scholarly journals High levels of toxigenic Clostridioides difficile contamination of hospital environments: a hidden threat in hospital-acquired infections in Kenya

2020 ◽  
Vol 2 (12) ◽  
Author(s):  
Erick Odoyo ◽  
Cecilia Kyanya ◽  
Winnie Mutai ◽  
Lillian Musila
Keyword(s):  
Type Species ◽  
Hospital Environment ◽  
Toxin Gene ◽  
Enrichment Broth ◽  
Content Type ◽  
Link Type ◽  
Clostridioides Difficile ◽  
Hospital Environments ◽  
Genes Encoding ◽  
Binary Toxins

Introduction. The contribution of Clostridioides difficile (formerly Clostridium difficile ) to the burden of hospital-associated infections (HAIs) remains undetermined in many African countries. Aim. This study aimed to identify a sensitive and readily adaptable C. difficile detection assay and to evaluate the C. difficile HAI risk in Kenya. Methodology. Sterile swabs in neutralizing buffer were used to sample equipment or surfaces that patients and clinical staff touched frequently. These swabs were either plated directly on chromogenic agar or cultured in an enrichment broth before plating. The swab suspensions, enrichment broth and plate cultures were screened by quantitative PCR (qPCR) to determine the most efficient detection method. The HAI risk was evaluated by testing the C. difficile -positive samples by qPCR for the A, B and binary toxins. Results. C. difficile was detected on 4/57 (7.0 %) equipment and surfaces by direct culture. The additional enrichment step increased the detection rate 10-fold to 43/57 (75.4 %). In total, 51/57 (89.5 %) environmental samples were positive for C. difficile detected through either culture or qPCR. The genes encoding the primary toxins, tcdA and tcdB, were detected on six surfaces, while the genes encoding the binary toxins, cdtA and cdtB, were detected on 2/57 (3.5 %) and 3/57 (5.3 %) surfaces, respectively. Different C. difficile toxin gene profiles were detected: the tcdA+/tcdB− gene profile on 4/10 (40 %) high-touch surfaces, tcdA−/tcdB+ on 3/10 (30 %) surfaces, tcdA+/tcdB+/cdtA+/cdtB+ on 2/10 (20 %) surfaces and tcdA−/tcdB+/cdtB+ on one high-touch surface. Conclusion. The widespread contamination of hospital environments by toxigenic C. difficile gives a strong indication of the high risk of C. difficile infections (CDIs). The two-step culture process described can easily be adapted for monitoring hospital environment contamination by C. difficile .

scholarly journals Characterization of Ba813 harbouring Bacillus cereus in patients with haematological malignancy and hospital environments at a medical centre in Japan

2020 ◽  
Vol 69 (7) ◽  
pp. 999-1004
Author(s):  
Tetsuji Aoyagi ◽  
Yasuhiro Kishihara ◽  
Miho Ogawa ◽  
Yuki Ito ◽  
Sakie Tanaka ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Type Species ◽  
Haematological Malignancy ◽  
Hospital Environment ◽  
Content Type ◽  
Link Type ◽  
Hospital Environments ◽  
Short Period ◽  
Clade 1

Introduction. Bacillus cereus harbouring Ba813, a specific chromosomal marker of Bacillus anthtacis, is found in patients with severe manifestations and causes nosocomial outbreaks. Aim. We assessed the genetic characteristics and virulence of Ba813(+) B. cereus in a hospital setting. Methodology. Three neutropenic patients with haematological malignancy developed B. cereus bacteraemia within a short period. Fifteen B. cereus were isolated from different sites in a haematology ward. A total of 18 isolates were evaluated for Ba813- and B. anthracis -related virulence, food poisoning-related virulence, genetic diversity, bacteria motility and biofilm formation. Results. Ba813(+) B. cereus was detected in 33 % (1/3) of patients and 66 % (9/15) of the hospital environment. The 18 strains were divided into 2 major clusters (clade 1 and clade 2), and 14 strains were classified into clade 1. All Ba813(+) strains, including four sequence types, were classified into clade 1/the cereus III lineage, which is most closely related to the anthracis lineage. Two strains belonging to clade 1/non-cereus III carried the B. anthracis -associated cap gene, but not Ba813. B. cereus, including Ba813(+) strains, had significantly lower prevalence of enterotoxin genes than clade 2 strains. In clade 1, B. cereus , Ba813(+) strains showed significantly higher swimming motility and biofilm formation ability than Ba813(−) strains. Conclusion. Ba813(+) B. cereus , which are genetically closely related to B. anthracis , were abundant in a haematological ward. Ba813(+) B. cereus with high motility and biofilm formation abilities may spread easily in hospital environments, and could become a hospital-acquired infection.

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

2020 ◽  
Vol 6 (10) ◽  
Author(s):  
Sandra Janezic ◽  
Kate Dingle ◽  
Joseph Alvin ◽  
Tomaž Accetto ◽  
Xavier Didelot ◽  
...  
Keyword(s):  
Type Species ◽  
Gene Evolution ◽  
Alpha Helix ◽  
Dimensional Structure ◽  
Binary Toxin ◽  
Toxin Gene ◽  
Content Type ◽  
Link Type ◽  
Toxin Genes ◽  
Clostridioides Difficile

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.

scholarly journals Transcriptomic analysis of cyanobacterial alkane overproduction reveals stress-related genes and inhibitors of lipid droplet formation

2020 ◽  
Vol 6 (10) ◽  
Author(s):  
Daisy B. Arias ◽  
Kevin A. Gomez Pinto ◽  
Kerry K. Cooper ◽  
Michael L. Summers
Keyword(s):  
Type Species ◽  
Transcriptomic Analysis ◽  
Lag Phase ◽  
Content Type ◽  
Link Type ◽  
Production Platform ◽  
Genes Encoding ◽  
Transcriptional Changes ◽  
Stress Related Genes ◽  
Upregulated Genes

The cyanobacterium Nostoc punctiforme can form lipid droplets (LDs), internal inclusions containing triacylglycerols, carotenoids and alkanes. LDs are enriched for a 17 carbon-long alkane in N. punctiforme , and it has been shown that the overexpression of the aar and ado genes results in increased LD and alkane production. To identify transcriptional adaptations associated with increased alkane production, we performed comparative transcriptomic analysis of an alkane overproduction strain. RNA-seq data identified a large number of highly upregulated genes in the overproduction strain, including genes potentially involved in rRNA processing, mycosporine-glycine production and synthesis of non-ribosomal peptides, including nostopeptolide A. Other genes encoding helical carotenoid proteins, stress-induced proteins and those for microviridin synthesis were also upregulated. Construction of N. punctiforme strains with several upregulated genes or operons on multi-copy plasmids resulted in reduced alkane accumulation, indicating possible negative regulators of alkane production. A strain containing four genes for microviridin biosynthesis completely lost the ability to synthesize LDs. This strain exhibited wild-type growth and lag phase recovery under standard conditions, and slightly faster growth under high light. The transcriptional changes associated with increased alkane production identified in this work will provide the basis for future experiments designed to use cyanobacteria as a production platform for biofuel or high-value hydrophobic products.

scholarly journals Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori

Microbiology ◽  
2020 ◽  
Vol 166 (8) ◽  
pp. 785-793
Author(s):  
Shou Miura ◽  
Yukino Tamamura ◽  
Mariko Takayasu ◽  
Miwa Sasaki ◽  
Natsuko Nishimura ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Type Species ◽  
Phage Type ◽  
Sos Response ◽  
Toxin Gene ◽  
Efficient Production ◽  
Prophage Induction ◽  
Content Type ◽  
Link Type ◽  
Quinolone Antibiotics

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori , which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori . Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

scholarly journals A global to local genomics analysis of Clostridioides difficile ST1/RT027 identifies cryptic transmission events in a northern Arizona healthcare network

2019 ◽  
Vol 5 (7) ◽  
Author(s):  
Charles H. D. Williamson ◽  
Nathan E. Stone ◽  
Amalee E. Nunnally ◽  
Heidie M. Hornstra ◽  
David M. Wagner ◽  
...  
Keyword(s):  
Type Species ◽  
Sequence Type ◽  
Infection Prevention And Control ◽  
Polymorphism Analysis ◽  
Whole Genome ◽  
Content Type ◽  
Northern Arizona ◽  
Healthcare Network ◽  
Link Type ◽  
Clostridioides Difficile

Clostridioides difficile is a ubiquitous, diarrhoeagenic pathogen often associated with healthcare-acquired infections that can cause a range of symptoms from mild, self-limiting disease to toxic megacolon and death. Since the early 2000s, a large proportion of C. difficile cases have been attributed to the ribotype 027 (RT027) lineage, which is associated with sequence type 1 (ST1) in the C. difficile multilocus sequence typing scheme. The spread of ST1 has been attributed, in part, to resistance to fluoroquinolones used to treat unrelated infections, which creates conditions ideal for C. difficile colonization and proliferation. In this study, we analysed 27 isolates from a healthcare network in northern Arizona, USA, and 1352 publicly available ST1 genomes to place locally sampled isolates into a global context. Whole genome, single nucleotide polymorphism analysis demonstrated that at least six separate introductions of ST1 were observed in healthcare facilities in northern Arizona over an 18-month sampling period. A reconstruction of transmission networks identified potential nosocomial transmission of isolates, which were only identified via whole genome sequence analysis. Antibiotic resistance heterogeneity was observed among ST1 genomes, including variability in resistance profiles among locally sampled ST1 isolates. To investigate why ST1 genomes are so common globally and in northern Arizona, we compared all high-quality C. difficile genomes and identified that ST1 genomes have gained and lost a number of genomic regions compared to all other C. difficile genomes; analyses of other toxigenic C. difficile sequence types demonstrate that this loss may be anomalous and could be related to niche specialization. These results suggest that a combination of antimicrobial resistance and gain and loss of specific genes may explain the prominent association of this sequence type with C. difficile infection cases worldwide. The degree of genetic variability in ST1 suggests that classifying all ST1 genomes into a quinolone-resistant hypervirulent clone category may not be appropriate. Whole genome sequencing of clinical C. difficile isolates provides a high-resolution surveillance strategy for monitoring persistence and transmission of C. difficile and for assessing the performance of infection prevention and control strategies.

scholarly journals Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile

2020 ◽  
Vol 2 (7) ◽  
Author(s):  
Yuta Okada ◽  
Shu Okugawa ◽  
Mahoko Ikeda ◽  
Tatsuya Kobayashi ◽  
Ryoichi Saito ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
In Silico ◽  
Type Species ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Accessory Gene ◽  
Content Type ◽  
Link Type ◽  
Accessory Gene Regulator ◽  
Clostridioides Difficile

Quorum sensing is known to regulate bacterial virulence, and the accessory gene regulator (agr) loci is one of the genetic loci responsible for its regulation. Recent reports examining Clostridioides difficile show that two agr loci, agr1 and agr2, regulate toxin production, but the diversity of agr loci and their epidemiology is unknown. In our study, in silico analysis was performed to research genetic diversity of agr, and C. difficile isolates from clinical samples underwent multilocus sequence typing (MLST) and PCR analysis of agr loci. To reveal the distribution of agr among different strains, phylogenetic analysis was also performed. In our in silico analysis, two different subtypes, named agr2R and agr2M, were found in agr2, which were previously reported. PCR analysis of 133 C . difficile isolates showed that 131 strains had agr1, 61 strains had agr2R, and 26 strains had agr2M; agr2R was mainly found in clade 1 or clade 2 organisms, whereas agr2M was only found in clade 4. With rare exception, agr1-negative sequence types (STs) belonged to clade C-Ⅰ and C-Ⅲ, and one clade 4 strain had agr2R. Our study revealed subtypes of agr2 not previously recognized, and the distribution of several agr loci in C. difficile . These findings provide a foundation for further functional and clinical research of the agr loci.

scholarly journals Clostridioides difficile strain-dependent and strain-independent adaptations to a microaerobic environment

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Andy Weiss ◽  
Christopher A. Lopez ◽  
William N. Beavers ◽  
Jhoana Rodriguez ◽  
Eric P. Skaar
Keyword(s):  
Type Species ◽  
Clinical Manifestations ◽  
Transcriptional Response ◽  
Low Oxygen ◽  
Independent Manner ◽  
Content Type ◽  
Link Type ◽  
Clostridioides Difficile ◽  
Conserved Genes ◽  
Oxygen Concentrations

Clostridioides difficile (formerly Clostridium difficile ) colonizes the gastrointestinal tract following disruption of the microbiota and can initiate a spectrum of clinical manifestations ranging from asymptomatic to life-threatening colitis. Following antibiotic treatment, luminal oxygen concentrations increase, exposing gut microbes to potentially toxic reactive oxygen species. Though typically regarded as a strict anaerobe, C. difficile can grow at low oxygen concentrations. How this bacterium adapts to a microaerobic environment and whether those responses to oxygen are conserved amongst strains is not entirely understood. Here, two C. difficile strains (630 and CD196) were cultured in 1.5% oxygen and the transcriptional response to long-term oxygen exposure was evaluated via RNA-sequencing. During growth in a microaerobic environment, several genes predicted to protect against oxidative stress were upregulated, including those for rubrerythrins and rubredoxins. Transcription of genes involved in metal homeostasis was also positively correlated with increased oxygen levels and these genes were amongst the most differentially transcribed. To directly compare the transcriptional landscape between C. difficile strains, a ‘consensus-genome’ was generated. On the basis of the identified conserved genes, basal transcriptional differences as well as variations in the response to oxygen were evaluated. While several responses were similar between the strains, there were significant differences in the abundance of transcripts involved in amino acid and carbohydrate metabolism. Furthermore, intracellular metal concentrations significantly varied both in an oxygen-dependent and oxygen-independent manner. Overall, these results indicate that C. difficile adapts to grow in a low oxygen environment through transcriptional changes, though the specific strategy employed varies between strains.

scholarly journals In vitro antivirulence activity of baicalin against Clostridioides difficile

2020 ◽  
Vol 69 (4) ◽  
pp. 631-639
Author(s):  
Abraham Joseph Pellissery ◽  
Poonam Gopika Vinayamohan ◽  
Kumar Venkitanarayanan
Keyword(s):  
Spore Germination ◽  
Type Species ◽  
Brain Heart Infusion ◽  
Toxin Production ◽  
Strictly Anaerobic ◽  
Content Type ◽  
Link Type ◽  
Clostridioides Difficile ◽  
Spore Outgrowth

Introduction. Clostridioides difficile is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize C. difficile pathogenicity and disease outcome in affected individuals. Aim. This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in C. difficile in vitro. Methodology. Hypervirulent C. difficile isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining C. difficile toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, C. difficile spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on C. difficile toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR. Results. The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, C. difficile genes critical for pathogenesis were significantly down-regulated in the presence of baicalin. Conclusion. Our results suggest that baicalin could potentially be used to control C. difficile , and warrant future studies in vivo.

scholarly journals Niche-specific adaptation of Lactobacillus helveticus strains isolated from malt whisky and dairy fermentations

2021 ◽  
Vol 7 (4) ◽  
Author(s):  
Yoshihiko Kido ◽  
Shintaro Maeno ◽  
Hiroki Tanno ◽  
Yuko Kichise ◽  
Yuh Shiwa ◽  
...  
Keyword(s):  
Type Species ◽  
Stop Codon ◽  
Lactobacillus Helveticus ◽  
Content Type ◽  
Link Type ◽  
Milk Fermentation ◽  
Genes Encoding ◽  
Metabolic Properties ◽  
Malt Whisky

Lactobacillus helveticus is a well characterized lactobacillus for dairy fermentations that is also found in malt whisky fermentations. The two environments contain considerable differences related to microbial growth, including the presence of different growth inhibitors and nutrients. The present study characterized L. helveticus strains originating from dairy fermentations (called milk strains hereafter) and malt whisky fermentations (called whisky strains hereafter) by in vitro phenotypic tests and comparative genomics. The whisky strains can tolerate ethanol more than the milk strains, whereas the milk strains can tolerate lysozyme and lactoferrin more than the whisky strains. Several plant-origin carbohydrates, including cellobiose, maltose, sucrose, fructooligosaccharide and salicin, were generally metabolized only by the whisky strains, whereas milk-derived carbohydrates, i.e. lactose and galactose, were metabolized only by the milk strains. Milk fermentation properties also distinguished the two groups. The general genomic characteristics, including genomic size, number of coding sequences and average nucleotide identity values, differentiated the two groups. The observed differences in carbohydrate metabolic properties between the two groups correlated with the presence of intact specific enzymes in glycoside hydrolase (GH) families GH1, GH4, GH13, GH32 and GH65. Several GHs in the milk strains were inactive due to the presence of stop codon(s) in genes encoding the GHs, and the inactivation patterns of the genes encoding specific enzymes assigned to GH1 in the milk strains suggested a possible diversification manner of L. helveticus strains. The present study has demonstrated how L. helveticus strains have adapted to their habitats.

scholarly journals Cryptic prophages within a Streptococcus pyogenes genotype emm4 lineage

2020 ◽  
Author(s):  
Alex Remmington ◽  
Samuel Haywood ◽  
Julia Edgar ◽  
Luke R. Green ◽  
Thushan de Silva ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Type Species ◽  
Gene Loss ◽  
Content Type ◽  
Regulatory Modules ◽  
Link Type ◽  
Genes Encoding ◽  
Variable Extent ◽  
The Usa ◽  
The Uk

The major human pathogen Streptococcus pyogenes shares an intimate evolutionary history with mobile genetic elements, which in many cases carry genes encoding bacterial virulence factors. During recent whole-genome sequencing of a longitudinal sample of S. pyogenes isolates in England, we identified a lineage within emm4 that clustered with the reference genome MEW427. Like MEW427, this lineage was characterized by substantial gene loss within all three prophage regions, compared to MGAS10750 and isolates outside of the MEW427-like lineage. Gene loss primarily affected lysogeny, replicative and regulatory modules, and to a lesser and more variable extent, structural genes. Importantly, prophage-encoded superantigen and DNase genes were retained in all isolates. In isolates where the prophage elements were complete, like MGAS10750, they could be induced experimentally, but not in MEW427-like isolates with degraded prophages. We also found gene loss within the chromosomal island SpyCIM4 of MEW427-like isolates, although surprisingly, the SpyCIM4 element could not be experimentally induced in either MGAS10750-like or MEW427-like isolates. This did not, however, appear to abolish expression of the mismatch repair operon, within which this element resides. The inclusion of further emm4 genomes in our analyses ratified our observations and revealed an international emm4 lineage characterized by prophage degradation. Intriguingly, the USA population of emm4 S. pyogenes appeared to constitute predominantly MEW427-like isolates, whereas the UK population comprised both MEW427-like and MGAS10750-like isolates. The degraded and cryptic nature of these elements may have important phenotypic and fitness ramifications for emm4 S. pyogenes , and the geographical distribution of this lineage raises interesting questions on the population dynamics of the genotype.

Sign in / Sign up

Export Citation Format

Share Document