Clostridioides difficile outbreak detection: Evaluation by ribotyping and whole-genome sequencing of a surveillance algorithm based on ward-specific cutoffs.

OBJECTIVE: We evaluated the performance of an early-warning algorithm, based on ward-specific incidence cutoffs for detecting Clostridioides difficile transmission in hospitals. We also sought to determine the frequency of intrahospital Clostridioides difficile transmission in our setting. DESIGN: Diagnostic performance of the algorithm was tested with confirmed transmission events as the comparison criterion. Transmission events were identified by a combination of high-molecular-weight typing, ward history, ribotyping, and whole-genome sequencing (WGS). SETTING: The study was conducted in 2 major and 2 minor secondary-care hospitals with adjacent catchment areas in western Sweden, comprising a total population of ∼480,000 and ∼1,000 hospital beds. PATIENTS: All patients with a positive PCR test for Clostridioides difficile toxin B during 2020 and 2021. METHODS: We conducted culturing and high-molecular-weight typing of all positive clinical samples. Ward history was determined for each patient to find possible epidemiological links between patients with the same type. Transmission events were determined by PCR ribotyping followed by WGS. RESULTS: We identified 4 clusters comprising a total of 10 patients (1.5%) among 673 positive samples that were able to be cultured and then typed by high-molecular-weight typing. The early-warning algorithm performed no better than chance; patient diagnoses were made at wards other than those where the transmission events likely occurred. CONCLUSIONS: In surveillance of potential transmission, it is insufficient to consider only the ward where diagnosis is made, especially in settings with high strain diversity. Transmission within wards occurs sporadically in our setting.

Molecular epidemiology of community- and hospital-associated Clostridioides difficile infections in Jönköping, Sweden, October 2017 - March 2018.

Clostridioides difficile infections (CDIs) in Sweden are mostly hospital-associated (HA) with limited knowledge regarding community-associated (CA) infections. Here, we investigated the molecular epidemiology of clinical isolates of CA-CDI and HA-CDI in a Swedish county. Data and isolates (n = 156) of CDI patients (n = 122) from Jönköping county, October 2017-March 2018, were collected and classified as CA (without previous hospital care or onset ≤2 days after admission or >12 weeks after discharge from hospital) or HA (onset >3 days after hospital admission or within 4 weeks after discharge). Molecular characterization of isolates included PCR ribotyping (n = 156 isolates) and whole genome sequencing with single nucleotide polymorphisms (SNP) analysis (n = 53 isolates). We classified 47 patients (39%) as CA-CDI and 75 (61%) as HA-CDI. Between CA-CDI and HA-CDI patients, we observed no statistically significant differences regarding gender, age, 30-day mortality or recurrence. Ribotype 005 (RR 3.1; 95% CI: 1.79-5.24) and 020 (RR 2.5; 95% CI: 1.31-4.63) were significantly associated with CA-CDI. SNP analysis identified seven clusters (0-2 SNP difference) involving 17/53 isolates of both CA-CDI and HA-CDI. Molecular epidemiology differed between CA-CDI and HA-CDI and WGS analysis suggests transmission of CDI within and between hospitals and communities.

An outbreak investigation of Legionella non-pneumophila Legionnaires' disease in Sweden, April to August 2018: Gardening and use of commercial bagged soil associated with infections.

In early June 2018, an increase in non-travel-related cases of Legionella non-pneumophila Legionnaires' disease (LD) was observed in Sweden and a national outbreak investigation was started. Outbreak cases were defined as notified confirmed or probable cases of L. non-pneumophila LD, with symptom onset after 1 April 2018. From April to August 2018, 41 cases were reported, 30 of whom were identified as L. longbeachae. We conducted a case-control study with 27 cases and 182 matched controls. Results from the case-control study indicated that gardening and handling commercial bagged soil, especially dusty dry soil, were associated with disease. L. longbeachae was isolated in soils from cases' homes or gardens, but joint analysis of soil and human specimens did not identify any genetic clonality. Substantial polyclonality was noted between and within soil samples, which made finding a genetic match between soil and human specimens unlikely. Therefore, whole genome sequencing may be of limited use to confirm a specific soil as a vehicle of transmission for L. longbeachae. Handling soil for residential gardening was associated with disease and the isolation of L. longbeachae in different soils provided further evidence for Legionella non-pneumophila infection from soil.

Impact of ribotype on Clostridioides difficile diagnostics.

This study investigates the performance of diagnostic methods for detection of Clostridioides difficile infection in Sweden, including impact of PCR ribotype on diagnostic performance. Between 2011 and 2016, a total of 17,878 stool samples from 26 laboratories were tested by either well-type enzyme immunoassays (EIAs), membrane bound EIAs, cell cytotoxicity neutralization assay (CTA), or nucleic acid amplification tests (NAATs) and subsequently cultured for C. difficile. Roughly half of the samples (9454/17878) were subjected to diagnostic testing both on the fecal sample and on the 1323 isolated C. difficile strains. All C. difficile isolates were typed by PCR ribotyping, and the isolates were classified as toxigenic or non-toxigenic based on the empirical knowledge of the association between toxin-positivity and ribotype. The overall sensitivity, specificity, and positive and negative predictive values were highest for NAATs and membrane EIAs. Ribotype-specific sensitivity varied greatly between methods and ribotypes. All methods had 100% sensitivity against ribotype 027 and 013. For other types, the sensitivity ranged from 33 to 85% in fecal samples and from 78 to 100% on isolates. For the most prevalent ribotypes (014, 020, and 001), the sensitivity varied between 38 and 100% in the fecal samples, with the lowest sensitivity observed for well-type EIAs and CTA. The large variation in diagnostic sensitivity implies that type distribution significantly affects the outcome when evaluating diagnostic performance. Furthermore, performing comparative studies of diagnostic tests in settings with high prevalence of ribotype 027 will overestimate the general performance of diagnostic tests.

National Surveillance for Clostridioides difficile Infection, Sweden, 2009-2016.

We report results from a national surveillance program for Clostridioides difficile infection (CDI) in Sweden, where CDI incidence decreased by 22% and the proportion of multidrug-resistant isolates decreased by 80% during 2012-2016. Variation in incidence between counties also diminished during this period, which might be attributable to implementation of nucleic acid amplification testing as the primary diagnostic tool for most laboratories. In contrast to other studies, our study did not indicate increased CDI incidence attributable the introduction of nucleic acid amplification testing. Our results also suggest that successful implementation of hygiene measures is the major cause of the observed incidence decrease. Despite substantial reductions in CDI incidence and prevalence of multidrug-resistant isolates, Sweden still has one of the highest CDI incidence levels in Europe. This finding is unexpected and warrants further investigation, given that Sweden has among the lowest levels of antimicrobial drug use.

Cases of human brucellosis in Sweden linked to Middle East and Africa.

BACKGROUND: Human brucellosis cases are still reported each year in Sweden despite eradication of the disease in animals. Epidemiological investigation has never been conducted to trace back the source of human infection in the country. The purpose of the study was to identify the source of infection for 16 human brucellosis cases that occurred in Sweden, during the period 2008-2012. RESULTS: The isolates were identified as Brucella melitensis and MLVA-16 genotyping revealed 14 different genotypes of East Mediterranean and Africa lineages. We also reported one case of laboratory-acquired brucellosis (LAB) that was shown to be epidemiological linked to one of the cases in the current study. CONCLUSIONS: Brucella melitensis was the only species diagnosed, confirming its highest zoonotic potential in the genus Brucella, and MLVA-16 results demonstrated that the cases of brucellosis in Sweden herein investigated, are imported and linked to travel in the Middle East and Africa. Due to its zoonotic concerns, any acute febrile illness linked to recent travel within those regions should be investigated for brucellosis and samples should be processed according to biosafety level 3 regulations.

High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of Clostridium difficile.

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.

Legionella norrlandica sp. nov., isolated from the biopurification systems of wood processing plants.

Fourteen isolates of an unknown species identified as belonging to the genus Legionella by selective growth on BCYE agar were isolated from the biopurification systems of three different wood processing plants. The mip gene sequence of all 14 isolates was identical and a close match alignment revealed 86 % sequence similarity with Legionella pneumophila serogroup 8. The whole genome of isolate LEGN(T) was sequenced, and a phylogenetic tree based on the alignment of 16S rRNA, mip, rpoB, rnpB and the 23S-5S intergenic region clustered LEGN(T) with L. pneumophila ATCC 33152(T). Analysis of virulence factors showed that strain LEGN(T) carries the majority of known L. pneumophila virulence factors. An amoeba infection assay performed to assess the pathogenicity of strain LEGN(T) towards Acanthamoeba castellanii showed that it can establish a replication vacuole in A. castellanii but does not significantly affect replication of amoebae. Taken together, the results confirm that strain LEGN(T) represents a novel species of the genus Legionella, for which the name Legionella norrlandica sp. nov. is proposed. The type strain is LEGN(T) ( = ATCC BAA-2678(T) = CCUG 65936(T)).

Rapid subtyping of Yersinia enterocolitica by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for diagnostics and surveillance.

In this study, an alternative to the current traditional bioserotyping techniques was developed for subtyping Y. enterocolitica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The most common pathogenic bioserotypes could easily be distinguished using only a few bioserotype-specific biomarkers. However, biochemical methods should still be used to distinguish biotype 1A from 1B.

Arabidopsis thaliana TERMINAL FLOWER2 is involved in light-controlled signalling during seedling photomorphogenesis.

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.

TFL2/LHP1 is involved in auxin biosynthesis through positive regulation of YUCCA genes.

TERMINAL FLOWER2 (TFL2) is the plant homologue of metazoan HETEROCHROMATIN PROTEIN1 (HP1) protein family. It is known that, unlike most HP1 proteins, TFL2 does not primarily localize to heterochromatin; instead it functions in regulation of specific genes in euchromatic regions. We show that the tfl2 mutant has a lower rate of auxin biosynthesis, resulting in low levels of auxin. In line with this, tfl2 mutants have lower levels of expression of auxin response genes and retain an auxin response. The reduced rate of auxin biosynthesis in tfl2 is correlated to the down-regulation of specific genes in the tryptophan-dependent auxin biosynthesis pathway, a sub-set of the YUCCA genes. In vivo, TFL2 is targeted to a number of the YUCCA genes in an auxin-dependent fashion revealing a role of TFL2 in auxin regulation, probably as a component of protein complexes affecting transcriptional control.

Cyclotide proteins and precursors from the genus Gloeospermum: filling a blank spot in the cyclotide map of Violaceae.

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.