 2022. Results We evaluated 1347 Enterobacterales isolates. Most isolates identified were Escherichia coli [n = 768 (57%)] and Klebsiella pneumoniae [n = 286(21.2%)]. The new TZP breakpoints reduced the percentage of strains that were previously judged to be susceptible by CLSI M100-S31 from 80.9%(1090/1347) to 50%(674/1347). The majority of isolates 674(50%) were susceptible; 416 (30.9%) isolates were susceptible-dose dependent and 257 (19.1%) were resistant to piperacillin/tazobactam using the new CLSI breakpoints. The results of the analysis of the source of infection found that the bacteria that had the greatest impact on the respiratory tract decreased from 43.6% to 12.7%, followed by the genitourinary tract infection decreased from 80.4% to 27%, bodily fluid infection decreased from 69.8% to 24.5%, and abscesses,wounds,pus decreased from 72.7% to 28.1%; The most insignificant are blood infection strains, from 83.1% to 81.3%. Conclusion Our results indicate an increase in resistance to Enterobacterales isolates tested. Non- susceptible to piperacillin/tazobactam from 19.1% to 50% using the revised CLSI breakpoints. The decreased susceptibilities make it clear that microbiology laboratories should accept new MIC breakpoints. Although our study has demonstrated decreased susceptibilities, there is no correlation with clinical outcomes. In conclusion, changes in breakpoints had a significant impact on the susceptibility of TZP for Enterobacterales isolates in our study. Understanding and evaluating the impact of the breakpoint changes is of paramount importance. Institutions should ensure that their breakpoints are up to date to allow for the most optimized treatment.

Patterns of Pathogens and Antimicrobial Susceptibility Profiles in Diabetic Foot Ulcers’ Infections among Patients at Bugando Medical Centre in Mwanza, Tanzania

Background: Diabetic foot ulcer (DFU) is an open sore or wound on the foot of a person with diabetes, and is most commonly located on the plantar surface, or bottom of the foot. The defect is relatively common in adult and elders with diabetes mellitus. Infected lower extremities wound with multi-drug resistant bacteria usually are associated with increased morbidity, mortality and long-term disabilities among diabetic patients. Although the burden of DFU is known in Tanzania (and Mwanza in particular), there is limited information on the patterns of pathogens associated with DFU in our setting, on bacterial and fungal pathogens which in turn limit specific management options to these patients. Methodology: Analytical cross section hospital-based study was conducted among patients with DFU admitted or attending outpatient clinics at BMC from May to July 2022. A structured questionnaire was used to collect socio-demographic, clinical and laboratory data from patients with DFU. Tissue culture was obtained from the base of the ulcer after cleaning with normal saline followed by removing of cellular debris and normal flora over the lesion. Finally, the obtained samples were subjected to culture methods to identify the presence of pathogens (bacteria and fungi) and antimicrobial susceptibility profiles of bacteria. Result: During the study period and based on inclusion criteria, a total of 71 patients, with a mean age of 59.2 ± 13.0 years were recruited. The majority of the participants were male 47 (66.2%). A total of 60 (84.5%) samples were culture positive, resulting into a total of 92 microorganisms isolated. More than half of cultures 34 (56.7%) revealed the presence of single microorganisms. Pathogens isolated were both bacterial 70 (76.1%) and fungi species 22 (23.9%), all bacterial isolates were aerobic. P. aeruginosa and E. coli were most frequent isolated gram-negative bacteria 12(23.5%) and 9(17.6%) respectively. On other hand common isolated gram-positive strains were S. aureus 13 (68.4%) out of all 19 gram-positive culture isolates. A total of 22 fungi spp were isolated, among them 7 (31.8%) were yeast C. albicans 3 (42.8%), A. fumigatus were frequent isolated 13 (86.7%). Polymicrobial growth was observed in 43.3% samples against 56.7% of monomicrobial growth. Most prevalent gram-negative bacteria Pseudomonas aeruginosa showed low resistance to ciprofloxacin, meropenem, gentamicin, piperacillin tazobactam (7.1%, 21.4%, 21.4% and 21.4 respectively) but more resistant to cephalosporins. Staphylococcus aureus showed low resistance to ciprofloxacin, gentamicin and clindamycin (30.8%, 15.4%, and 38.5% respectively), Total MDR bacteria isolates were 64.3%, where methicillin resistance staphylococcus aureus (MRSA) were 76.9%. More than 85.7% of the patient with higher grade ulcer, stage 3 and 4 according to Wagner’s classification were positive to diabetic foot infection and 14.3% of patients with Wagner’s stage 3 and 4 were not infected on their ulcers. Conclusion: Gram negative bacteria were most commonly isolated than gram positive bacteria in causing DFU infections. For gram negative spp the most effective antibiotic were ciprofloxacin, gentamicin, piperaccilin tazobactam and meropenem. Third generation cephalosporins, amoxicillin clavulanate showed poor effectiveness. All cases of DFU infection should therefore be subjected to culture and antimicrobial sensitivity testing for targeted infection management. More studies involving anaerobic pathogens and antifungal susceptibility patterns

Comparative genomic analysis of Fusobacterium nucleatum reveals high intra-species diversity and cgmlst marker construction

BackgroundFusobacterium nucleatum is a one of the most important anaerobic opportunistic pathogens in the oral and intestinal tracts of human and animals. It can cause various diseases such as infections, Lemierre's syndrome, oral cancer and colorectal cancer. The comparative genomic studies on the population genome level, have not been reported.ResultsWe analyzed all publicly available Fusobacterium nucleatums’ genomic data for a comparative genomic study, focusing on the pan-genomic features, virulence genes, plasmid genomes and developed cgmlst molecular markers. We found the pan-genome shows a clear open tendency and most of plasmids in Fusobacterium nucleatum are mainly transmitted intraspecifically.ConclusionsOur comparative analysis of Fusobacterium nucleatum systematically revealed the open pan-genomic features and phylogenetic tree based on cgmlst molecular markers. What’s more, we also identified common plasmid typing among genomes. We hope that our study will provide a theoretical basis for subsequent functional studies.

Porphyromonas gingivalis suppresses oral squamous cell carcinoma progression by inhibiting MUC1 expression and remodeling the tumor microenvironment.

Bacteria are the causative agents of various infectious diseases; however, the anti-tumor effect of some bacterial species has attracted the attention of many scientists. The human oral cavity is inhabited by abundant and diverse bacterial communities and some of these bacterial communities could play a role in tumor suppression. Therefore, it is crucial to find oral bacterial species that show anti-tumor activity on oral cancers. In the present study, we found that a high abundance of Porphyromonas gingivalis, an anaerobic periodontal pathogen, in the tumor microenvironment (TME) was positively associated with the longer survival of patients with oral squamous cell carcinoma (OSCC). An in vitro assay confirmed that P. gingivalis accelerated the death of OSCC cells by inducing cell cycle arrest at the G2/M phase, thus exerting its anti-tumor effect. We also found that P. gingivalis significantly decreased tumor growth in a 4-nitroquinoline-1-oxide-induced in situ OSCC mouse model. The transcriptomics data demonstrated that P. gingivalis suppressed the biosynthesis of mucin O-glycan and other O-glycans, as well as the expression of chemokines. Validation experiments further confirmed the downregulation of mucin-1 (MUC1) and C-X-C motif chemokine 17 (CXCL17) expression by P. gingivalis treatment. Flow cytometry analysis showed that P. gingivalis successfully reversed the immunosuppressive TME, thereby suppressing OSCC growth. In summary, the findings of the present study indicated that the rational use of P. gingivalis could serve as a promising therapeutic strategy for OSCC.

Isolation of Facultative Anaerobic Bacterial Pathogens from Canned Food and Use of Lactobacillus Plantarum as A Bio-Control Agent

Preserved foods can play a significant role in causing food poisoning when they are not handled, processed, or stored properly. Objective: To investigate facultative anaerobic foodborne bacterial pathogens from canned foods and to control their growth Lactobacillus plantarum was used as a bio-control agent. Methods: Different canned food samples were collected to isolate and identify facultative anaerobic bacterial pathogens. Results: Out of n=65 samples, n=13 samples cultured positive as facultative anaerobes. They were further confirmed with biochemical and molecular identifications as foodborne bacterial pathogens with a ratio of 62% Escherichia coli, 30% Salmonella typhimurium, and 8% Vibrio cholerae. During bio-control studies, the results revealed possible inhibition of facultative anaerobic bacterial pathogens by using purified compounds of Lactobacillus plantarum. Conclusions: The use of probiotics in canned foods requires careful consideration, as factors such as the specific strain, food matrix, processing conditions, and storage practices can influence its effectiveness.

Aggressive abscesses in the head and neck region of probable odontogenic origin – two case reports

Odontogenic infections are the most common type of infections of the head and neck among adults. They usually spread continuously from the mandible and/or maxilla into the sublingual, submandibular or masticatory spaces, then directly into the parapharyngeal space. They require comprehensive diagnostic and therapeutic management, as they may potentially lead to life-threatening complications. We present two clinical manifestations of aggressive abscesses in the head and neck region of probable odontogenic origin and discuss different therapeutic approaches. The first patient, a 31-year-old man, was admitted because of a tumor-like lesion within the right maxillary sinus. He complained of purulent nasal discharge of two weeks duration despite antibiotic therapy. In the Caldwell-Luc operation a cyst filled with puss was displayed, the sac of the cyst and the polypoid mass were removed. The second patient, a 28-year-old man, was admitted due to an abscess of the left half of the neck and viscerocranium, with a large edema of this area, neck pain, fever, dyspnea and lockjaw. The patient was qualified for surgery. During intubation, a purulent collection emptied into the oral cavity and throat. In both cases, simultaneously with the surgery, an empirical broad-spectrum antibiotic therapy was administered. Microbiological analysis of the material collected during the operations revealed Prevotella melaninogenica in the first case, and Streptococcus group C in the second. Treatment of aggressive odontogenic infections must take a comprehensive approach, including both surgical intervention and antibiotic therapy covering Gram-positive as well as Gram-negative aerobic and anaerobic pathogens.

Peran photodynamic therapy dalam perawatan periodontal non-bedah

Introduction: The main of non-surgical periodontal treatment is mechanical debridement. However, the treatment success rate is greatly influenced by ease of mechanical instrument access. Therefore, adjunctive antimicrobial therapy is indicated for complete removal of periodontal pathogen. Since long term systemic antimicrobial treatment can cause resistency, local antimicrobial treatment such as photodynamic therapy may serve as a better choice. Literature review: Photodynamic therapy (PDT) consists of 3 elements: photosensitizer, oxygen, and visible light. After photosensitizer binds with target cells, they are activated by visible light with suitable wavelength, thus producing singlet oxygen and Reactive Oxygen Species (ROS) to create toxic environment for anaerobic periodontal pathogen. PDT increases clinical attachment gain in both chronic and aggressive periodontitis treatment. It also decreases bleeding on probing and pocket depth. Discussion: The advantages of PDT are non invasive, short treatment time required, and reduced possibility of antimicrobial resistency. The disadvantages of PDT are tissue thermogenesis effect and temporary pigmentation of gingiva by photosensitizer. Several precautions should be made while using PDT such as adequate eye protection, irrigation, and isolation. Conclusion: Combination of mecahnical debridement and PDT is proven effective to increase clinical parameters and decrease bacterial burden in peridontitis treatment.

Species Characteristics of Causative Agents of Acute Appendicitis in Children and Determination of Their Susceptibility to Antibiotics

In pediatric surgery, acute appendicitis is considered one of the most common problems requiring surgical intervention. Among the causes of this disease, microorganisms are of primary importance. The specificity of postoperative treatment depends both on the degree of virulence of the pathogen and on the microbial load that caused the inflammatory process. The increase in the use of antimicrobial agents is of great concern because of the emergence of antibiotic-resistant bacteria. Therefore, the issue of rational postoperative antibiotic therapy remains relevant, as excessive, often unjustified use and incorrect dosage of drugs have become the cause of many medical problems. The aim of the research was to determine the species structure and analyze antibiotic resistance of microorganisms in biomaterial obtained from children after appendectomy for acute appendicitis. Methods. We studied biomaterial obtained from 74 patients aged 2—18 years who were treated at the MNCE Ternopil Regional Children Clinical Hospital TRC in the period from September 2021 to March 2022. After appendectomy, the samples were placed in a transport medium for further laboratory research, which involved staining smears according to the Gram method, sowing microorganisms on nutrient media such as blood agar, salt agar, sugar broth and serum agar, and Endo medium for enterobacteria as well as for anaerobic pathogens — thioglycolic medium and Kitta-Tarozzi medium, and identifying by morphological, tinctorial, cultural and biochemical properties. The sensitivity of selected pathogenic microorganisms to antibiotics was determined using the Kirby-Bauer method. Statistical processing of digital data was carried out using Excel software (Microsoft, USA) and the Statistica 10.0 program. Results. 74 children aged from 2 to 18 years were involved in the study. E. coli (28.4% of all examined), S. aureus (21.6%), and P. aeruginosa (14.9%) were found during the laboratory study of biomaterial. E. faecalis, Klebsiella spp., S. epidermidis, and S. viridans occurred much less often (from 9.4% to 4.5%). The study of antibiotic resistance showed that the isolated microorganisms differed significantly in their sensitivity both to different groups of antimicrobial agents and to generations of drugs within the same group. Ceftriaxone was the most effective inhibitor of all detected microorganisms. E. coli, S. aureus, P. aeruginosa, and S. epidermidis showed 100% sensitivity to it, and the others — within 75—50%. Bacteria Klebsiella spp. and S. epidermidis were sensitive to amikacin, strains of E. coli — 90.5%, P. aeruginosa and S. aureus — 81.8% and 81.2%, respectively. Amoxiclav and ampisulbin had weak inhibitory activity, except for 100% of Klebsiella spp. and 75% of E. faecalis, which were inhibited only by amoxiclav. However, almost all studied microorganisms were partially sensitive to azithromycin. The activity of this antibiotic ranged from 100—81.8% (S. epidermidis, S. aureus) to 36.4% (P. aeruginosa). Conclusions. The most common causative agents of acute appendicitis among children are the following microorganisms: E. coli, S. aureus, P. aeruginosa, and sporadically — S. saprophiticus, Micrococus spp., S. viridans, and Candida spp. Among the antibiotics used for the treatment of postoperative complications in various forms of appendicitis, the most effective were ceftriaxone — cephalosporins of the III generation — 100—66.7%, amikacin — aminoglycosides — 100—57.1%, and furagin — nitrofuran derivatives — 100—60%. The studied microorganisms were the least sensitive to penicillins and antibiotics of the II generation of the fluoroquinolone group. Resistance of microorganisms to certain antibiotics was also found, in particular: P. aeruginosa and E. faecalis — to cefuroxime and cefazolin; E. coli and S. aureus — to tobramycin, S. epidermidis — to ofloxacin and amoxiclav.

Phagocyte extracellular traps formation contributes to host defense against Clostridium perfringens infection

Clostridium perfringens (C. perfringens) is an important Gram-positive anaerobic spore-forming pathogen that provokes life-threatening gas gangrene and acute enterotoxaemia, although it colonizes as a component of the symbiotic bacteria in humans and animals. However, the mechanisms by which C. perfringens is cleared from the host remains poorly understood, thereby impeding the development of novel strategies for control this infection. Here, we uncover a beneficial effect of extracellular traps (ETs) formation on bacterial killing and clearance by phagocytes. C. perfringens strain ATCC13124, and wild-type isolates CP1 and CP3 markedly trigger ETs formation in macrophages and neutrophils. As expected, visualization of DNA decorated with histone, myeloperoxidase (MPO) and neutrophils elastase (NE) in C. perfringens-triggered classical ETs structures. Notably, the bacteria-induced ETs formation is an ERK1/2-, P38 MAPK-, store-operated calcium entry (SOCE)-, NADPH oxidase-, histone-, NE-, and MPO-dependent process, and is independent of LDH activity. Meanwhile, the defect of bactericidal activity is mediated by impairing ETs formation in phagocytes. Moreover, In vivo studies indicated that degradation of ETs by DNase I administration leads to a defect in the protection against experimental gas gangrene, with higher mortality rates, exacerbated tissue damage, and more bacterial colonization. Together, these results suggest that phagocyte ETs formation is essential for the host defense against C. perfringens infection.

Emergence of Third Generation Tetracyclines: New Magic Bullets to Tackle Antibiotic Resistance in the Post-antibiotic Era

Tetracyclines are broad-spectrum antibiotics effective against a wide variety of microorganisms including bacteria, both Gram-positive and Gram-negative, mycoplasmas, rickettsiae, chlamydiae, and protozoan parasites. Owing to their broad-spectrum antimicrobial activities and inexpensiveness, tetracyclines have been used extensively in both human and animal infections and in animal feed as growth promoters. Owing to this, the global prevalence of antibiotic resistance, particularly of tetracyclines, for Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae and Gram-negative extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella species is high. Indeed, the acquisition of tetracycline-specific resistance genes, mutations within the ribosomal binding site, and/or chromosomal mutations lead to both class-specific and intrinsic antimicrobial resistance (AMR) mechanisms. As drug resistance increases globally, rendering diseases difficult to manage and eventually to mortality, antibiotics are becoming progressively less effective. Therefore, the discovery and development of novel antibiotics with appropriate indications is of the utmost importance. Among all antibiotics, the tetracyclines have acquired much attention due to the optimization of their chemical structures that paved the way to develop and introduce modern tetracyclines, referred to as third generation, namely tigecycline in the recent past, followed by omadacycline, eravacycline, and sarecycline very recently. Intriguingly, these novel tetracyclines are unique in two ways, first, these are highly effective against pathogens that acquired tetracycline-class resistance, and second, these agents exhibit either narrow or broad spectrum of in vitro activity against Gram-positive, Gram-negative, anaerobic, and atypical pathogens, including many drug-resistant strains lead to approval for limited use and unique indications. These beneficial effects represent a new era in the rational use of newer tetracyclines, the new magic bullets to tackle AMR in the post-antibiotic era. The present review focuses on third-generation tetracyclines emphasizing their safety, efficacy, and therapeutic choices in various clinical conditions of in-patient and out-patient settings.

Next-generation DNA sequencing offers diagnostic advantages over traditional culture testing.

While the clinical utility of next-generation DNA sequencing (NGS) as a diagnostic tool for infections in humans and traditional pets has been demonstrated, there is a lack of data regarding its utility for exotic animals. For exotic patients, traditional culturing is especially challenging for anaerobic and fungal pathogens. Therefore, diagnosis often relies on PCR, which provides a high degree of sensitivity and specificity, although it targets only a predetermined, finite pathogen panel. NGS provides the same benefits as PCR, while also offering de novo identification and quantification of all bacteria and fungi present in a clinical sample, including novel pathogen discovery. Clinical samples from 78 exotic animal patients were collected simultaneously for conventional culture testing and NGS analysis. Results provided by each laboratory were compared for the presence and absence of bacterial and fungal pathogens and commensals. Results showed large bacterial and fungal species diversity in the study cohort and a lack of sensitivity of microbial culture testing. Culture failed to grow 15% of putative bacterial and 81% of putative fungal pathogens that were identified by NGS. The probability of a "no growth" diagnosis was 14% higher for bacteria and 49% higher for fungi with culture versus NGS testing if fungal culture was conducted. Culture testing failed to diagnose a substantial number of both bacterial and fungal pathogens, which were detected by NGS. This highlights the limitations of traditional culture-based testing and displays the clinically advanced utility of NGS-based diagnostics in exotic animal medicine.

Role of the Escherichia coli ubiquinone-synthesizing UbiUVT pathway in adaptation to changing respiratory conditions.

Isoprenoid quinones are essential for cellular physiology. They act as electron and proton shuttles in respiratory chains and various biological processes. Escherichia coli and many α-, β-, and γ-proteobacteria possess two types of isoprenoid quinones: ubiquinone (UQ) is mainly used under aerobiosis, while demethylmenaquinones (DMK) are mostly used under anaerobiosis. Yet, we recently established the existence of an anaerobic O2-independent UQ biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes. Here, we characterize the regulation of ubiTUV genes in E. coli. We show that the three genes are transcribed as two divergent operons that are both under the control of the O2-sensing Fnr transcriptional regulator. Phenotypic analyses using a menA mutant devoid of DMK revealed that UbiUV-dependent UQ synthesis is essential for nitrate respiration and uracil biosynthesis under anaerobiosis, while it contributes, though modestly, to bacterial multiplication in the mouse gut. Moreover, we showed by genetic study and 18O2 labeling that UbiUV contributes to the hydroxylation of ubiquinone precursors through a unique O2-independent process. Last, we report the crucial role of ubiT in allowing E. coli to shift efficiently from anaerobic to aerobic conditions. Overall, this study uncovers a new facet of the strategy used by E. coli to adjust its metabolism on changing O2 levels and respiratory conditions. This work links respiratory mechanisms to phenotypic adaptation, a major driver in the capacity of E. coli to multiply in gut microbiota and of facultative anaerobic pathogens to multiply in their host. IMPORTANCE Enterobacteria multiplication in the gastrointestinal tract is linked to microaerobic respiration and associated with various inflammatory bowel diseases. Our study focuses on the biosynthesis of ubiquinone, a key player in respiratory chains, under anaerobiosis. The importance of this study stems from the fact that UQ usage was for long considered to be restricted to aerobic conditions. Here we investigated the molecular mechanism allowing UQ synthesis in the absence of O2 and searched for the anaerobic processes that UQ is fueling in such conditions. We found that UQ biosynthesis involves anaerobic hydroxylases, that is, enzymes able to insert an O atom in the absence of O2. We also found that anaerobically synthesized UQ can be used for respiration on nitrate and the synthesis of pyrimidine. Our findings are likely to be applicable to most facultative anaerobes, which count many pathogens (Salmonella, Shigella, and Vibrio) and will help in unraveling microbiota dynamics.

Genomic Insights into Virulence Factors and Multi-Drug Resistance in Clostridium perfringens IRMC2505A.

Clostridium perfringens is a spore-forming, Gram-positive anaerobic pathogen that causes several disorders in humans and animals. A multidrug-resistant Clostridium strain was isolated from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic exposure and diarrhea. The strain was identified by 16s rRNA sequencing as Clostridium perfringens. The strain's pathogenesis was analyzed through its complete genome, specifically antimicrobial resistance-related genes. The Clostridium perfringens IRMC2505A genome contains 19 (Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, and S12p) antibiotic-susceptible genetic species according to the k-mer-based detection of antimicrobial resistance genes. Genome mapping using CARD and VFDB databases revealed significant (p-value = 1 × 10-26) genes with aligned reads against antibiotic-resistant genes or virulence factors, including phospholipase C, perfringolysin O, collagenase, hyaluronidase, alpha-clostripain, exo-alpha-sialidase, and sialidase activity. In conclusion, this is the first report on C. perfringens from Saudi Arabia that conducted whole genome sequencing of IRMC2505A and confirmed the strain as an MDR bacterium with several virulence factors. Developing control strategies requires a detailed understanding of the epidemiology of C. perfringens, its virulence factors, and regional antimicrobial resistance patterns.

Investigating the role of Candida albicans as a universal substrate for oral bacteria using a transcriptomic approach: implications for interkingdom biofilm control?

Candida albicans is frequently identified as a colonizer of the oral cavity in health and has recently been termed a "keystone" commensal due to its role on the bacterial communities. However, the role that C. albicans plays in such interactions is not fully understood. Therefore, this study aimed to identify the relationship between C. albicans and bacteria associated with oral symbiosis and dysbiosis. To do this, we evaluated the ability of C. albicans to support the growth of the aerobic commensal Streptococcus gordonii and the anaerobic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis in the biofilm environment. RNA-Sequencing with the Illumina platform was then utilized to identify C. albicans gene expression and functional pathways involved during such interactions in dual-species and a 4-species biofilm model. Results indicated that C. albicans was capable of supporting growth of all three bacteria, with a significant increase in colony counts of each bacteria in the dual-species biofilm (p < 0.05). We identified specific functional enrichment of pathways in our 4-species community as well as transcriptional profiles unique to the F. nucleatum and S. gordonii dual-species biofilms, indicating a species-specific effect on C. albicans. Candida-related hemin acquisition and heat shock protein mediated processes were unique to the organism following co-culture with anaerobic and aerobic bacteria, respectively, suggestive that such pathways may be feasible options for therapeutic targeting to interfere with these fungal-bacterial interactions. Targeted antifungal therapy may be considered as an option for biofilm destabilization and treatment of complex communities. Moving forward, we propose that further studies must continue to investigate the role of this fungal organism in the context of the interkingdom nature of oral diseases.

Regulatory Role of Anti-Sigma Factor RsbW in Clostridioides difficile Stress Response, Persistence, and Infection.

The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σB) is employed to modulate gene transcription, and σB is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW), in which σB is assumed to be "always on," was generated. ΔrsbW did not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. ΔrsbW was defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand the unique phenotype of ΔrsbW showed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σB-controlled regulators, including the pleiotropic regulator sinRR'. While these profiles were distinct to ΔrsbW, changes in some σB-controlled stress-associated genes were similar to those reported in the absence of σB. Further analysis of ΔrsbW showed unexpected lower intracellular levels of σB, suggesting an additional post-translational control mechanism for σB in the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses in C. difficile. IMPORTANCE Pathogens like Clostridioides difficile face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σB) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provide C. difficile with the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a rsbW mutant in growth, persistence, and virulence and suggest alternate σB control mechanisms in C. difficile. Understanding C. difficile responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.

Abstract 2483: Attenuated Salmonella blocks cancer metastasis by activating NK cells in an IFN-γ-dependent manner

Abstract Introduction and Objectives: The recent remarkable clinical results of cancer immunotherapies, such as immune checkpoint blockade and chimeric antigen receptor T cells (CAR-T), has sparked a great deal of enthusiasm in manipulating the immune response in the fight against cancer. Bacterial cancer therapies represent an alternative approach to targeting solid tumors for evoking anti-tumor immune responses. As a facultative anaerobic pathogen that can penetrate and colonize solid tumors, Salmonella typhimurium gained lot of interest in the investigation of bacterial cancer therapies and can exert its intrinsic antitumor activity by directly inducing systemic immune responses. However, most bacterial cancer therapies, including salmonella-based cancer therapies, have mainly focused on the suppression of already established tumors rather than metastasis. Metastasis accounts for 90% of cancer related deaths and blocking of metastatic cascade has critical clinical impact. However, the clinical drug development for cancer treatment, including cancer immunotherapies, is evaluated largely depending on their ability to cause tumor shrinkage and ignores the effect on metastasis as it has proven challenging to target. Therefore, there is an urgent need for novel therapeutic strategies and agents targeting metastasis. Experimental Procedures and Outcomes: Using an attenuated Salmonella typhimurium strain YB1 engineered by our lab, we have found a potent suppressive effect of attenuated salmonella on cancer metastasis, regardless of cancer types and genetic background, by evoking strong anti-metastatic immune response. Further study showed that suppression of cancer metastasis by attenuated Salmonella only requires the innate immune response. Among the many induced cytokines, we identified IFN-γ as an indispensable factor for inhibiting cancer metastasis. Based on CyTOF (mass cytometry or cytometry by time of flight) analysis of the innate immune responses after Salmonella treatment and antibody-mediated cell depletion, we further demonstrated that NK cells are the major factor involved in Salmonella-provoked metastasis suppression. We found that IFN-γ was mainly produced by NK cells during early Salmonella infection, and in turn, IFN-γ promoted the accumulation, activation and cytotoxicity of NK cells. The IFN-γ-dependent NK cells directly eliminated newly accumulated cancer cells in the lung to block the cancer metastasis cascade in response to the Salmonella treatment. Citation Format: Li Rong, Qiubin Lin, Shuhai Lin, Guo Fu, Jiandong Huang. Attenuated Salmonella blocks cancer metastasis by activating NK cells in an IFN-γ-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2483.

Novel Twin-Crystal Nanosheets with MnO2 Modification to Combat Bacterial Biofilm against Periodontal Infections via Multipattern Strategies.

Nowadays the multifunctional approaches to kill oral bacteria based on various nanocomposites have made great progress against periodontal infections, while the material structure and its functional integration are still insufficient. Herein, this work proposes a therapeutic strategy of chemodynamical therapy (CDT) and photothermal therapy (PTT) in monocrystals to effectively enhance the synergistic treatment. The CuS/MnS@MnO2 consisting of hexagonal CuS/MnS nano-twin-crystal with a shell layer of MnO2 is developed. In this nanosystem, the purpose of synergistic treatment of periodontitis by combining PTT/CDT is achieved within a CuS/MnS monocrystal, where CuS serves to achieve photothermal conversion, dissipate the biofilm and transfer the heat in situ to the integrated MnS, thus promoting the Mn2+ -mediated CDT process. Meanwhile, the CDT process can generate the highly toxic hydroxyl radical to destroy extracellular DNA by utilization of endogenous H2 O2 produced by Streptococci in the oral biofilm, cooperating with PTT to dissipate the bacterial biofilm. With the design of the outer shell of MnO2 , the selective bacteria-killing can be realized by producing oxygen which can protect the periodontal non-pathogenic aerobic bacteria and threaten the survival of anaerobic pathogens. Therefore, such design via multipattern strategies to combat microorganisms would provide a bright prospect for the clinical treatment of bacterial infections.

Peculiarities of Periodontal Pocket Microbiome in Patients with Generalized Periodontitis in the Post-COVID Period

The oral cavity, like the lungs, is often referred to as the «ecological niche of commensal, symbiotic, and pathogenic organisms», and the emigration and elimination of microbes between them are constant, ensuring a healthy distribution of saprophytic microorganisms that maintains organ, tissue, and immune homeostasis. The prolonged hospital stays due to COVID-19 complications, cross-infection, oxygenation therapy through the mask or incubation, and long-term intravenous infusions limit the patient’s ability to care about the oral cavity, regularly clean teeth, floss interdental, etc., which creates extremely favorable conditions for colonization by aerobic and anaerobic pathogens of the oral cavity and periodontal pockets and leads to the rapid progression of chronic generalized periodontitis in this category of patients in the future. The goal of the study was to assess the state of the microbiome of the periodontal pockets of dental patients in the post-covid period. Methods. The object of the study was 140 patients with generalized periodontitis of the I and II stages of development in the chronic course (GP), among which 80 patients had coronavirus disease in the closest past. The patients were randomized by age, sex, and stage of GP development. The diagnosis of periodontal disease was established according to the classifi cation by Danilevskyi. The bacteriological material for aerobic and facultative anaerobic microflora and yeast-like fungi was collected from periodontal pockets with a calibrated bacteriological loop and immediately seeded on blood agar. Results. Significant qualitative and quantitative changes in the nature of the oral microbiocenosis were observed in patients with GP after the recent coronavirus disease, compared with similar patients who did not suffer from COVID-19. We have noticed almost complete disappearance of bacteria that belong to the transient representatives of the oral microflora such as Neisseria, corynebacteria (diphtheria), micrococci, and lactobacilli. The main resident representatives of the oral microflora, i.e., α-hemolytic Streptococci of the mitis group, were found in all healthy individuals and patients of groups A and C, but in 30.0±4.58% of patients in group B, α-hemolytic streptococci in the contents of periodontal pockets are present in quantities not available for detection by the applied method (&lt;2.7 lg CCU/mL). In terms of species, Streptococcus oralis and Streptococcus salivarius are more characteristic in gingival crevicular fluid in healthy individuals (93.8% of selected strains). In 68.4±3.32% of patients in group A, 64.0±3.43% of patients in group B, and 67.5±3.76% of patients in group C, the dominant species were Streptococcus gordonii and Streptococcus sanguinis (p&lt;0.01), which increased pathogenic potential as they produce streptolysin-O, inhibit complement activation, bind to fibronectine, actively form biofilms on the surface of tooth enamel and gum epithelial surface, and can act as an initiator of adhesion of periodontal pathogens. The other representatives of the resident microflora of the oral cavity — Stomatococcus mucilaginosus and Veillonella parvula for the patients of group C are also found in periodontal pockets with a signifi cantly lower index of persistence and minimal population level. In the post-covid period, both the population level and the frequency of colonization of periodontal pockets by Staphylococci and β-hemolytic Streptococci decreases rapidly. For these patient groups, unlike for those that did not suff er from COVID-19, we did not find any case of colonization with Staphylococcus aureus, as well as β-hemolytic Streptococci and epidermal Staphylococcus were also absent. The most characteristic in the post-covid period is a decrease in the proportion of α-hemolytic Streptococci, an increase in the proportion of yeast-like fungi of Candida species, as well as the appearance of a significant number of gram-negative rod-shaped bacteria (Enterobacteria and Pseudomonads). In periodontal patients, the microbial count is approximately 2 orders of magnitude lower than in those with GP who did not suffer from COVID-19 (p&lt;0.05). Conclusions. The overpassed coronavirus disease due to intensive antibiotic therapy leads to a marked decrease in the number of viable saprophytic microorganisms in the periodontal pockets of patients with GP. In the post-covid period for the patients with GP, there is a decrease in the level of colonization of periodontal pockets by species of resident oral microflora — α-hemolytic Streptococci, reduction of resident microorganism’s species, and almost complete disappearance of transient microfl ora. On the other hand, the frequency of colonization of periodontal pockets by fungi species, enterobacteria, and pseudomonads significantly increases. There are more expressed disorders in the periodontal pocket’s microbiome for the patients with a severe and complicated course of coronavirus disease, such as post-covid pulmonary fibrosis, which requires reconsideration of approaches to therapeutic and pharmacological treatment in this category of patients.

Fusobacterium nucleatum, a key pathogenic factor and microbial biomarker for colorectal cancer.

Colorectal cancer (CRC), one of the most prevalent cancers, has complex etiology. The dysbiosis of intestinal bacteria has been highlighted as an important contributor to CRC. Fusobacterium nucleatum, an oral anaerobic opportunistic pathogen, is enriched in both stools and tumor tissues of patients with CRC. Therefore, F. nucleatum is considered to be a risk factor for CRC. This review summarizes the biological characteristics and the mechanisms underlying the regulatory behavior of F. nucleatum in the tumorigenesis and progression of CRC. F. nucleatum as a marker for the early warning and prognostic prediction of CRC, and as a target for prevention and treatment, is also described.

Nonredundant Dimethyl Sulfoxide Reductases Influence Salmonella enterica Serotype Typhimurium Anaerobic Growth and Virulence.

Facultative anaerobic enteric pathogens can utilize a diverse array of alternate electron acceptors to support anaerobic metabolism and thrive in the hypoxic conditions within the mammalian gut. Dimethyl sulfoxide (DMSO) is produced by methionine catabolism and can act as an alternate electron acceptor to support anaerobic respiration. The DMSO reductase complex consists of three subunits, DmsA, DmsB, and DmsC, and allows bacteria to grow anaerobically with DMSO as an electron acceptor. The genomes of nontyphoidal Salmonella enterica encode three putative dmsABC operons, but the impact of the apparent genetic redundancy in DMSO reduction on the fitness of nontyphoidal S. enterica during infection remains unknown. We hypothesized that DMSO reduction would be needed for S. enterica serotype Typhimurium to colonize the mammalian gut. We demonstrate that an S. Typhimurium mutant with loss of function in all three putative DMSO reductases (ΔdmsA3) poorly colonizes the mammalian intestine when the microbiota is intact and when inflammation is absent. DMSO reduction enhances anaerobic growth through nonredundant contributions of two of the DMSO reductases. Furthermore, DMSO reduction influences virulence by increasing expression of the type 3 secretion system 2 and reducing expression of the type 3 secretion system 1. Collectively, our data demonstrate that the DMSO reductases of S. Typhimurium are functionally nonredundant and suggest DMSO is a physiologically relevant electron acceptor that supports S. enterica fitness in the gut.