Interactions Between Clostridioides difficile and Fecal Microbiota in in Vitro Batch Model: Growth, Sporulation, and Microbiota Changes

Disturbance in gut microbiota is crucial for the development of Clostridioides difficile infection (CDI). Different mechanisms through which gut microbiota influences C. difficile colonization are known. However, C. difficile could also affect gut microbiota balance as previously demonstrated by cultivation of fecal microbiota in C. difficile conditioned medium. In current study, the interactions of C. difficile cells with gut microbiota were addressed. Three different strains (ribotypes 027, 014/020, and 010) were co-cultivated with two types of fecal microbiota (healthy and dysbiotic) using in vitro batch model. While all strains showed higher sporulation frequency in the presence of dysbiotic fecal microbiota, the growth was strain dependent. C. difficile either proliferated to comparable levels in the presence of dysbiotic and healthy fecal microbiota or grew better in co-culture with dysbiotic microbiota. In co-cultures with any C. difficile strain fecal microbiota showed decreased richness and diversity. Dysbiotic fecal microbiota was more affected after co-culture with C. difficile than healthy microbiota. Altogether, 62 OTUs were significantly changed in co-cultures of dysbiotic microbiota/C. difficile and 45 OTUs in co-cultures of healthy microbiota/C. difficile. However, the majority of significantly changed OTUs in both types of microbiota belonged to the phylum Firmicutes with Lachnospiraceae and Ruminococcaceae origin

An overview of molecular markers for identification of non-human fecal pollution sources

Identifying primary sources of fecal pollution is important for assessing public health risks and implementing effective remediation strategies. To date, one of the main molecular approaches for identifying sources of fecal pollution relies on detecting molecular markers within bacterial, viral, or mitochondrial nucleic acids, that are indicative of a particular host. With a primary focus on identifying fecal pollution originating from humans, the field of fecal source tracking often places less emphasis on livestock sources, frequently leaving the problem of wildlife fecal pollution unaddressed. In this review, we summarize 55 previously published and validated molecular assays and describe methods for the detection of molecular markers that are indicative of non-human hosts. They cover a range of 15 animal species/groups with a primary focus on domestic animals including cattle, pigs, dogs, and poultry. Among assays associated with wild animals, the majority are designed to detect bird feces, while the availability of assays for detecting feces of other wild animals is limited. Both domestic and wild animals can represent a zoonotic reservoir of human enteropathogens, emphasizing the importance of their role in public health. This review highlights the need to address the complexity of fecal contamination and to include a broader range of animal species into assay validation and marker identification

Clostridium difficile genotypes other than ribotype 078 that are prevalent among human, animal and environmental isolates

<p>Abstract</p> <p>Background</p> <p>Characterising the overlap of <it>C. difficile </it>genotypes in different reservoirs can improve our understanding of possible transmission routes of this pathogen. Most of the studies have focused on a comparison of the PCR ribotype 078 isolated from humans and animals. Here we describe for the first time a comparison of <it>C. difficile </it>genotypes isolated during longer time intervals from different sources including humans, animals and the non-hospital environment.</p> <p>Results</p> <p>Altogether 786 isolates from time interval 2008-2010 were grouped into 90 PCR ribotypes and eleven of them were shared among all host types and the environment. Ribotypes that were most common in humans were also present in water and different animals (014/020, 002, 029). Interestingly, non-toxigenic isolates were very common in the environment (30.8%) in comparison to humans (6.5%) and animals (7.7%). A high degree of similarity was observed for human and animal isolates with PFGE. In human isolates resistance to erithromycin, clindamycin and moxifloxacin was detected, while all animal isolates were susceptible to all antibiotics tested.</p> <p>Conclusion</p> <p>Our results show that many other types in addition to PCR Ribotype 078 are shared between humans and animals and that the most prevalent genotypes in humans have the ability to survive also in the environment and several animal hosts. The genetic relatedness observed with PFGE suggests that transmission of given genotype from one reservoir to the other is likely to occur.</p

MRSA diversity and the emergence of LA-MRSA in a large teaching hospital in Slovenia

The methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. One of the most prominent changes in the MRSA epidemiology is the emergence of livestock-associated MRSA (LA-MRSA) strains in the human population. The aim of this study was to follow the MRSA epidemiology in a large teaching hospital during an 8-year time period (2006–2013). Altogether 519 MRSA, cultured from screening or clinical samples, were distributed into 77 spa types, of which three (t003 and t001, associated with CC5; and t015; associated with CC45) were the most common. LA-MRSA-associated spa types (t011, t034, t108, t899; associated with CC398) started to emerge in the year 2009 and continued to be found annually at a frequency from 3.9% to 12.7% of all MRSA strains examined. Only 6 of 27 LA-MRSA strains were associated with infections

Cameral Conscription of the Našice District from 1723

U Hrvatskom državnom arhivu u fondu Acta urbarialia et conscriptiones bonorum u fasciklu broj 132 čuva se komorski popis našičkog vlastelinstva iz 1723. godine, koji je nastao u svibnju 1723. u Požegi. U fasciklu 132 postoje dva popisa s brojem 28, koji se znatno razlikuju. Naime, jedan popis je koncept koji je očito nastao tijekom izvršenja popisa (to je u fondu drugi popis, stranice 125-162), a drugi popis je čistopis koncepta (u fondu je to prvi popis, stranice 88-124), odnosno original koji se razlikuje u načinu pisanja pojedinih sela, toponima, redoslijedu pojedinih riječi, upotrebi pojedinih izraza (npr. cedunt - cadunt) te u iznosima popisivanih dobara. Priređujući ovaj popis, odlučili smo se prirediti čistopis jer smo smatrali da je on nastao nakon povratka s terena te da su u taj tekst uneseni ispravci pogrešaka nastalih tijekom obavljanja popisa. Također, taj popis je imao pravnu snagu te je korišten pri određivanju poreznih obaveza podanika na području našičkog okruga, odnosno budućeg vlastelinstva. S druge strane, koncept nije imao pravnu valjanost te nije imao pravnog učinka na podanike našičkog okruga, odnosno budućeg vlastelinstva, pa se nije koristio, primjerice, za određivanje poreznih obaveza njegovih podanika. Međutim, treba napomenuti da je koncept u nekim slučajevima točnije bilježio toponime i antroponime, što vjerojatno treba pripisati činjenici da je popisivač uistinu čuo imena na terenu, dok prepisivač nije imao iskustva ni s jezikom ni s prostorom. Ovaj je popis nastao svega nekoliko mjeseci prije darovnice kralja Karla III., kojom je 29. listopada 1723. darovao našički okrug knezu Svetog Rimskog Carstva Njemačke Narodnosti Franji Antunu Lampergu i muškim potomcima, a ženskima u trajno nasljedstvo uz naknadu od 27.328,40 forinti. Budući je ovaj popis nastao dvije godine poslije komorskog popisa iz 1721. , značajan je jer omogućuje budućim istraživačima gospodarske povijesti našičkog područja uspoređivanje podataka iz tih dvaju popisa te analizu promjena u gospodarskom i demografskom razvoju na našičkom području prvih godina trećeg desetljeća 18. stoljeća

Typing Clostridium difficile strains based on tandem repeat sequences

Background: Genotyping of epidemic Clostridium difficile strains is necessary to track their emergence and spread. Portability of genotyping data is desirable to facilitate inter-laboratory comparisons and epidemiological studies. Results: This report presents results from a systematic screen for variation in repetitive DNA in the genome of C. difficile. We describe two tandem repeat loci, designated \u27TR6\u27 and \u27TR10\u27, which display extensive sequence variation that may be useful for sequence-based strain typing. Based on an investigation of 154 C. difficile isolates comprising 75 ribotypes, tandem repeat sequencing demonstrated excellent concordance with widely used PCR ribotyping and equal discriminatory power. Moreover, tandem repeat sequences enabled the reconstruction of the isolates\u27 largely clonal population structure and evolutionary history. Conclusion: We conclude that sequence analysis of the two repetitive loci introduced here may be highly useful for routine typing of C. difficile. Tandem repeat sequence typing resolves phylogenetic diversity to a level equivalent to PCR ribotypes. DNA sequences may be stored in databases accessible over the internet, obviating the need for the exchange of reference strains

Surface-layer (S-layer) of Human and Animal Clostridium Difficile Strains and Their Behaviour in Adherence to Epithelial Cells and Intestinal Colonization

Clostridium difficile is a frequent cause of severe, recurrent post-antibiotic diarrhoea and pseudomembranous colitis. The surface layer (S-layer) is the predominant outer surface component of C. difficile which is involved in pathogen-host interactions critical to pathogenesis. In this study, we characterized the S-layer protein A (SIpA) of animal and human strains belonging to different PCR-ribotypes (PR) and compared the in vitro adherence and in vivo colonization properties of strains showing different SIpA variants. Since each SIpA variant has been recently associated with an S-layer cassette, we were able to deduce the cassette for each of our strains. In this study, an identity of 99-100% was found among the SIpA of isolates belonging to PR 012, 014/020, 045 and 078. One exception was the SIpA of a poultry isolate, PR 014/020, which showed 99% identity with that of strain 0160, another PR 014/020 which contains an S-layer cassette 6. Interestingly, this cassette has also been found in a PR 018 strain, an emerging virulent type currently predominant in Italy. Five other SIpA variants (v014/020a-e) were identified in strains PR 014/020. In vitro adherence assays and in vivo colonization experiments were performed on five PR 014/020 strains: human 1064 (v014/020e), human 4684/08 (v014/020b), human Ill 106 (v078a), poultry P30 (v014/020d) and poultry PB90 (v014/020b) strains. Adhesion assays indicate that C. difficile strains vary in their capacity to adhere to cells in culture and that adhesion seems to be independent of the SIpA variant. Colonization properties were assessed in vivo using a dixenic mouse model of colonization. The kinetics of faecal shedding and caecal colonization were similar when human 4684/08 (v014/020b) strain was compared with human 1064 (v014/020e) and poultry PB90 (v014/02013) strain. In contrast, poultry P30 (v014/020d) strain outcompeted both human 4684/08 (v014/020b) and IT1106 (v078a) strains and its adherence to caeca at day 7 was significantly higher. The peculiar characteristics of C. difficile P30 seem to advantage it in colonizing the intestinal mice niche, increasing its ability to compete and adapt. The results obtained underline the need of an increased attention to the genetic evolution of C. difficile to prevent and limit the consequences of the emergence of increasingly virulent strains

Comparative genomics of Clostridioides difficile toxinotypes identifies module-based toxin gene evolution

Clostridioides difficile is a common cause of nosocomial diarrhoea. Toxins TcdA and TcdB are considered to be the main virulence factors and are encoded by the PaLoc region, while the binary toxin encoded in the CdtLoc region also contributes to pathogenicity. Variant toxinotypes reflect the genetic diversity of a key toxin-encoding 19 kb genetic element (the PaLoc). Here, we present analysis of a comprehensive collection of all known major C. difficile toxinotypes to address the evolutionary relationships of the toxin gene variants, the mechanisms underlying the origin and development of variability in toxin genes and the PaLoc, and the relationship between structure and function in TcdB variants. The structure of both toxin genes is modular, composed of interspersed blocks of sequences corresponding to functional domains and having different evolutionary histories, as shown by the distribution of mutations along the toxin genes and by incongruences of domain phylogenies compared to overall C. difficile cluster organization. In TcdB protein, four mutation patterns could be differentiated, which correlated very well with the type of TcdB cytopathic effect (CPE) on cultured cells. Mapping these mutations to the three-dimensional structure of the TcdB showed that the majority of the variation occurs in surface residues and that point mutation at residue 449 in alpha helix 16 differentiated strains with different types of CPE. In contrast to the PaLoc, phylogenetic trees of the CdtLoc were more consistent with the core genome phylogenies, but there were clues that CdtLoc can also be exchanged between strains