The majority of antimicrobial susceptibility testing (AST) in Dutch medical microbiology laboratories (MMLs) is performed using the automated system VITEK2 or Phoenix. One of the Phoenix users... Show moreThe majority of antimicrobial susceptibility testing (AST) in Dutch medical microbiology laboratories (MMLs) is performed using the automated system VITEK2 or Phoenix. One of the Phoenix users noted a high percentage of gentamicin resistance in Proteus mirabilis compared to national resistance data. We therefore performed a descriptive analysis comparing gentamicin and tobramycin minimum inhibitory concentration (MIC) distributions for Enterobacterales and non-fermenters as measured using VITEK2 and Phoenix. Routine susceptibility data from 5 MMLs using Phoenix and 31 using VITEK2 with complete data in the Dutch national surveillance system for antimicrobial resistance from January 2016 to December 2021 were included. A panel of 12 discrepant isolates was sent to Becton Dickinson for confirmation. In general, Phoenix measured higher MICs, with discrepancies being most prevalent for Proteus, Providencia, Morganella, Serratia, and Acinetobacter species (borderline susceptibility for gentamicin ranging from 1% for VITEK2 to 67% for Phoenix systems), and less, but still clinically relevant, for Escherichia coli (1%-12%). Furthermore, we observed a yearly increase in resistance for Proteus and Providencia species measured by Phoenix. Similar discrepancies were found for tobramycin. The company confirmed our findings on all strains. Significantly more false aminoglycoside borderline susceptible and resistant Enterobacterales were found using Phoenix compared to the VITEK2 system. These findings should be taken into consideration in the development of clinical treatment guidelines for patients with sepsis.IMPORTANCEAntimicrobial sensitivity data are important to guide antimicrobial therapy. In microbiological laboratories, routine sensitivity measurements are typically performed with automated testing systems such as VITEK2 and Phoenix. Using data from the Dutch national surveillance system for antimicrobial resistance over a 6-year period, we found that the measured minimum inhibitory concentrations for aminoglycosides in Enterobacterales and non-fermenters were too high for the Phoenix system. In addition, we observed a yearly increase in resistance for several species measured by Phoenix. These findings might have consequences for clinical treatment of patients with sepsis.Antimicrobial sensitivity data are important to guide antimicrobial therapy. In microbiological laboratories, routine sensitivity measurements are typically performed with automated testing systems such as VITEK2 and Phoenix. Using data from the Dutch national surveillance system for antimicrobial resistance over a 6-year period, we found that the measured minimum inhibitory concentrations for aminoglycosides in Enterobacterales and non-fermenters were too high for the Phoenix system. In addition, we observed a yearly increase in resistance for several species measured by Phoenix. These findings might have consequences for clinical treatment of patients with sepsis. Show less
Schouls, L.M.; Witteveen, S.; Santen-Verheuvel, M. van; Haan, A. de; Labdman, F.; Heide, H. van der; ... ; Steingrover, R. 2023
Background.Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage... Show moreBackground.Although the Netherlands is a country with a low endemic level, methicillin-resistant Staphylococcus aureus (MRSA) poses a significant health care problem. Therefore, high coverage national MRSA surveillance has been in place since 1989. To monitor possible changes in the type-distribution and emergence of resistance and virulence, MRSA isolates are molecularly characterized.Methods.All 43,321 isolates from 36,520 persons, collected 2008-2019, were typed by multiple-locus variable number tandem repeats analysis (MLVA) with simultaneous PCR detection of the mecA, mecC and lukF-PV genes, indicative for PVL. Next-generation sequencing data of 4991 isolates from 4798 persons were used for whole genome multi-locus sequence typing (wgMLST) and identification of resistance and virulence genes.Results.We show temporal change in the molecular characteristics of the MRSA population with the proportion of PVL-positive isolates increasing from 15% in 2008-2010 to 25% in 2017-2019. In livestock-associated MRSA obtained from humans, PVL-positivity increases to 6% in 2017-2019 with isolates predominantly from regions with few pig farms. wgMLST reveals the presence of 35 genogroups with distinct resistance, virulence gene profiles and specimen origin. Typing shows prolonged persistent MRSA carriage with a mean carriage period of 407 days. There is a clear spatial and a weak temporal relationship between isolates that clustered in wgMLST, indicative for regional spread of MRSA strains.Conclusions.Using molecular characterization, this exceptionally large study shows genomic changes in the MRSA population at the national level. It reveals waxing and waning of types and genogroups and an increasing proportion of PVL-positive MRSA.A group of bacteria that cause difficult-to-treat infections in humans is methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to monitor changes in the spread of MRSA, their disease causing potential and resistance to antibiotics used to treat MRSA infections. MRSA from patients and their contacts in the Netherlands were collected over a period of 12 years and characterized. This revealed new types of MRSA emerged and others disappeared. An increasing number of MRSA produces a protein called PVL toxin, enabling MRSA to cause more severe infections. Also, some people appear to carry MRSA without any disease for more than a year. These findings suggest an increasing disease potential of MRSA and possible unnoticed sources of infection. Consequently, it is important to maintain monitoring of these infections to minimize MRSA spread.Schouls et al. characterize 43,321 methicillin-resistant Staphylococcus aureus (MRSA) isolates obtained between 2008 and 2019 in the Netherlands. Genomic changes occur in the MRSA population, with increases in the proportion of PVL-positive MRSA. Show less
Objectives: To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting.Methods: A multi-centre study was conducted at four... Show moreObjectives: To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting.Methods: A multi-centre study was conducted at four hospitals located across the Netherlands. Newly admitted patients were screened for CDC. The risk of development of Clostridioides difficile infection (CDI) during admission and 1-year follow-up was assessed in patients with and without colonization. C. difficile isolates from patients with colonization were compared with isolates from incident CDI cases using core genome multi-locus sequence typing to determine whether onwards transmission had occurred.Results: CDC was present in 108 of 2211 admissions (4.9%), whereas colonization with a toxigenic strain (toxigenic Clostridoides difficile colonization [tCDC]) was present in 68 of 2211 admissions (3.1%). Among these 108 patients with colonization, diverse PCR ribotypes were found and no 'hypervirulent' PCR ribotype 027 (RT027) was detected (95% CI, 0-0.028). None of the patients with colonization developed CDI during admission (0/49; 95% CI, 0-0.073) or 1-year follow-up (0/38; 95% CI, 0-0.93). Core genome multi-locus sequence typing identified six clusters with genetically related isolates from patients with tCDC and CDI; however, in these clusters, only one possible transmission event from a patient with tCDC to a patient with CDI was identified based on epidemiological data.Conclusion: In this endemic setting with a low prevalence of 'hypervirulent' strains, screening for CDC at admission did not detect any patients with CDC who progressed to symptomatic CDI and detected only one possible transmission event from a patient with colonization to a patient with CDI. Thus, screening for CDC at admission is not useful in this setting. Monique J.T. Crobach, Clin Microbiol Infect 2023;29:891 & COPY; 2023 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/). Show less
Chernova, V.O.; Terveer, E.M.; Prehn, J. van; Kuijper, E.J.; Keller, J.J.; Jong, A.E.V.; ... ; Contarino, M.F. 2023
We report a patient with a 5-year diagnosis of akinetic-rigid Parkinson's disease under treatment with Levodopa-Carbidopa Intestinal Gel therapy through a PEG-J tube due to motor complications, in... Show moreWe report a patient with a 5-year diagnosis of akinetic-rigid Parkinson's disease under treatment with Levodopa-Carbidopa Intestinal Gel therapy through a PEG-J tube due to motor complications, in which, in the context of a clinical study, we successfully and safely administered fecal microbiota transplant through a PEG-J. Show less
Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut... Show moreClostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 mu g/mL, compared to the EUCAST breakpoint of 2 mu g/mL) and contained a vanR(Cd) 343A>G mutation resulting in a Thr115Ala substitution in the VanR(Cd) response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC >= 128 mu g/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanR(Cd) Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance.IMPORTANCE The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanR(Cd) 343A>G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates of C. difficile obtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets. Show less
Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut... Show moreClostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 μg/mL, compared to the EUCAST breakpoint of 2 μg/mL) and contained a vanRCd 343A>G mutation resulting in a Thr115Ala substitution in the VanRCd response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC ≥ 128 μg/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanRCd Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance. Show less
Objectives: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the... Show moreObjectives: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization.Methods: Isolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs).Results: A cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance.Conclusions: Novel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria. Show less
Ducarmon, Q.R.; Bruggen, T. van der; Harmanus, C.; Sanders, I.M.J.G.; Daenen, L.G.M.; Fluit, A.C.; ... ; Smits, W.K. 2023
Objectives: We report a patient case of pseudomembranous colitis associated with a monotoxinproducing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR)... Show moreObjectives: We report a patient case of pseudomembranous colitis associated with a monotoxinproducing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype (RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151. Methods: Illumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades CeI and C-II, which includes the patient isolate. Results: We found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C eI and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novelpathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test. Discussion: This study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted. Quinten R. Ducarmon, Clin Microbiol Infect 2023;29:538.e1-538.e6 (c) 2022 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Show less
Ducarmon, Q.R.; Bruggen, T. van der; Harmanus, C.; Sanders, I.M.J.G.; Daenen, L.G.M.; Fluit, A.C.; ... ; Smits, W.K. 2023
ObjectivesWe report a patient case of pseudomembranous colitis associated with a monotoxin-producing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR)... Show moreObjectivesWe report a patient case of pseudomembranous colitis associated with a monotoxin-producing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype (RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151.MethodsIllumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades C–I and C-II, which includes the patient isolate.ResultsWe found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C–I and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novel-pathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test.DiscussionThis study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted. Show less
Short-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being... Show moreShort-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being unclear. We aimed to investigate the role of gut microbiota in the induction of metabolic benefits of dietary butyrate. We performed antibiotic-induced microbiota depletion of the gut and fecal microbiota transplantation (FMT) in APOE*3-Leiden.CETP mice, a well-established translational model for developing human-like metabolic syndrome, and revealed that dietary butyrate reduced appetite and ameliorated high-fat diet-induced (HFD-induced) weight gain dependent on the presence of gut microbiota. FMT from butyrate-treated lean donor mice, but not butyrate-treated obese donor mice, into gut microbiota-depleted recipient mice reduced food intake, attenuated HFD-induced weight gain, and improved insulin resistance. 16S rRNA and metagenomic sequencing on cecal bacterial DNA of recipient mice implied that these effects were accompanied by the selective proliferation of Lachnospiraceae bacterium 28-4 in the gut as induced by butyrate. Collectively, our findings reveal a crucial role of gut microbiota in the beneficial metabolic effects of dietary butyrate as strongly associated with the abundance of Lachnospiraceae bacterium 28-4. Show less
Short-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being... Show moreShort-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being unclear. We aimed to investigate the role of gut microbiota in the induction of metabolic benefits of dietary butyrate. We performed antibiotic-induced microbiota depletion of the gut and fecal microbiota transplantation (FMT) in APOE*3-Leiden.CETP mice, a well-established translational model for developing human-like metabolic syndrome, and revealed that dietary butyrate reduced appetite and ameliorated high-fat diet–induced (HFD-induced) weight gain dependent on the presence of gut microbiota. FMT from butyrate-treated lean donor mice, but not butyrate-treated obese donor mice, into gut microbiota–depleted recipient mice reduced food intake, attenuated HFD-induced weight gain, and improved insulin resistance. 16S rRNA and metagenomic sequencing on cecal bacterial DNA of recipient mice implied that these effects were accompanied by the selective proliferation of Lachnospiraceae bacterium 28-4 in the gut as induced by butyrate. Collectively, our findings reveal a crucial role of gut microbiota in the beneficial metabolic effects of dietary butyrate as strongly associated with the abundance of Lachnospiraceae bacterium 28-4. Show less
Background: The gut microbiome plays an important role in immune modulation. Specifically, presence or absence of certain gut bacterial taxa has been associated with better antitumor immune... Show moreBackground: The gut microbiome plays an important role in immune modulation. Specifically, presence or absence of certain gut bacterial taxa has been associated with better antitumor immune responses. Furthermore, in trials using fecal microbiota transplantation (FMT) to treat melanoma patients unresponsive to immune checkpoint inhibitors (ICI), complete responses (CR), partial responses (PR), and durable stable disease (SD) have been observed. However, the underlying mechanism determining which patients will or will not respond and what the optimal FMT composition is, has not been fully elucidated, and a discrepancy in microbial taxa associated with clinical response has been observed between studies. Furthermore, it is unknown whether a change in the microbiome itself, irrespective of its origin, or FMT from ICI responding donors, is required for reversion of ICI-unresponsiveness. To address this, we will transfer microbiota of either ICI responder or nonresponder metastatic melanoma patients via FMT. Methods: In this randomized, double-blinded phase Ib/IIa trial, 24 anti-PD1-refractory patients with advanced stage cutaneous melanoma will receive an FMT from either an ICI responding or nonresponding donor, while continuing anti-PD-1 treatment. Donors will be selected from patients with metastatic melanoma treated with anti-PD-1 therapy. Two patients with a good response (& GE; 30% decrease according to RECIST 1.1 within the past 24 months) and two patients with progression (& GE; 20% increase according to RECIST 1.1 within the past 3 months) will be selected as ICI responding or nonresponding donors, respectively. The primary endpoint is clinical benefit (SD, PR or CR) at 12 weeks, confirmed on a CT scan at 16 weeks. The secondary endpoint is safety, defined as the occurrence of grade & GE; 3 toxicity. Exploratory endpoints are progression-free survival and changes in the gut microbiome, metabolome, and immune cells. Discussion: Transplanting fecal microbiota to restore the patients' perturbed microbiome has proven successful in several indications. However, less is known about the potential role of FMT to improve antitumor immune response. In this trial, we aim to investigate whether administration of FMT can reverse resistance to anti-PD-1 treatment in patients with advanced stage melanoma, and whether the ICI-responsiveness of the feces donor is associated with its effectiveness. Show less
A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential... Show moreA subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology. Show less
Clostridioides difficile infection (CDI) is an important cause of diarrhea in hospitals worldwide. The incidence of CDI in Latin America has not yet been standardized. To fill this gap, the present... Show moreClostridioides difficile infection (CDI) is an important cause of diarrhea in hospitals worldwide. The incidence of CDI in Latin America has not yet been standardized. To fill this gap, the present study performed a daily active surveillance, for three months, between April to July of 2021, at a quaternary referral university hospital in Brazil. The incidence density was 9.2 cases per 10,000 patient-days. Cases were associated mostly with ribotypes 014 and 106 (44% and 22%, respectively). Ribotype 027 was not identified. The findings strongly reinforce the need for broad epidemiological studies on the incidence of CDI in Brazilian hospitals to increase the understanding, prevention, and treatment of this infection.(c) 2022 Elsevier Ltd. All rights reserved. Show less
The plasmid pCD-METRO confers metronidazole re-sistance in Clostridioides difficile. We showed high se-quence similarity among pCD-METRO plasmids from different isolates and identified pCD-METRO... Show moreThe plasmid pCD-METRO confers metronidazole re-sistance in Clostridioides difficile. We showed high se-quence similarity among pCD-METRO plasmids from different isolates and identified pCD-METRO and as-sociated metronidazole-resistant isolates in clinical and veterinary reservoirs in the Americas. We recommend using PCR or genomic assays to detect pCD-METRO in metronidazole-resistant C. difficile. Show less
Wiese, M.; Schuren, F.H.J.; Smits, W.K.; Kuijper, E.J.; Ouwens, A.; Heerikhuisen, M.; ... ; Vossen, J.M.B.M. van der 2022
BackgroundClostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly... Show moreBackgroundClostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly transmitted as endospores, which germinate to produce the pathogenic vegetative cells under suitable conditions in the gut. To efficiently screen novel therapeutic- interventions against the proliferation of C. difficile within a complex microbial community, platforms are needed that facilitate parallel experimentation. In order to allow for screening of novel interventions a medium-to-high throughput in vitro system is desirable. To this end, we have developed the 96-well CDi-screen platform that employs an adapted simulated ileal effluent medium (CDi-SIEM) and allows for culturing of pathogenic C. difficile. MethodsC. difficile strain ATCC 43599 was inoculated in the form of vegetative cells and spores into the CDi-screen in the presence and absence of a cultured fecal microbiota and incubated for 48h. To demonstrate its utility, we investigated the effect of the human milk oligosaccharide 2'-Fucosyllactose (2'-FL) at 4 and 8 mg/mL on C. difficile outgrowth and toxin production in the CDi-screen. The test conditions were sampled after 24 and 48 hours. C. difficile -specific primers were used to monitor C. difficile growth via qPCR and barcoded 16S rRNA gene amplicon sequencing facilitated the in-depth analysis of gut microbial community dynamics. ResultsC. difficile ATCC 43599 proliferated in CDi-SIEM, both when inoculated as spores and as vegetative cells. The strain reached cell numbers expressed as C. difficile genome equivalents of up to 10 (8) cells per mL after 24h of incubation. 2'-FL significantly inhibited the outgrowth of the ATTC 43599 strain within a complex human gut microbial community in the CDi-screen. In addition, a dose-dependent modulation of the gut microbial community composition by 2'-FL supplementation was detected, with a significant increase in the relative abundance of the genus Blautia in the presence of 2'-FL. ConclusionThe CDi-screen is suitable for studying C. difficile proliferation in a complex gut ecosystem and for screening for anti-pathogenic interventions that target C. difficile directly and/or indirectly through interactions with the gut microbiota. Different doses of compounds such as in this study the dose of the human milk oligosaccharide 2'-FL can be screened for efficacy in the inhibition of C. difficile proliferation. Show less
Aims: Here we used a mature seven -day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or... Show moreAims: Here we used a mature seven -day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or antibiotics or non-contact induction heating, and which combinations could eradicate S. aureus in mature biofilms. Methods: Mature biofilms of S. aureus (ATCC 29213) were grown on titanium alloy (Ti6Al4V) coupons for seven days and were subjected to the following treatments or their combinations: antibiotics, mechanical cleaning, or heat shock by induction heating of 60 & DEG;C for one minute. Experiments were repeated at least five times. Results: In the untreated biofilm, growth up to 1.8x1011 colony-forming units (CFU)/cm2 was observed. Treatment with ciprofloxacin, flucloxacillin, vancomycin, cefuroxime, and amoxicillin all with rifampicin gave 6.0 log, 6.1 log, 1.4 log, 4.8 log, and 3.6 log reduction in CFU/cm2, respectively. Mechanical cleaning alone resulted in 4.9 log reduction and induc-tion heating in 7.3 log reduction. There was an additional effect of ciprofloxacin, fluclox-acillin, and induction heating when used in combinations. There was no additional effect for mechanical cleaning. No bacterial growth could be detected after induction heating followed by seven days of ciprofloxacin with rifampicin. Conclusion: Mechanical cleaning, antibiotics, and non-contact induction heating reduced the bacterial load of mature S. aureus biofilms with approximately 5 log or more as a single treatment. The effect of mechanical cleaning on mature S. aureus biofilms was limited when used in combination with antibiotics and/or induction heating. Show less
Background: Clostridioides difficile is the most common causative agent of antibiotic-acquired diarrhea in hospitalized patients associated with substantial morbidity and mortality. The global... Show moreBackground: Clostridioides difficile is the most common causative agent of antibiotic-acquired diarrhea in hospitalized patients associated with substantial morbidity and mortality. The global epidemic of CDI (Clostridioides difficile infection) began in the early 20th century with the emergence of the hypervirulent and resistant ribotype 027 strains, and requires an urgent search for new therapeutic agents. Objective: The aim of this study is to investigate the antibacterial activity of the three essential oils isolated from spice herbs (wild oregano, garlic and black pepper) against C. difficile clinical isolates belonging to 6 different PCR ribotypes and their potential inhibitory effect on the biofilm production in in vitro conditions. Results: Wild oregano essential oil showed strong inhibitory activity in concentrations 0.02-1.25 mg/mL and bactericidal activity in concentrations from 0.08 to 10 mg/mL. Garlic essential oil was effective in the concentration range of 0.02-40 mg/mL, and 0.16 -> 40 mg/mL. MIC and MBC for black pepper oil ranged from 0.04 to 40 mg/mL, and 0.08 -> 40 mg/mL, respectively. All the tested oils reduced in vitro biofilm production, with the best activity of oregano oil. Conclusion: Essential oils of wild oregano, black pepper and garlic are candidates for adjunctive therapeutics in the treatment of CDI. Oregano oil should certainly be preferred due to the lack of selectivity of action in relation to the ribotype, the strength of the produced biofilm and/or antibiotic-susceptibility patterns.(c) 2022 Elsevier Ltd. All rights reserved. Show less