Antimicrobial resistance surveillance of Bacteroides fragilis isolated from blood cultures, Europe, 2022 (ReSuBacfrag).

BACKGROUND: Bacteroides fragilis is the most frequent cause of anaerobic bacteraemia. Although recent data suggest a rise in antimicrobial resistance (AMR) of this and other anaerobic bacteria, surveillance remains limited due to a lack of both data availability and comparability. However, a newly introduced standardised method for antimicrobial susceptibility testing (AST) of anaerobic bacteria has made larger scale surveillance possible for the first time. AIM: To investigate phenotypic AMR of Bacteroides fragilis isolates from bacteraemia across Europe in 2022. METHODS: In a multicentre approach, clinical microbiology laboratories in Europe were invited to contribute results of AST for Bacteroides fragilis blood culture isolates (including only the first isolate per patient and year). AST of a selection of four antibiotics was performed locally by participating laboratories in a prospective or retrospective manner, using the new EUCAST disc diffusion method on fastidious anaerobe agar (FAA-HB). RESULTS: A total of 16 European countries reported antimicrobial susceptibilities in 449 unique isolates of Bacteroides fragilis from blood cultures in 2022. Clindamycin demonstrated the highest resistance rates (20.9%, range 0 - 63.6%), followed by piperacillin-tazobactam (11.1%, 0 - 54.5%), meropenem (13.4%, 0 - 45.5%), and metronidazole (1.8%, 0 - 20.0%), all with wide variation between countries. CONCLUSION: Considering that the mean resistance rates across Europe were higher than expected for three of the four anti-anaerobic antibiotics under surveillance, both local AST of clinically relevant isolates of Bacteroides fragilis and continued surveillance on an international level is warranted.

Detection of the antibiotic resistance genes content of intestinal Bacteroides, Parabacteroides and Phocaeicola isolates from healthy and carbapenem-treated patients from European countries.

BACKGROUND: Bacteroides fragilis group (BFG) species are the most significant anaerobic pathogens and are also the most antibiotic-resistant anaerobic species. Therefore, surveying their antimicrobial resistance levels and investigating their antibiotic resistance mechanisms is recommended. Since their infections are endogenous and they are important constituents of the intestinal microbiota, the properties of the intestinal strains are also important to follow. The aim of this study was to investigate the main antibiotic gene content of microbiota isolates from healthy people and compare them with the gene carriage of strains isolated from infections. RESULTS: We detected 13, mainly antibiotic resistance determinants of 184 intestinal BFG strains that were isolated in 5 European countries (Belgium, Germany, Hungary, Slovenia and Turkey) and compared these with values obtained earlier for European clinical strains. Differences were found between the values of this study and an earlier one for antibiotic resistance genes that are considered to be mobile, with higher degrees for cfxA, erm(F) and tet(Q) and with lower degrees for msrSA, erm(B) and erm(G). In addition, a different gene prevalence was found depending on the taxonomical groups, e.g., B. fragilis and NBFB. Some strains with both the cepA and cfiA ß-lactamase genes were also detected, which is thought to be exceptional since until now, the B. fragilis genetic divisions were defined by the mutual exclusion of these two genes. CONCLUSIONS: Our study detected the prevalences of a series of antibiotic resistance genes in intestinal Bacteroides strains which is a novelty. In addition, based on the current and some previous data we hypothesized that prevalence of some antibiotic resistance genes detected in the clinical and intestinal BFG strains were different, which could be accounted with the differential composition of the Bacteroides microbiota and/or the MGE mobilities at the luminal vs. mucosal sites of the intestine.

Comparative evaluation of culture results and composition of microbiome of removed tonsils due to distant focal disease or other reasons. A prospective pilot study.

The aim of this prospective pilot study was to compare culture and microbiome results of the removed tonsils of patients with assumed distant focal disease (11 patients) and those who underwent a tonsillectomy, due to other reasons, such as recurrent tonsillitis, tonsil stones or snoring (nine patients). Aerobic culture was carried out for samples taken from the surface of the tonsils by swabs before tonsillectomy for all 20 patients. The squeezed detritus and the tissue samples of removed tonsils, taken separately for the right and left tonsils, were incubated aerobically and anaerobically. The microbiome composition of tissue samples of removed tonsils was also evaluated. Based on the culture results of the deep samples Staphylococcus aureus was the dominating pathogen, besides a great variety of anaerobic and facultative anaerobic bacteria present in the oral microbiota in those patients who underwent tonsillectomy due to distant focal diseases. Microbiome study of the core tissue samples showed a great diversity on genus and species level among patients of the two groups however, S. aureus and Prevotella nigrescens were present in higher proportion in those, whose tonsils were removed due to distant focal diseases. Our results may support previous findings about the possible triggering role of S. aureus and P. nigrescens leading to distant focal diseases. Samples taken by squeezing the tonsils could give more information about the possible pathogenic/triggering bacteria than the surface samples cultured only aerobically.

Proteomics-Based RT-qPCR and Functional Analysis of 18 Genes in Metronidazole Resistance of Bacteroides fragilis.

Previously, we reported that metronidazole MICs are not dependent on the expression levels of nim genes in B. fragilis strains and we compared the proteomes of metronidazole-resistant laboratory B. fragilis strains to those of their susceptible parent strains. Here, we used RT-qPCR to correlate the expression levels of 18 candidate genes in a panel of selected, clinical nim gene-positive and -negative B. fragilis strains to their metronidazole MICs. Metronidazole MICs were correlated with the expression of certain tested genes. Specifically, lactate dehydrogenase expression correlated positively, whereas cytochrome fumarate reductase/succinate dehydrogenase, malate dehydrogenase, phosphoglycerate kinase redox and gat (GCN5-like acetyltransferase), and relA (stringent response) regulatory gene expressions correlated negatively with metronidazole MICs. This result provides evidence for the involvement of carbohydrate catabolic enzymes in metronidazole resistance in B. fragilis. This result was supported by direct substrate utilization tests. However, the exact roles of these genes/proteins should be determined in deletion-complementation tests. Moreover, the exact redox cofactor(s) participating in metronidazole activation need to be identified.

Novel and rare ß-lactamase genes of Bacteroides fragilis group species: Detection of the genes and characterization of their genetic backgrounds.

OBJECTIVES: This study screened the prevalence of rare ß-lactamase genes in Bacteroides fragilis group strains from clinical specimens and normal microbiota and examined the genetic properties of the strains carrying these genes. METHODS: blaHGD1, blaOXA347, cblA, crxA, and pbbA were detected by real-time polymerase chain reaction in collections of Bacteroides strains from clinical (n = 406) and fecal (n = 184) samples. To examine the genetic backgrounds of the samples, end-point PCR, FT-IR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used. RESULTS: All B. uniformis isolates were positive for cblA in both collections. Although crxA was B. xylanisolvens-specific and associated with carbapenem resistance, it was only found in six fecal and three clinical B. xylanisolvens strains. Moreover, the crxA-positive strains were not clonal among B. xylanisolvens (contrary to cfiA in B. fragilis), implicating a rate of mobility or emergence by independent evolutionary events. The Phocaeicola (B.) vulgatus/P. dorei-specific gene blaHGD1 was detected among all P. vulgatus/P. dorei isolates from fecal (n = 36) and clinical (n = 26) samples. No blaOXA347-carrying isolate was found from European collections, but all US samples (n = 6) were positive. For three clinical isolates belonging to B. thetaiotaomicron (n = 2) and B. ovatus (n = 1), pbbA was detected on mobile genetic elements, and pbbA-positive strains displayed non-susceptibility to piperacillin or piperacillin/tazobactam phenotypically. CONCLUSIONS: Based on these observations, ß-lactamases produced by rare ß-lactamase genes in B. fragilis group strains should not be overlooked because they could encode important resistance phenotypes.

Collateral sensitivity: An evolutionary trade-off between antibiotic resistance mechanisms, attractive for dealing with drug-resistance crisis.

Background: The discovery and development of antimicrobial drugs were one of the most significant advances in medicine, but the evolution of microbial resistance limited the efficiency of these drugs. Aim: This paper reviews the collateral sensitivity in bacteria and its potential and limitation as a new target for treating infections. Results and Discussion: Knowledge mechanisms of resistance to antimicrobial agents are useful to trace a practical approach to treat and control of resistant pathogens. The effect of a resistance mechanism to certain antibiotics on the susceptibility or resistance to other drugs is a key point that may be helpful for applying a strategy to control resistance challenges. In an evolutionary trade-off known as collateral sensitivity, the resistance mechanism to a certain drug may be mediated by the hypersensitivity to other drugs. Collateral sensitivity has been described for different drugs in various bacteria, but the molecular mechanisms affecting susceptibility are not well demonstrated. Collateral sensitivity could be studied to detect its potential in the battle against resistance crisis as well as in the treatment of pathogens adapting to antibiotics. Collateral sensitivity-based antimicrobial therapy may have the potential to limit the emergence of antibiotic resistance.

The effects of identical nim gene-insertion sequence combinations on the expression of the nim genes and metronidazole resistance in Bacteroides fragilis strains.

In this study we examined whether the same nim gene-insertion sequence (IS) element combinations give rise to the same expression levels as they harbor shared IS element-borne promoters. From our quantitative analysis, we found that the expressions of the nimB and nimE genes with their cognate IS elements were similar, but the metronidazole resistance of these strains were more diverse.

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis.

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.

Machine learning-based typing of Salmonella enterica O-serogroups by the Fourier-Transform Infrared (FTIR) Spectroscopy-based IR Biotyper system.

BACKGROUND: Salmonella enterica is among the major burdens for public health at global level. Typing of salmonellae below the species level is fundamental for different purposes, but traditional methods are expensive, technically demanding, and time-consuming, and therefore limited to reference centers. Fourier transform infrared (FTIR) spectroscopy is an alternative method for bacterial typing, successfully applied for classification at different infra-species levels. AIM: This study aimed to address the challenge of subtyping Salmonella enterica at O-serogroup level by using FTIR spectroscopy. We applied machine learning to develop a novel approach for S. enterica typing, using the FTIR-based IR Biotyper® system (IRBT; Bruker Daltonics GmbH & Co. KG, Germany). We investigated a multicentric collection of isolates, and we compared the novel approach with classical serotyping-based and molecular methods. METHODS: A total of 958 well characterized Salmonella isolates (25 serogroups, 138 serovars), collected in 11 different centers (in Europe and Japan), from clinical, environmental and food samples were included in this study and analyzed by IRBT. Infrared absorption spectra were acquired from water-ethanol bacterial suspensions, from culture isolates grown on seven different agar media. In the first part of the study, the discriminatory potential of the IRBT system was evaluated by comparison with reference typing method/s. In the second part of the study, the artificial intelligence capabilities of the IRBT software were applied to develop a classifier for Salmonella isolates at serogroup level. Different machine learning algorithms were investigated (artificial neural networks and support vector machine). A subset of 88 pre-characterized isolates (corresponding to 25 serogroups and 53 serovars) were included in the training set. The remaining 870 samples were used as validation set. The classifiers were evaluated in terms of accuracy, error rate and failed classification rate. RESULTS: The classifier that provided the highest accuracy in the cross-validation was selected to be tested with four external testing sets. Considering all the testing sites, accuracy ranged from 97.0% to 99.2% for non-selective media, and from 94.7% to 96.4% for selective media. CONCLUSIONS: The IRBT system proved to be a very promising, user-friendly, and cost-effective tool for Salmonella typing at serogroup level. The application of machine learning algorithms proved to enable a novel approach for typing, which relies on automated analysis and result interpretation, and it is therefore free of potential human biases. The system demonstrated a high robustness and adaptability to routine workflows, without the need of highly trained personnel, and proving to be suitable to be applied with isolates grown on different agar media, both selective and unselective. Further tests with currently circulating clinical, food and environmental isolates would be necessary before implementing it as a potentially stand-alone standard method for routine use.

Initial expression levels of nimA are decisive for protection against metronidazole in Bacteroides fragilis.

OBJECTIVES: In the genus Bacteroides, the nim genes are resistance determinants for metronidazole, a nitroimidazole drug widely used against anaerobic pathogens. The Nim proteins are considered to act as nitroreductases. However, data from several studies suggest that the expression levels of Nim do not increase with increasing resistance which is conflicting with this notion. The impact of Nim protein levels on low-level metronidazole resistance, however, representing the early stage of induced resistance in the laboratory, has not been assessed as yet. METHODS: The nimA gene was cloned into two different plasmids and introduced into B. fragilis strain 638R. Expression levels of nimA mRNA were measured by RT-qPCR and compared to those in strain 638R harbouring plasmid pI417, the original clinical plasmid harbouring IS element IS1168 with the nimA gene. Further, metronidazole susceptibility was assessed by Etest and the activity of pyruvate:ferredoxin oxidoreductase (PFOR) was measured in all strains after induction of high-level metronidazole resistance. RESULTS: The level of protection against metronidazole by nimA correleated with the level of expression of nimA mRNA. Further, the activity of PFOR in highly-resistant B. fragilis 638R was only preserved when expression levels of nimA were high. CONCLUSIONS: Although the development of high-level metronidazole resistance in B. fragilis strains with a nimA gene is not caused by an increase of nimA expression as compared to the less resistant parent strains, nimA expression levels might be of decisive importance in the early stage of resistance development. This has potential implications for metronidazole resistance in clinical isolates.

Corynebacterium striatum-Got Worse by a Pandemic?

The role of Corynebacterium striatum has been demonstrated in different nosocomial infections. An increasing number of publications have demonstrated its virulence in the respiratory tract, especially in the immunosuppressed patient population. The number of these patients has increased significantly during the COVID-19 pandemic. For this reason, we aimed to investigate the prevalence and antimicrobial resistance pattern of this species between 2012 and 2021 at the Clinical Center of the University of Szeged, Hungary. Altogether, 498 positive samples were included from 312 patients during the study period. On the isolates, 4529 antibiotic susceptibility tests were performed. Our data revealed that the prevalence of C. striatum increased during the COVID-19 pandemic, the rise occurred in respiratory, blood culture, and superficial samples. During the study period, the rifampicin resistance significantly increased, but others have also changed dynamically, including linezolid. The species occurred with diverse and changing co-pathogens in the COVID-19 era. However, the increasing rifampicin and linezolid resistance of C. striatum was probably not due to the most commonly isolated co-pathogens. Based on resistance predictions, vancomycin is likely to remain the only effective agent currently in use by 2030.

Carbapenem resistance in Bacteroides fragilis: A review of molecular mechanisms.

Carbapenems are an applicable subclass of ß-lactam drugs in the antibiotic therapy of anaerobic infections, especially for poly-microbial cases, due to their broad antimicrobial spectrum on aerobic and anaerobic bacteria. Bacteroides fragilis is the most commonly recovered anaerobic bacteria in the clinical laboratories from mono- and poly-microbial infections. B. fragilis is relatively non-susceptible to different antibiotics, including ß-lactams, tetracyclines, fluoroquinolones, and macrolides. Carbapenems are among the most effective drugs against B. fragilis strains with high-level resistance to different antibiotics. Increased antibiotic resistance of B. fragilis strains has been reported following the overuse of an antimicrobial agent. Earlier contact with carbapenems is linked with increased resistance to them that limits the options for treatment of B. fragilis caused infections, especially in cases caused by multidrug-resistant strains. Several molecular mechanisms of resistance to carbapenems have been described for different carbapenem-resistant bacteria. Understanding the mechanisms of resistance to antimicrobial agents is necessary for selecting alternative antimicrobial agents and the application of control strategies. In the present study, we reviewed the mechanisms contributing to resistance to carbapenems in B. fragilis strains.

Modulation of Iron Import and Metronidazole Resistance in Bacteroides fragilis Harboring a nimA Gene.

Bacteroides fragilis is a commensal of the human gut but can also cause severe infections when reaching other body sites, especially after surgery or intestinal trauma. Bacteroides fragilis is an anaerobe innately susceptible to metronidazole, a 5-nitroimidazole drug that is prescribed against the majority of infections caused by anaerobic bacteria. In most of the cases, metronidazole treatment is effective but a fraction of B. fragilis is resistant to even very high doses of metronidazole. Metronidazole resistance is still poorly understood, but the so-called nim genes have been described as resistance determinants. They have been suggested to encode nitroreductases which reduce the nitro group of metronidazole to a non-toxic aminoimidazole. More recent research, however, showed that expression levels of nim genes are widely independent of the degree of resistance observed. In the search for an alternative model for nim-mediated metronidazole resistance, we screened a strain carrying an episomal nimA gene and its parental strain 638R without a nim gene for physiological differences. Indeed, the 638R daughter strain with the nimA gene had a far higher pyruvate-ferredoxin oxidoreductase (PFOR) activity than the parental strain. High PFOR activity was also observed in metronidazole-resistant clinical isolates, either with or without a nim gene. Moreover, the strain carrying a nimA gene fully retained PFOR activity and other enzyme activities such as thioredoxin reductase (TrxR) after resistance had been induced. In the parental strain 638R, these were lost or very strongly downregulated during the development of resistance. Further, after induction of high-level metronidazole resistance, parental strain 638R was highly susceptible to oxygen whereas the daughter strain with a nimA gene was hardly affected. Ensuing RT-qPCR measurements showed that a pathway for iron import via hemin uptake is downregulated in 638R with induced resistance but not in the resistant nimA daughter strain. We propose that nimA primes B. fragilis toward an alternative pathway of metronidazole resistance by enabling the preservation of normal iron levels in the cell.

Phenotypic and Molecular Characterization of Carbapenem-Heteroresistant Bacteroides fragilis Strains.

Carbapenem-resistant Bacteroides fragilis strains usually emerge by an insertion sequence (IS) jump into the upstream region of the cfiA carbapenemase gene. However, intermediate or fully resistant cfiA-positive strains also exist. These do not have such IS element activations, but usually have heterogeneous resistance (HR) phenotypes, as detected by a disc diffusion or gradient tests. Heteroresistance is a serious antibiotic resistance problem, whose molecular mechanisms are not fully understood. We aim to characterize HR and investigate diagnostic issues in the set of cfiA-positive B. fragilis strains using phenotypic and molecular methods. Of the phenotypic methods used, the population analysis profile (PAP) and area under curve (AUC) measurements were the best prognostic markers for HR. PAP AUC, imipenem agar dilution and imipenemase production corresponded well with each other. We also identified a saturation curve parameter (quasi-PAP curves), which correlated well with these phenotypic traits, implying that HR is a stochastic process. The genes, on a previously defined 'cfiA element', act in a complex manner to produce the HR phenotype, including a lysine-acetylating toxin and a lysine-rich peptide. Furthermore, imipenem HR is triggered by imipenem. The two parameters that most correlate with the others are imipenemase production and 'GNAT' expression, which prompted us to suspect that carbapenem heteroresistance of the B. fragilis strains is stochastically regulated and is mediated by the altered imipenemase production.

A Porcine Sepsis Model With Numerical Scoring for Early Prediction of Severity.

Introduction: Sepsis can lead to organ dysfunctions with disturbed oxygen dynamics and life-threatening consequences. Since the results of organ-protective treatments cannot always be transferred from laboratory models into human therapies, increasing the translational potential of preclinical settings is an important goal. Our aim was to develop a standardized research protocol, where the progression of sepsis-related events can be characterized reproducibly in model experiments within clinically-relevant time frames. Methods: Peritonitis was induced in anesthetized minipigs injected intraperitoneally with autofeces inoculum (n = 27) or with saline (sham operation; n = 9). The microbial colony-forming units (CFUs) in the inoculum were retrospectively determined. After awakening, clinically relevant supportive therapies were conducted. Nineteen inoculated animals developed sepsis without a fulminant reaction. Sixteen hours later, these animals were re-anesthetized for invasive monitoring. Blood samples were taken to detect plasma TNF-α, IL-10, big endothelin (bET), high mobility group box protein1 (HMGB1) levels and blood gases, and sublingual microcirculatory measurements were conducted. Hemodynamic, respiratory, coagulation, liver and kidney dysfunctions were detected to characterize the septic status with a pig-specific Sequential Organ Failure Assessment (pSOFA) score and its simplified version (respiratory, cardiovascular and renal failure) between 16 and 24 h of the experiments. Results: Despite the standardized sepsis induction, the animals could be clustered into two distinct levels of severity: a sepsis (n = 10; median pSOFA score = 2) and a septic shock (n = 9; median pSOFA score = 8) subgroup at 18 h of the experiments, when the decreased systemic vascular resistance, increased DO2 and VO2, and markedly increased ExO2 demonstrated a compensated hyperdynamic state. Septic animals showed severity-dependent scores for organ failure with reduced microcirculation despite the adequate oxygen dynamics. Sepsis severity characterized later with pSOFA scores was in correlation with the germ count in the induction inoculum (r = 0.664) and CFUs in hemocultures (r = 0.876). Early changes in plasma levels of TNF-α, bET and HMGB1 were all related to the late-onset organ dysfunctions characterized by pSOFA scores. Conclusions: This microbiologically-monitored, large animal model of intraabdominal sepsis is suitable for clinically-relevant investigations. The methodology combines the advantages of conscious and anesthetized studies, and mimics human sepsis and septic shock closely with the possibility of numerical quantification of host responses.

A novel Bacteroides metallo-ß-lactamase (MBL) and its gene (crxA) in Bacteroides xylanisolvens revealed by genomic sequencing and functional analysis.

OBJECTIVES: We sought to characterize the carbapenem resistance mechanism of Bacteroides xylanisolvens 14880, an imipenem-resistant strain from Germany, and assess its prevalence. METHODS: Antimicrobial susceptibilities were determined using agar dilution or Etest methodology and specific imipenemase activity was detected. The genomic sequence of B. xylanisolvens 14880 was determined and analysed for antibiotic resistance genes and genomic islands. We also used gene transfer to a carbapenem susceptible host, along with 5'-RACE, conventional PCR with capillary sequencing and RT-PCR-based screening. RESULTS: B. xylanisolvens 14880 displayed resistance to carbapenems and produced high specific imipenemase activity. Its genomic sequence was 6.1 Mbp and a class B1 ß-lactamase gene (termed crxA) was detected in it. crxA was carried on a putative genomic island with insertion sequence (IS) elements and a putative GNAT (Gcn5-like acetyltransferase) toxin gene. Promoter localization by 5'-RACE and gene targeting to an imipenem-susceptible Bacteroides host indicated that it is activated by an IS1380-like IS element and it can confer carbapenem resistance. The PCR screening of Bacteroides strains showed that crxA was specific to B. xylanisolvens with a carriage rate of 16.7%. CONCLUSIONS: B. xylanisolvens strains can harbour a carbapenem resistance gene, which has many similarities to the 'cfiA system': metallo-ß-lactamase (MBL), IS element activation, carriage of a GNAT toxin gene, specific for a unique Bacteroides species with a significant prevalence.

Characterization of the components of the thioredoxin system in Bacteroides fragilis and evaluation of its activity during oxidative stress.

OBJECTIVES: Bacteroides fragilis has a pronounced ability to survive prolonged exposure to atmospheric oxygen. The major objective of this study was to biochemically characterize the components of the thioredoxin system in B. fragilis. The nitroreductase activity of TrxR was also assayed. METHODS: Components of the thioredoxin system were expressed in E. coli and used in a disulfide reductase activity assay. Activity of TrxR was measured with purified recombinant enzyme or with cell extracts after or without exposure to oxygen or hydrogen peroxide, respectively. RESULTS: Of all six thioredoxins tested, only thioredoxins A, D, and F were reduced by recombinant TrxR and natural TrxR present in B. fragilis cell extracts. Exposure to oxygen and hydrogen peroxide increased the activity of TrxR. Further, B. fragilis TrxR acts as a nitroreductase with furazolidone or 1-Chloro-2,4-dinitrobenzene as substrates but cannot reduce metronidazole. CONCLUSION: TrxR shows an increase in activity under the conditions of oxidative stress and exerts nitroreductase activity.

Haemin deprivation renders Bacteroides fragilis hypersusceptible to metronidazole and cancels high-level metronidazole resistance.

BACKGROUND: Infections with Bacteroides fragilis are routinely treated with metronidazole, a 5-nitroimidazole antibiotic that is active against most anaerobic microorganisms. Metronidazole has remained a reliable treatment option, but resistance does occur, including in B. fragilis. OBJECTIVES: In this study we tested whether haemin, a growth supplement for B. fragilis in vivo and in vitro, had an influence on the susceptibility of resistant B. fragilis strains to metronidazole. We further tested whether haemin-deprived B. fragilis would be more susceptible to oxygen and oxidative stress. Metronidazole has been described to cause oxidative stress, which we argued would be exacerbated in haemin-deprived B. fragilis because the bacteria harness haemin, and the iron released from it, in antioxidant enzymes such as catalase and superoxide dismutase. METHODS: Haemin was omitted from growth media and the effect on metronidazole susceptibility was monitored in susceptible and resistant B. fragilis strains. Further, haemin-deprived B. fragilis were tested for resistance to aeration and hydrogen peroxide and the capacity for the removal of oxygen. RESULTS: Omission of haemin from the growth medium rendered metronidazole-resistant B. fragilis strains, including an MDR isolate from the UK, highly susceptible to metronidazole. Haemin deprivation further rendered B. fragilis highly susceptible to oxygen, which was further exacerbated in resistant strains. B. fragilis was incapable of scavenging oxygen when haemin was omitted. CONCLUSIONS: We propose that haemin deprivation overrules resistance mechanisms by rendering B. fragilis hypersusceptible to metronidazole due to a compromised antioxidant defence. Monitoring of haemin concentrations is imperative when conducting metronidazole susceptibility testing in B. fragilis.

A comparison of the antimicrobial resistance of fecal Bacteroides isolates and assessment of the composition of the intestinal microbiotas of carbapenem-treated and non-treated persons from Belgium and Hungary.

The antimicrobial susceptibilities of Bacteroides strains isolated from the feces of imipenem-treated patients from Belgium and Hungary were compared with those isolated from the normal microbiota from these two and five other European countries and assessed. Of the 10 antibiotics tested, highly significant differences were found with cefoxitin (decrease for Belgium and for this two and the five countries from the previous study), clindamycin (decrease for Belgium and for this two and the five countries from the previous study) and moxifloxacin (increase for Belgium and for this two and the five countries from the previous study) relative to normal microbiota strains reported earlier. Imipenem treatment brought about modest, but notable differences in the compositions of the microbiomes where there was less diversity in the treated group relative to the non-treated group.

The microbial composition of the initial insult can predict the prognosis of experimental sepsis.

We hypothesized that the composition of sepsis-inducing bacterial flora influences the course of fecal peritonitis in rodents. Saline or fecal suspensions with a standardized dose range of bacterial colony-forming units (CFUs) were injected intraperitoneally into Sprague-Dawley rats. The qualitative composition of the initial inoculum and the ascites was analyzed separately by MALDI-TOF mass spectrometry. Invasive monitoring was conducted in separate anesthetized groups (n = 12-13/group) after 12, 24, 48 and 72 h to determine rat-specific organ failure assessment (ROFA) scores. Death and ROFA scores peaked at 24 h. At this time, 20% mortality occurred in animals receiving a monomicrobial E. coli suspension, and ROFA scores were significantly higher in the monomicrobial subgroup than in the polymicrobial one (median 6.5; 5.0-7.0 and 5.0; 4.75-5.0, respectively). ROFA scores dropped after 48 h, accompanied by a steady decrease in ascites CFUs and a shift towards intra-abdominal monomicrobial E. coli cultures. Furthermore, we found a relationship between ascites CFUs and the evolving change in ROFA scores throughout the study. Hence, quantitatively identical bacterial loads with mono- or polymicrobial dominance lead to a different degree of sepsis severity and divergent outcomes. Initial and intraperitoneal microbiological testing should be used to improve translational research success.