Characterization of a clinical Clostridioides difficile isolate with markedly reduced fidaxomicin susceptibility and a V1143D mutation in rpoB.

Objectives: The identification and characterization of clinical Clostridioides difficile isolates with reduced fidaxomicin susceptibility. Methods: Agar dilution assays were used to determine fidaxomicin MICs. Genome sequence data were obtained by single-molecule real-time (SMRT) sequencing in addition to amplicon sequencing of rpoB and rpoC alleles. Allelic exchange was used to introduce the identified mutation into C. difficile 630Δerm. Replication rates, toxin A/B production and spore formation were determined from the strain with reduced fidaxomicin susceptibility. Results: Out of 50 clinical C. difficile isolates, isolate Goe-91 revealed markedly reduced fidaxomicin susceptibility (MIC >64 mg/L). A V1143D mutation was identified in rpoB of Goe-91. When introduced into C. difficile 630Δerm, this mutation decreased fidaxomicin susceptibility (MIC >64 mg/L), but was also associated with a reduced replication rate, low toxin A/B production and markedly reduced spore formation. In contrast, Goe-91, although also reduced in toxin production, showed normal growth rates and only moderately reduced spore formation capacities. This indicates that the rpoBV1143D allele-associated fitness defect is less pronounced in the clinical isolate. Conclusions: To the best of our knowledge, this is the first description of a pathogenic clinical C. difficile isolate with markedly reduced fidaxomicin susceptibility. The lower-than-expected fitness burden of the resistance-mediating rpoBV1143D allele might be an indication for compensatory mechanisms that take place during in vivo selection of mutants.

Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile.

BACKGROUND: Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media. RESULTS: The three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon. CONCLUSION: Our findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut.

Corynebacterium gottingense sp. nov., isolated from a clinical patient.

A Gram-positive bacterial strain, 99221/2016T, was isolated from blood of a patient with bacteraemia at the Institute of Medical Microbiology, Göttingen, Germany. The strain was rod-shaped with a palisade arrangement of cells, non-spore-forming, non-lipophilic, catalase-positive and oxidase-negative. It grew well at 37 °C on Columbia blood agar and showed good growth under aerobic, microaerophilic and anaerobic conditions. The colonies were white-cream, circular and convex with a shiny, smooth surface. The predominant respiratory quinones were MK-8(H2) and MK-9(H2). The polar lipids profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. Two unidentified phospholipids and several unidentified lipids were also detected. The prevalent cellular fatty acids comprised cis-9-octadecenoic acid (C18 : 1 ω9c), hexadecanoic acid (C16 : 0) and pentadecanoic acid (C15 : 0). Corynemycolates with 28-36 carbons in length were present. The whole-cell hydrolysate contained meso-diaminopimelic acid and arabinose, glucose, galactose and ribose as major sugars. Analysis of the 16S rRNA gene sequence identities revealed that the strain is most closely related to Corynebacterium imitans DSM 44264T (98.0 %), Corynebacterium lipophiloflavum DSM 44291T (96.9 %), Corynebacterium afermentans subsp. afermentans DSM 44280T (96.9 %) and Corynebacterium afermentans subsp. lipophilum DSM 44282T (96.8 %). The identity with Corynebacterium diphtheriae DSM 44123T, the type species of the genus, was 94 %. The DNA G+C content was 69.2 mol%. DNA-DNA hybridization with Corynebacterium imitans DSM 44264T revealed a value of 34 %, confirming that the strain represents a novel species. The type strain 99221/2016T (DSM 103494T=JCM 31931T) is proposed to represent a novel species of the genus Corynebacterium with the name Corynebacterium gottingense.

Prevalence of pregnancy-relevant infections in a rural setting of Ghana.

BACKGROUND: Although infectious diseases still account for a high burden of morbidity and mortality in sub-Saharan Africa, simultaneous investigations on multiple infections affecting maternal and child health are missing. METHODS: We conducted a cross-sectional, single-centre pilot study in a rural area of Ghana to assess the infectiological profile during pregnancy. Screening of 180 expectant mothers was done by vaginal swabs and serology to detect the most common pregnancy-relevant infections. They were also interviewed for potential risk factors, outcome of previous pregnancies, and socio-economic aspects. RESULTS: We found a high prevalence of infections caused by hepatitis B virus (16.7% HBs antigen positive). In contrast, infections caused by hepatitis C virus (1.1% anti-HCV) and HIV (0.6%) were rare. Maternal malaria was frequent (10.6%), despite increasing acceptance of intermittent preventive treatment during pregnancy (IPTp). Group B streptococci were present in 10.6% of all pregnant women. Absence of antibodies against varicella zoster virus in 43.2%, Toxoplasma gondii in 26.8%, parvovirus B19 in 20.0%, and rubella virus in 15.7% makes a significant proportion of pregnant women susceptible for acquiring primary infections. Whereas all study participants had specific IgG antibodies against human cytomegalovirus, infections with Listeria, Brucella, or Neisseria gonorrhoeae as well as active syphilis were absent. CONCLUSIONS: Our pilot study in a rural community in Ghana indicates an urgent need for action in dealing at least with high-prevalent pregnancy-relevant infections, such as hepatitis B, malaria and those caused by group B streptococci. In addition, the resulting prevalence rates of various other infections may offer guidance for health officials to prioritize possible future intervention schemes.

Effect of fiber insertion depth on antibacterial efficacy of photodynamic therapy against Enterococcus faecalis in rootcanals.

OBJECTIVES: This in vitro study evaluated the effect of fiber insertion depth on antimicrobial efficacy of antimicrobial photodynamic therapy (aPDT) using a photosensitizer (PS; toluidine blue) and a red light-emitting diode (LED) in root canals infected with Enterococcus faecalis. MATERIALS AND METHODS: Single-rooted extracted teeth were prepared with nickel-titanium-instruments, sterilized, contaminated with E. faecalis, and incubated for 72 h. Roots were randomly divided into four experimental groups: PS only, LED only, aPDT with LED in the apical third, aPDT with LED in the coronal third, as well as into infection and sterile controls (each n = 10). Samples were taken by collecting standardized dentine shavings from the root canal walls. After serial dilution and culturing on blood agar, colony-forming units (CFU) were counted. RESULTS: Both aPDT groups showed a CFU reduction of 1-2 log10 steps compared with the infection control, whereas the effect of fiber insertion depth was negligible (<0.5 log10 steps). CFU reduction of approximately 0.5 log10 steps for PS alone was detected compared with the infection control, but PS alone was less effective than both aPDT groups. No antibacterial effect was detected for LED alone. CONCLUSIONS: aPDT reduced E. faecalis within the root canal, whereas fiber insertion depth had a negligible influence on antimicrobial effectiveness of aPDT. CLINICAL RELEVANCE: The insertion depth of the light-emitting diode may not influence the antibacterial efficacy of photodynamic therapy against E. faecalis in straight root canals.

High prevalence of nontoxigenic Clostridium difficile isolated from hospitalized and non-hospitalized individuals in rural Ghana.

Since data about Clostridium difficile infection in sub-Saharan Africa are scarce, we determined its epidemiology and risk factors in a cross-sectional study in Eikwe, a rural community in Ghana. We tested stool samples from 176 hospitalized patients with diarrhoea and from 131 asymptomatic non-hospitalized individuals for C. difficile and some other enteric pathogens. The overall prevalence rate of C. difficile was 4.9% with ribotype 084 being predominant. With 75% of the isolates, a high rate of nontoxigenic strains was present in symptomatic patients, most of whom had no other identified enteric pathogens. All strains were susceptible against metronidazole and vancomycin, respectively. Data on lifestyle and medical history showed that age <5years (p=0.004), and use of ceftriaxone (p=0.023) were the most important risk factors for C. difficile carriage status. Although our data suggest that C. difficile is currently not a major cause of diarrhoea in this setting, the epidemiology of C. difficile in sub-Saharan Africa awaits further investigation.

Linezolid resistance in clinical isolates of Staphylococcus epidermidis from German hospitals and characterization of two cfr-carrying plasmids.

OBJECTIVES: This study was a detailed investigation of Staphylococcus epidermidis clinical isolates exhibiting linezolid resistance. METHODS: Thirty-six linezolid-resistant S. epidermidis from eight German hospitals, including isolates from suspected hospital-associated outbreaks between January 2012 and April 2013, were analysed with respect to their antimicrobial susceptibility and the presence of cfr and/or mutations in the 23S rRNA, rplC, rplD and rplV genes. Relatedness of isolates was estimated by MLST and SmaI macrorestriction analysis. Characterization of cfr plasmids was carried out by means of Illumina sequencing. RESULTS: The MICs of linezolid varied substantially between the isolates. No apparent correlation was detected between the level of resistance, the presence of cfr and ribosomal target site mutations. S. epidermidis isolates from two hospitals were confirmed as clonally related, indicating the spread of the respective clone over a period of 1 year. Next-generation sequencing revealed two different categories of cfr-expressing plasmids, both of them varying in genetic arrangement and composition from previously published cfr plasmids: p12-00322-like plasmids showed incorporation of cfr into a pGO1-like backbone and displayed capabilities for intra- and inter-species conjugational transfer. CONCLUSIONS: To date, linezolid-resistant S. epidermidis have rarely been isolated from human clinical sources in Germany. Here, we describe the emergence and outbreaks of these strains. We detected previously described and novel point mutations in the 23S ribosomal genes. The cfr gene was only present in six isolates. However, this is the first known description of cfr incorporation into conjugative vectors; under selective pressure, these vectors could give reasonable cause for concern.

Isolated ocular Jarisch-Herxheimer reaction after initiating tuberculostatic therapy in a child.

After being exposed to a kindergarten teacher with infectious pulmonary tuberculosis, a7-year-old girl with a positive tuberculin skin test was treated with isoniazid. 3 days after initiation of the tuberculostatic therapy, the girl was referred to our hospital with an acute onset of blurred vision. Visual acuity (VA) was 20/200 in both eyes. Examination revealed mild anterior chamber inflammation, optic disc swelling, cystoid macular edema and periphlebitis in both eyes. However, although active tuberculosis was ruled out, the interferon-gamma release assay was positive. The anti-tuberculosis therapy was intensified with pyrazinamide, isoniazid, rifampicin and methylprednisolone. Within 10 days we saw a resolution of the macular edema and VA was 20/25. The paradoxical worsening of the patient's condition after initiation of tuberculostatic therapy with isoniazid and the prompt response to systemic steroids are typical for Jarisch-Herxheimer reaction (JHR). Our patient presented no symptoms before the isoniazid therapy was started and the reaction was ocular without any generalized symptoms. This is unique among all other reported cases of ocular JHR.

A comparative pilot study on Gram-negative bacteria contaminating the hands of children living in urban and rural areas of Indonesia versus Germany - A suitable monitoring strategy for diarrhea risk assessment?

Diarrhea is the second leading cause of death mainly effecting young children. Often it is the result of fecal-oral pathogen transmission. We aimed to investigate whether monitoring the prevalence of Gram-negative bacteria on the hands of asymptomatic children is suitable as an indicator of fecal contamination of the environment in their playground. We compared the prevalence of Gram-negative bacteria on the hands of children, who live in the German city of Göttingen, an urban area in a high-income country, with the situation in Medan as an urban area and Siberut as a rural area both in the middle-income country Indonesia. A total of 511 children at the age of 3 months to 14 years were asked to put their thumb print on MacConkey agar, which was used to screen for the presence of Gram-negative bacteria. These were subsequently identified by using MALD-TOF mass spectrometry and classified into the order Enterobacterales, Pseudomonadales, and others. The highest burden of hand contamination was found in children from rural Siberut (66.7%) followed by children from urban Medan (53.9%), and from urban Göttingen (40.6%). In all three study sites, hand contamination was lower in the youngest (<1 year) and oldest age groups (10-14 years) and highest in the age group 5-9 years. Bacteria of the order Enterobacterales possibly indicating fecal contamination were most prevalent in Siberut (85.1%) followed by Medan (62.9%) and Göttingen (21.5%). Most facultative and obligate gastrointestinal pathogens such as Escherichia coli (n = 2) and Providencia rettgeri (n = 7), both being members of the order Enterobacterales, as well as Aeromonas caviae (n = 5), and Vibrio cholerae (n = 1) both belonging to other orders were nearly exclusively identified on the hands of children in Siberut. This result was not surprising, because hygienic conditions were lowest in Siberut. Only one isolate of A. caviae was found in Medan, and no facultative gastrointestinal pathogen was identified on the hands of children from Göttingen. Our pilot study therefore indicates that investigating hands of children for the prevalence of Gram-negative bacteria using selective media are a helpful method to monitor hygienic conditions, and thereby assess the risk for diarrhea-causing bacterial pathogens in the environment.

Neonatal infection in Sub-Saharan Africa: a cross-sectional pilot study on bacterial pathogens and maternal risk factors.

Introduction: Although child morbidity and mortality could be reduced in Sub-Saharan Africa during the last years both remain high. Since neonatal infections play a major role, we conducted a cross-sectional pilot study in the lake region of Western Tanzania in order to analyze not only the prevalence of neonatal infection with its bacterial etiology including antimicrobial resistance pattern but also to detect potential maternal risk factors. Methods: We screened 156 women for potential risk factors and examined their neonates for clinical signs of an infection including microbiological verification. All women were interviewed for medical history and their socio-economic background. High-vaginal swabs (HVS) of pregnant women and blood cultures of sick infants were investigated for bacterial pathogens using culture followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) or polymerase-chain-reaction (PCR)-based assays. Antimicrobial resistances were determined using a disk diffusion test and verified by VITEK 2. Maternal malaria, blood glucose, and hemoglobin levels were determined by rapid tests and helminth infections by stool microscopy. Results and discussion: Our results showed a prevalence of 22% for neonatal infections. In total, 57% of them had culture-positive bloodstream infections with Gram-negative bacteria being the most prevalent. All these expressed resistance against ampicillin. The prevalence of maternal infection with helminths or Plasmodium was low, indicating that anti-worming strategies and intermittent preventive treatment of malaria for pregnant women (IPTp) are effective. The study identified maternal urinary tract infection (UTI) and an elevated blood glucose level as potential maternal risk factors for early neonatal infection, an elevated blood glucose level, and maternal anemia for a late-onset infection. Conclusion: Our study, therefore, indicates that monitoring maternal UTI in the last trimester as well as levels of maternal hemoglobin and blood glucose might be important to predict and eventually manage neonatal infections. As Gram-negative bacteria with resistance to ampicillin were most prevalent in culture-proven neonatal sepsis, WHO recommendations for calculated antibiosis in the sick young infant should be discussed.

Surface-associated motility, a common trait of clinical isolates of Acinetobacter baumannii, depends on 1,3-diaminopropane.

While flagella-independent motility has long been described in representatives of the genus Acinetobacter, the mechanism of motility remains ambiguous. Acinetobacter baumannii, a nosocomial pathogen appearing increasingly multidrug-resistant, may profit from motility during infection or while persisting in the hospital environment. However, data on the frequency of motility skills among clinical A. baumannii isolates is scarce. We have screened a collection of 83 clinical A. baumannii isolates of different origin and found that, with the exception of one isolate, all were motile on wet surfaces albeit to varying degrees and exhibiting differing morphologies. Screening a collection of transposon mutants of strain ATCC 17978 for motility defects, we identified 2 akinetic mutants carrying transposon insertions in the dat and ddc gene, respectively. These neighbouring genes contribute to synthesis of 1,3-diaminopropane (DAP), a polyamine ubiquitously produced in Acinetobacter. Supplementing semi-solid media with DAP cured the motility defect of both mutants. HPLC analyses confirmed that DAP synthesis was abolished in ddc and dat mutants of different A. baumannii isolates and was re-established after genetic complementation. Both, the dat and ddc mutant of ATCC 17978 were attenuated in the Galleria mellonella caterpillar infection model. Taken together, surface-associated motility is a common trait of clinical A. baumannii isolates that requires DAP and may play a role in its virulence.

Risk Factors of Patients With Diarrhea for Having Clostridioides (Clostridium) difficile Infection.

Nosocomial infections with Clostridioides (Clostridium) difficile have become an emergent health threat. We sought to define risk factors for a C. difficile infection (CDI) beyond the widely known ones, such as antibiotic use and prior hospital stay. We therefore focused on a group of patients with diarrhea in order to identify risk factors for C. difficile infection among this symptomatic cohort. A total of 121 hospitalized patients from Seesen/Germany with diarrhea were included who submitted a stool sample and were interviewed about their socio-demographic background, lifestyle and state of health using a standardized questionnaire. Antibiotic potential of diuretics was examined by agar diffusion test. C. difficile was identified in 29 patients resulting in a prevalence of 24.0%. The infection was hospital-acquired in most cases (p < 0.001, 82.1%; n = 23/28, versus 29/91, 31.9%). The generally accepted risk factor previous antibiotic use was confirmed in this study (p = 0.002, n = 23/28 CDI patients, 82.1%, versus n = 44/91 non-CDI patients, 48.4%). The following additional risk factors were identified: regular consumption of proton pump inhibitors; PPI (p = 0.011, n = 24/29, 82.8% vs. n = 52/92, 56.5%), CDI patients ate less vegetables (p = 0.001, n = 12/29, 41.4% vs. 69/92, 75.0%). The intake of the diuretic agent torasemid in patients with CDI (p = 0.005, n = 18/29, 62.1%) was higher than in patients without (n = 30/92, 32.6%). More patients with CDI had to undergo a surgery in the previous year (p = 0.022, n = 13/29, 44.8% vs. n = 21/92, 22.8%) and held more birds (p = 0.056, n = 4/29, 13.8%) than individuals of the negative group (n = 3/92, 3.3%). In conclusion, although no antibiotic potential was detected in diuretics, especially torasemid seems to have significant influence for the occurrence of a CDI as well as a nutrition poor in vegetables. A diet rich in vegetables represented a fourfold lower risk for a CDI (OR 0.240, CI (0.0720 - 0.796]).

A Case Report and Review of the Literature: Reactive Arthritis Caused by Clostridioides difficile ribotype 027.

With an annual incidence of 250-300 per 100,000 inhabitants, reactive arthritis is not uncommon. However, the fact that Clostridioides difficile infection (CDI) can also lead to this complication is largely unknown. We report on a 69-years-old man who developed reactive arthritis of his right knee joint one week after antibiotic-associated diarrhea with evidence of C. difficile of the hypervirulent ribotype 027. His female partner also became infected with C. difficile ribotype 027, but did not develop reactive arthritis. The further investigation showed that the patient - in contrast to his partner - was HLA-B27 positive and had strong antibody levels against C. difficile. The case history together with the review of 45 other cases described so far shows that C. difficile can also lead to reactive arthritis. C. difficile-associated reactive arthritis (CDARA) is characterized by the fact that patients suffer from diarrhea or colitis after taking antibiotics, toxigenic C. difficile or only the toxins are detectable in the stool and there are no other explanations for the arthritis and diarrhea.

Bacteremia and antimicrobial drug resistance over time, Ghana.

Bacterial distribution and antimicrobial drug resistance were monitored in patients with bacterial bloodstream infections in rural hospitals in Ghana. In 2001-2002 and in 2009, Salmonella enterica serovar Typhi was the most prevalent pathogen. Although most S. enterica serovar Typhi isolates were chloramphenicol resistant, all isolates tested were susceptible to ciprofloxacin.

Antimicrobial Resistance Patterns in Clostridioides difficile Strains Isolated from Neonates in Germany.

Young children are frequently colonized with Clostridioides (C.) difficile. Depending on their resistance patterns, antibiotic treatment can facilitate gastrointestinal spreading in colonized individuals, potentially leading to transmission to others. C. difficile was isolated from stool samples from infants born in two hospitals in Göttingen and Darmstadt, Germany. All isolates were subjected to phenotypic antimicrobial resistance testing, PCR-based screening for toxin genes and mass spectrometry-based exclusion of ribotypes 027 and 176. Within an initial cohort of 324 neonates with a longitudinal survey of C. difficile, 137 strains were isolated from 48 individuals. Antimicrobial resistance was recorded against metronidazole in one (0.7%), erythromycin in 16 (11.7%) and moxifloxacin in 2 (1.5%) of the strains, whereas no resistance was observed against vancomycin (0.0%) or rifampicin (0.0%). Newly observed resistance against erythromycin in children with detection of previously completely sensitive isolates was reported for C. difficile isolates from 2 out of 48 children. In 20 children (42%), non-toxigenic strains were detected, and from 27 children (56%), toxigenic strains were isolated, while both toxigenic and non-toxigenic strains were recorded for 1 child (2%). Ribotypes 027 or 176 were not observed. In conclusion, the German C. difficile strains isolated from the children showed mild to moderate resistance with predominance of macrolide resistance, a substance class which is frequently applied in children. The observed switches to the dominance of macrolide-resistant isolates suggests likely selection of resistant C. difficile strains already in children.

Characterization of Clostridioides difficile DSM 101085 with A-B-CDT+ Phenotype from a Late Recurrent Colonization.

During the last decades, hypervirulent strains of Clostridioides difficile with frequent disease recurrence and increased mortality appeared. Clostridioides difficile DSM 101085 was isolated from a patient who suffered from several recurrent infections and colonizations, likely contributing to a fatal outcome. Analysis of the toxin repertoire revealed the presence of a complete binary toxin locus and an atypical pathogenicity locus consisting of only a tcdA pseudogene and a disrupted tcdC gene sequence. The pathogenicity locus shows upstream a transposon and has been subject to homologous recombination or lateral gene transfer events. Matching the results of the genome analysis, neither TcdA nor TcdB production but the expression of cdtA and cdtB was detected. This highlights a potential role of the binary toxin C. difficile toxin in this recurrent colonization and possibly further in a host-dependent virulence. Compared with the C. difficile metabolic model strains DSM 28645 (630Δerm) and DSM 27147 (R20291), strain DSM 101085 showed a specific metabolic profile, featuring changes in the threonine degradation pathways and alterations in the central carbon metabolism. Moreover, products originating from Stickland pathways processing leucine, aromatic amino acids, and methionine were more abundant in strain DSM 101085, indicating a more efficient use of these substrates. The particular characteristics of strain C. difficile DSM 101085 may represent an adaptation to a low-protein diet in a patient with recurrent infections.

A publicly accessible database for Clostridioides difficile genome sequences supports tracing of transmission chains and epidemics.

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10-4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile.

Effectiveness of caries-preventing agents on initial carious lesions within the scope of orthodontic therapy.

OBJECTIVE: To evaluate the effectiveness of three different caries-preventing agents on artificial caries in a Streptococcus mutans-based caries model. METHODS: Sixty-five caries-free human molar enamel blocks were treated with a demineralization solution and a remineralization solution. The specimens were assigned to the following groups according to the caries-protective product applied: group A, chlorhexidine varnish; group B, fluoride-releasing chemically cured sealant; group C, fluoride-releasing lightcured sealant; group D, positive control (specimens that were subjected to de- and remineralization cycles without treatment with any caries-protective agents); and group E, negative control (specimens that were not subjected to de- and remineralization cycles). Samples in groups A-D were stored in demineralization solution with S. mutans and thereafter in artificial saliva. This procedure was performed for 30 days. Average fluorescence loss (ΔF) and surface size of the lesions were measured using quantitative light-induced fluorescence at baseline and on the 7th, 14th, and 30th days. RESULTS: After 30 days, group A demonstrated a significant increase in ΔF and the surface size of the lesions, no significant difference in comparison with the positive control group, and a significant difference in comparison with the negative control group. Group B showed no significant changes in both parameters at any of the measurement points. While group C showed increased ΔF after 14 days, no significant fluorescence change was observed after 30 days. CONCLUSIONS: Both fluoride-releasing sealants (chemically or light-cured) show anti-cariogenic effects, but the use of chlorhexidine varnish for the purpose of caries protection needs to be reconsidered.

Proteotyping of Clostridioides difficile as Alternate Typing Method to Ribotyping Is Able to Distinguish the Ribotypes RT027 and RT176 From Other Ribotypes.

Clostridioides difficile, a Gram-positive spore-forming bacterium, is the leading cause of nosocomial diarrhea worldwide and therefore a substantial burden to the healthcare system. During the past decade, hypervirulent PCR-ribotypes (RT) e.g., RT027 or RT176 emerged rapidly all over the world, associated with both, increased severity and mortality rates. It is thus of great importance to identify epidemic strains such as RT027 and RT176 as fast as possible. While commonly used diagnostic methods, e.g., multilocus sequence typing (MLST) or PCR-ribotyping, are time-consuming, proteotyping offers a fast, inexpensive, and reliable alternative solution. In this study, we established a MALDI-TOF-based typing scheme for C. difficile. A total of 109 ribotyped strains representative for five MLST clades were analyzed by MALDI-TOF. MLST, based on whole genome sequences, and PCR-ribotyping were used as reference methods. Isoforms of MS-detectable biomarkers, typically ribosomal proteins, were related with the deduced amino acid sequences and added to the C. difficile proteotyping scheme. In total, we were able to associate nine biomarkers with their encoding genes and include them in our proteotyping scheme. The discriminatory capacity of the C. difficile proteotyping scheme was mainly based on isoforms of L28-M (2 main isoforms), L35-M (4 main isoforms), and S20-M (2 main isoforms) giving rise to at least 16 proteotyping-derived types. In our test population, five of these 16 proteotyping-derived types were detected. These five proteotyping-derived types did not correspond exactly to the included five MLST-based C. difficile clades, nevertheless the subtyping depth of both methods was equivalent. Most importantly, proteotyping-derived clade B contained only isolates of the hypervirulent RT027 and RT176. Proteotyping is a stable and easy-to-perform intraspecies typing method and a promising alternative to currently used molecular techniques. It is possible to distinguish the group of RT027 and RT176 isolates from non-RT027/non-RT176 isolates using proteotyping, providing a valuable diagnostic tool.