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
Carnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial... Show moreCarnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial interest in using C. divergens as biopreservatives and/or probiotics, some strains are known to be fish pathogens, and the uncontrolled growth of C. divergens has been associated with food spoilage. Bacteriophages offer a selective approach to identify and control the growth of bacteria; however, to date, few phages targeting C. divergens have been reported. In this study, we characterize bacteriophage cd2, which we recently isolated from minced beef. A detailed host range study reveals that phage cd2 infects certain phylogenetic groups of C. divergens. This phage has a latent period of 60 min and a burst size of ~28 PFU/infected cell. The phage was found to be acid and heat sensitive, with a complete loss of phage activity when stored at pH 2 or heated to 60°C. Electron microscopy shows that phage cd2 is a siphophage, and while it shares the B3 morphotype with a unique cluster of Listeria and Enterococcus phages, a comparison of genomes reveals that phage cd2 comprises a new genus of phage, which we have termed as Carnodivirus. Show less
Schmitz, D.; Zwagemaker, F.; Veer, B. van der; Vennema, H.; Laros, J.F.J.; Koopmans, M.P.G.; ... ; Kroneman, A. 2023
Norovirus is the primary cause of viral gastroenteritis (GE). To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of complete genomes.... Show moreNorovirus is the primary cause of viral gastroenteritis (GE). To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of complete genomes. To investigate the potential of shotgun metagenomic sequencing on the Illumina platform for whole-genome sequencing, 71 reverse transcriptase quantitative PCR (RT-qPCR) norovirus positive-feces (threshold cycle [C-T], <30) samples from norovirus surveillance within The Netherlands were subjected to metagenomic sequencing. Data were analyzed through an in-house next-generation sequencing (NGS) analysis workflow. Additionally, we assessed the potential of metagenomic sequencing for the surveillance of off-target viruses that are of importance for public health, e.g., sapovirus, rotavirus A, enterovirus, parechovirus, aichivirus, adenovirus, and bocaparvovirus. A total of 60 complete and 10 partial norovirus genomes were generated, representing 7 genogroup I capsid genotypes and 12 genogroup II capsid genotypes. In addition to the norovirus genomes, the metagenomic approach yielded partial or complete genomes of other viruses for 39% of samples from children and 6.7% of samples from adults, including adenovirus 41 (N = 1); aichivirus 1 (N = 1); coxsackievirus A2 (N = 2), A4 (N = 2), A5 (N = 1), and A16 (N = 1); bocaparvovirus 1 (N = 1) and 3 (N = 1); human parechovirus 1 (N = 2) and 3 (N = 1); Rotavirus A (N = 1); and a sapovirus GI.7 (N = 1). The sapovirus GI.7 was initially not detected through RT-qPCR and warranted an update of the primer and probe set. Metagenomic sequencing on the Illumina platform robustly determines complete norovirus genomes and may be used to broaden gastroenteritis surveillance by capturing off-target enteric viruses.IMPORTANCE Viral gastroenteritis results in significant morbidity and mortality in vulnerable individuals and is primarily caused by norovirus. To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of full genomes. Using surveillance samples sent to the Dutch National Institute for Public Health and the Environment (RIVM), we compared metagenomics against conventional techniques, such as RT-qPCR and Sanger-sequencing, with norovirus as the target pathogen. We determined that metagenomics is a robust method to generate complete norovirus genomes, in parallel to many off-target pathogenic enteric virus genomes, thereby broadening our surveillance efforts. Moreover, we detected a sapovirus that was not detected by our validated gastroenteritis RT-qPCR panel, which exemplifies the strength of metagenomics. Our study shows that metagenomics can be used for public health gastroenteritis surveillance, the generation of reference-sets for molecular epidemiology, and how it compares to current surveillance strategies.Viral gastroenteritis results in significant morbidity and mortality in vulnerable individuals and is primarily caused by norovirus. To investigate norovirus epidemiology, there is a need for whole-genome sequencing and reference sets consisting of full genomes. Show less
Salgado-Benvindo, C.; Leijs, A.A.; Thaler, M.; Tas, A.; Arbiser, J.L.; Snijder, E.J.; Hemert, M.J. van 2023
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019, and the resulting pandemic has already caused the death of over 6 million people. There are currently few antivirals... Show moreSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019, and the resulting pandemic has already caused the death of over 6 million people. There are currently few antivirals approved for treatment of the 2019 coronavirus disease (COVID-19), and more options would be beneficial, not only now but also to increase our preparedness for future coronavirus outbreaks. Honokiol is a small molecule from magnolia trees for which several biological effects have been reported, including anticancer and anti-inflammatory activities. Honokiol has also been shown to inhibit several viruses in cell culture. In this study, we determined that honokiol protected Vero E6 cells from SARS-CoV-2-mediated cytopathic effect, with a 50% effective concentration of 7.8 mu M. In viral load reduction assays, honokiol decreased viral RNA copies as well as viral infectious progeny titers. The compound also inhibited SARS-CoV-2 replication in the more relevant human A549 cells expressing angiotensin converting enzyme 2 and transmembrane protease serine 2. Time-of-addition and other assays showed that honokiol inhibited virus replication at a post-entry step of the replication cycle. Honokiol was also effective against more recent variants of SARS-CoV-2, including Omicron, and it inhibited other human coronaviruses as well. Our study suggests that honokiol is an interesting molecule to be evaluated further in animal studies and, when successful, maybe even in clinical trials to investigate its effect on virus replication and pathogenic (inflammatory) host responses.IMPORTANCE Honokiol is a compound that shows both anti-inflammatory and antiviral effects, and therefore its effect on SARS-CoV-2 infection was assessed. This small molecule inhibited SARS-CoV-2 replication in various cell-based infection systems, with up to an similar to 1,000-fold reduction in virus titer. In contrast to earlier reports, our study clearly showed that honokiol acts on a postentry step of the replication cycle. Honokiol also inhibited different recent SARS-CoV-2 variants and other human coronaviruses (Middle East respiratory syndrome CoV and SARS-CoV), demonstrating its broad spectrum of antiviral activity. The anticoronavirus effect, combined with its anti-inflammatory properties, make honokiol an interesting compound to be further explored in animal coronavirus infection models. 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
Shitut, S.S.; Shen, M.; Claushuis, B.; Derks, R.J.E.; Giera, M.; Rozen, D.E.; ... ; Kros, A. 2022
Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different... Show moreCell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery.Fusion of cells is an important and common biological process that leads to the mixing of cellular contents and the formation of multinuclear cells. Cell fusion occurs when distinct membranes are brought into proximity of one another and merge to become one. Fusion holds promise for biotechnological innovations, for instance, for the discovery of urgently needed new antibiotics. Here, we used antibiotic-producing bacteria that can proliferate without their cell wall as a model to investigate cell-cell fusion. We found that fusion between genetically distinct cells yields heterokaryons that are viable, contain multiple selection markers, and show increased antimicrobial activity. The rate of fusion induced using physical and chemical methods was dependent on membrane fluidity, which is related to lipid composition as a function of cellular age. Finally, by using an innovative system of synthetic membrane-associated lipopeptides, we achieved targeted fusion between distinctly marked cells to further enhance fusion efficiency. These results provide a molecular handle to understand and control cell-cell fusion, which can be used in the future for the discovery of new drugs. IMPORTANCE Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery. We present here wall-deficient cells as a platform for the same. We identify the fluidity of the membrane as an important characteristic for the process of fusion. We demonstrate a cell-specific approach for fusion using synthetically designed peptides yielding cells with modified antibiotic production profiles. Overall, wall-deficient cells can be a chassis for innovative metabolite production by providing an alternative method for cell-cell fusion. Show less
Gorkom, T. van; Voet, W.; Arkel, G.H.J. van; Heron, M.; Hoeve-Bakker, B.J.A.; Thijsen, S.F.T.; Kremer, K. 2022
The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated... Show moreThe diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking.Laboratory diagnosis of Lyme neuroborreliosis (LNB) is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Random forest (RF) modeling was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine cerebrospinal fluid (CSF) parameters (i.e., leukocyte count, total protein, blood-CSF barrier functionality, and intrathecal total antibody synthesis), two-tier serology on serum, the CSF level of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13), and a Borrelia species PCR on CSF. In total, 156 patients were included who were classified as definite LNB (n = 10), possible LNB (n = 7), or non-LNB patient (n = 139) according to the criteria of the European Federation of Neurological Societies using a consensus strategy for intrathecal Borrelia-specific antibody synthesis. The seven antibody assays showed sensitivities ranging from 47.1% to 100% and specificities ranging from 95.7% to 100%. RF modeling demonstrated that the sensitivities of most antibody assays could be improved by including other parameters to the diagnostic repertoire for diagnosing LNB (range: 94.1% to 100%), although with slightly lower specificities (range: 92.8% to 96.4%). The most important parameters for LNB diagnosis are the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. In conclusion, this study shows that LNB diagnosis is best supported using multiparameter analysis. Furthermore, a collaborative prospective study is proposed to investigate if a standardized diagnostic algorithm can be developed for improved LNB diagnosis. IMPORTANCE The diagnosis of LNB is established by clinical symptoms, pleocytosis, and proof of intrathecal synthesis of Borrelia-specific antibodies. Laboratory diagnosis of LNB is challenging, and validated diagnostic algorithms are lacking. Therefore, this retrospective cross-sectional study aimed to compare the diagnostic performance of seven commercial antibody assays for LNB diagnosis. Multiparameter analysis was conducted to investigate whether the diagnostic performance using the antibody assays could be improved by including several routine (CSF) parameters. The results of this study show that LNB diagnosis is best supported using the detection of intrathecally produced Borrelia-specific antibodies, two-tier serology on serum, CSF-CXCL13, Reibergram classification, and pleocytosis. Furthermore, we propose a collaborative prospective study to investigate the potential role of constructing a diagnostic algorithm using multiparameter analysis for improved LNB diagnosis. Show less
Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We studied the effects of coinfections on the... Show moreCoinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis, and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water, and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/ infected with only one type of parasite (P <= 0.05). There was a positive association between exposure/infection with P. falciparum and exposure/infection with helminths or the number of helminth species, and vice versa (P <= 0.001). In addition, children coexposed/coinfected tended (P = 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host.IMPORTANCE Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites because they induce different immune profiles. We compared the antibody profile between groups of Mozambican individuals defined by P. falciparum and helminth previous exposure and/or current infection. Our results show a significant increase in antibody responses in individuals coexposed/coinfected with P. falciparum and helminths in comparison with individuals exposed/infected with only one of these parasites, and suggest that this increase is due to a more permissive immune environment to infection in the host. Importantly, this study takes previous exposure into account, which is particularly relevant in endemic areas where continuous infections imprint and shape the immune system. Deciphering the implications of coinfections deserves attention because accounting for the real interactions that occur in nature could improve the design of integrated disease control strategies. Show less
