This study aimed to evaluate the fecal shedding of C. difficile in calves on farms in Sao Paulo State, Brazil.
Fecal samples (n=300) were collected from diarrheic (n=78) and nondiarrheic (n=222) ...calves less than 60 days of age from 20 farms. Fecal samples were inoculated into enrichment broth supplemented with taurocholate and cultured under anaerobic conditions. Colonies suspected to be C. difficile were harvested for DNA extraction and then multiplex PCR for the detection of genes encoding toxins A and B and binary toxins. All toxigenic isolates were ribotyped and tested for antimicrobial susceptibility, and five selected strains were subjected to whole-genome sequencing to determine their sequence type.
C. difficile was isolated from 29.3% (88/300) of the samples. All toxigenic isolates (17/88, 19.3%) were classified as ribotypes RT046 (13/17 -79.47%, A
B
CDT
) and RT126 (4/17=20.53%, A
B
CDT
). The sequenced strains from RT046 were classified as ST35 (Clade 1), while those from RT126 were classified as ST11 (Clade 5). No associations between the epidemiological factors in any of the groups and C. difficile isolation were observed. Most of the toxigenic isolates (16/17=94.41%) were classified as multidrug-resistant. Calves can be an important source of toxigenic C. difficile strains, including multidrug-resistant isolates from ribotypes commonly observed in humans.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile ...typing but lacks the discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N = 630; 100 unique ribotypes RTs), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere and EnteroBase), whole-genome MLST (wgMLST) (EnteroBase), and single-nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (more than six allele differences) was observed in 82 of 100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known to have low intra-RT allele differences) showed no distinction between outbreak and nonoutbreak strains in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize, and offers higher discrimination. However, adjusted cutoff thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold of three alleles to identify outbreaks.
Whether cross-infection of respiratory pathogens between patients with non-cystic fibrosis bronchiectasis occurs is debated. Investigation with traditional microbiological culture risks simplifying ...the lung microbiome. We demonstrate the use of culture-independent Multilocus sequence typing to screen for Haemophilus influenzae strain types in a cohort of twenty-eight patients with non-cystic fibrosis bronchiectasis.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring
of emerging serovars is essential to adopt appropriate control strategies. Multi-Locus Sequence
Typing (MLST) was ...reported to be less expensive compared to other cumbersome methods like
whole genome sequencing. The present study was conducted to obtain isolates of Leptospira from
infected animals and rats and for the identification of serovars using MLST. A total of 205 blood
samples (dog, cat, cattle, goat), 43 urine samples (dog, cattle) and post-mortem kidney samples
from various animals such as dog (n=12), cattle (n=2) and rat (n=25) were collected and subjected
to polymerase chain reaction (PCR) using G1/G2 primers to identify the pathogenic Leptospira.
Fifteen samples were found to be positive, these samples when inoculated in the Ellinghausen-
McCullough-Johnson-Harris (EMJH) semi-solid medium to obtain ten isolates. These ten isolates
were further subjected to secY, icdA and GyraseB PCR and sequenced. The obtained sequences
were analysed using BLAST and were fed into specified MLST database of Leptospira scheme-3,
the allelic profile and sequence type were generated. The MLST results obtained in the study
indicated that the isolates S24 and S33 belonged to serovar Canicola, S40 and 47 were Sejroe
and S19, S27, S55, S69 and S71 were Bataviae, Autumnalis, Pomona, Icterohaemorraghiae and
Australis, respectively. It was concluded that MLST is a convenient method for identifying leptospiral
serovars.
Enterococcus faecium is emerging as an important cause of multidrug resistance and hospital acquired infections, special attention being paid to the vancomycin resistant species. Therefore, the ...characterization of pathogenic strains/isolates plays an important role in the epidemiology of infectious diseases. The enterococcal rate was determined from wastewaters in Cluj-Napoca area. As presence of E. faecium was detected, a number of isolates from wastewater, birds and humans were epidemiologically analyzed according to the MLST website. Comparisons were performed against a collection of available isolates, with multiple origins, contained in the MLST database. Out of the Enterococcus isolates collected from wastewater, 11 were identified as E. faecalis (40.74%); 8 as E. casseliflavus (29.62%); 5 as E. faecium (18.50%); 2 as E. gallinarum (7.40%) and one isolate as E. durans. Based on the MLST data and using the eBURST algorithm, the isolates of E. faecium sampled from Romania were split in three groups: one group comprised isolates from human hosts and wastewater (Cj316, 106/6, Cj197, Cj22, 129/6, Cj117, Cj24, 284/7, and 43/7), while the second (G9, G10-2, G7, G3-2, and G9-1) and the third group (G8, G6, and 40/7) originated from bird hosts. The rest of the isolates were not joined in a particular group, assuming the lack of a phylogenetic bond between these isolates. The obtained data suggested the existence of at least two phylogenetic lines of E. faecium in Romania: a line that had mainly human host prevalence, while in the other line the animal hosts dominated.
Full text
Available for:
IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, UL, UM, UPUK
As WGS is increasingly used by food industry to characterize pathogen isolates, users are challenged by the variety of analysis approaches available, ranging from methods that require extensive ...bioinformatics expertise to commercial software packages. This study aimed to assess the impact of analysis pipelines (i.e., different hqSNP pipelines, a cg/wgMLST pipeline) and the reference genome selection on analysis results (i.e., hqSNP and allelic differences as well as tree topologies) and conclusion drawn. For these comparisons, whole genome sequences were obtained for 40
isolates collected over 18 years from a cold-smoked salmon facility and 2 other isolates obtained from different facilities as part of academic research activities; WGS data were analyzed with three hqSNP pipelines and two MLST pipelines. After initial clustering using a k-mer based approach, hqSNP pipelines were run using two types of reference genomes: (i) closely related closed genomes ("closed references") and (ii) high-quality
assemblies of the dataset isolates ("draft references"). All hqSNP pipelines identified similar hqSNP difference ranges among isolates in a given cluster; use of different reference genomes showed minimal impacts on hqSNP differences identified between isolate pairs. Allelic differences obtained by wgMLST showed similar ranges as hqSNP differences among isolates in a given cluster; cgMLST consistently showed fewer differences than wgMLST. However, phylogenetic trees and dendrograms, obtained based on hqSNP and cg/wgMLST data, did show some incongruences, typically linked to clades supported by low bootstrap values in the trees. When a hqSNP cutoff was used to classify isolates as "related" or "unrelated," use of different pipelines yielded a considerable number of discordances; this finding supports that cut-off values are valuable to provide a starting point for an investigation, but supporting and epidemiological evidence should be used to interpret WGS data. Overall, our data suggest that cgMLST-based data analyses provide for appropriate subtype differentiation and can be used without the need for preliminary data analyses (e.g., k-mer based clustering) or external closed reference genomes, simplifying data analyses needs. hqSNP or wgMLST analyses can be performed on the isolate clusters identified by cgMLST to increase the precision on determining the genomic similarity between isolates.
(KP) is a common nosocomial pathogen. Capsules are an important component of KP's virulence, among which the K1, K2, K5, K20, K54, and K57 serotypes are predominant and exhibit varying degrees of ...virulence.
The capsule and virulence genes of 150 carbapenem-resistant
(CRKP) and 213 carbapenem-sensitive
(CSKP) isolates were examined by polymerase chain reaction (PCR). The isolates were tested for hypermucoviscosity by string tests. Phylogenetic relationships between KP isolates were analyzed using multilocus sequence typing (MLST) and a
infection model confirmed the differences in virulence.
A total of 111 of 363 isolates of KP were detected, the highest detected serotypes were K1, K5, and K2, and CSKP was detected more frequently than CRKP. There was a greater prevalence of K1 and K2 serotypes in CSKP, while in CRKP, K5 serotypes were more prevalent. K1 isolates had the highest detection rates for hypermucoviscosity
(hmKP) and hypervirulent
(hvKP), and carried the most virulence genes. K54 isolates had the lowest detection rate of hmKP while K5 isolates had the lowest detection rate of hvKP and carried the fewest virulence genes. MLST results for serotypes K1, K20, and K57 showed significant homogeneity, while those for serotypes K2, K5, and K54 showed diversity. The
infection model showed that the K1 serotype was the most virulent and the K54 serotype was the weakest.
CSKP isolates were detected more frequently than CRKP isolates for capsular serotype detection. K1 isolates had the most virulence gene and strongest virulence, K5 isolates carried the fewest virulence genes, and K54 isolates had the weakest virulence. Furthermore, significant homogeneity was observed among K1, K20, and K57 isolates.
Multilocus sequence typing (MLST) has become a useful tool for studying the genetic diversity of important public health pathogens, such as
(
). Four MLST schemes have been proposed for
(data ...available from Chlamydiales MLST databases). However, the lack of a sole standardized scheme represents the greatest limitation regarding typing this species. This study was thus aimed at evaluating the usefulness of the four MLST schemes available for
, describing each molecular marker's pattern and its contribution toward a description of intra-specific genetic diversity and population structure. The markers for each scheme, showed a variable power of dicrimination, exhibiting in some cases over estimation in the determination of Sequence Types (STs). However, individual analysis of each locus's typing efficiency and discrimination power led to identifying 8 markers as having a suitable pattern for intra-specific typing. analyzing the 8 candidate markers gave a combination of 3 of these loci as an optimal scheme for identifying a large amount of STs, maximizing discrimination power whilst maintaining suitable typing efficiency. One scheme was compared against core genome phylogenies, finding a higher typing resolution through the last approach. These results confirm once again that although complete genome data, in particular from core genome MLST (cgMLST) allow a high resolution clustering for
isolates. There are combinations of molecular markers that could generate equivalent results, with the advantage of representing an easy implementation strategy and lower costs leading to contribute to the monitoring and molecular epidemiology of
.
PDF
