Sequence Typing Research Articles

Characterization of Streptococcus pyogenes Strains from Tonsillopharyngitis and Scarlet Fever Resurgence, 2023—FIRST Detection of M1UK in Bulgaria

Recently a resurgence of Streptococcus pyogenes infections has arisen, with concerns around the highly virulent M1UK lineage. Our aim was to characterize S. pyogenes, the immune responses it causes, and to determine the presence of the M1UK lineage in Sofia, Bulgaria. In our study, the infections were confirmed by culture testing or rapid antigen test. Identification was performed by MALDI-TOF and was followed up by antibiotic susceptibility testing (EUCAST). Virulence factors were identified using multiplex PCR and whole genome sequencing (WGS). Immune responses were measured through detection of serum complement levels, lymphocyte subsets, and cytokine profiling. Out of 82 children, 38 had scarlet fever and the rest had streptococcal pharyngitis. Strains were susceptible to penicillin (β-lactams), macrolides, clindamycin, tetracyclines, co-trimoxazole, fluoroquinolones, and linezolid. Superantigen profiles were identified: SpeA + SpeJ (45%), SpeC, and SpeI + SpeH (27.5% each). A novel Multilocus sequence typing (MLST) haplotype in the mutS gene (d90b) was found in four strains. The M1UK lineage was detected for the first time in Bulgaria. We observed an increase in complement fractions C3 and C4 and a decrease in T lymphocytes. A significant increase in the levels of IFN-γ, IL-6, and IL-10 with corresponding reduction in IL-17A were revealed. In conclusion, the studied S. pyogenes strains were characterized by their susceptibility to antibiotics and the predominance of SpeA superantigen; for the first time in Bulgaria the presence of M1UK and a novel SNP variation in the mutS gene (d90b) were found. A mixed pattern of pro- and anti-inflammatory immune responses in patients was observed.

Genomic characterization of pathotype diversity and drug resistance among generic Escherichia coli isolated from broiler chickens in Canada

Escherichia coli is a Gram-negative bacterium that is ubiquitous in animals and humans, with some strains capable of causing disease. The aim of this study was to perform a comparative genomic analysis of 2,732 generic E. coli isolates that were recovered from poultry samples collected from six regions in Canada as part of the National Microbiological Baseline study in Broiler Chicken. Isolates were subjected to whole genome sequencing and a subset (1,122/2,732) were tested for phenotypic resistance to fifteen antimicrobials. These E. coli isolates were highly diverse, representing 376 serotypes, 236 Sequence Types and twenty-one pathotypes, of which 19 were hybrid pathotypes. A high concordance (>85%) between resistance phenotype and the presence of antimicrobial resistance genes and point mutations (resistance determinants) was observed for 13/15 antimicrobials. Over 95% of the β-lactam, fluoroquinolone, and phenicol resistance genes were predicted to be plasmid-borne. The number of resistance determinants per genome was highest in Quebec, while resistance genes associated with β-lactam resistance were more frequently detected in isolates from British Columbia. Generic E. coli in Canadian poultry are highly diverse, can carry pathotype-associated virulence factors and resistance determinants of clinical significance with a risk of emerging into pathogenic strains.

Global insights into the genome dynamics of Clostridioides difficile associated with antimicrobial resistance, virulence, and genomic adaptations among clonal lineages

BackgroundClostridioides difficile is a significant cause of healthcare-associated infections, with rising antimicrobial resistance complicating treatment. This study offers a genomic analysis of C. difficile, focusing on sequence types (STs), global distribution, antibiotic resistance genes, and virulence factors in its chromosomal and plasmid DNA.MethodsA total of 19,711 C. difficile genomes were retrieved from GenBank. Prokka was used for genome annotation, and multi-locus sequence typing (MLST) identified STs. Pan-genome analysis with Roary identified core and accessory genes. Antibiotic resistance genes, virulence factors, and toxins were detected using the CARD and VFDB databases, and the ABRicate software. Statistical analyses and visualizations were performed in R.ResultsAmong 366 identified STs, ST1 (1,326 isolates), ST2 (1,141), ST11 (893), and ST42 (763) were predominant. Trends of genome streamlining included reductions in chromosomal length, gene count, protein-coding genes, and pseudogenes. Common antibiotic resistance genes—cdeA (99.46%), cplR (99.63%), and nimB (99.67%)—were nearly ubiquitous. Rare resistance genes like blaCTX-M-2, cfxA3, and blaZ appeared in only 0.005% of genomes. Vancomycin susceptibility-reducing vanG cluster genes were detected at low frequencies. Virulence factors showed variability, with highly prevalent genes such as zmp1 (99.62%), groEL (99.60%), and rpoB/rpoB2 (99.60%). Moderately distributed genes included cwp66 (54.61%) and slpA (79.02%). Toxin genes tcdE (91.26%), tcdC (89.67%), and tcdB (89.06%) were widespread, while binary toxin genes cdtA (26.19%) and cdtB (26.26%) were less common. Toxin gene prevalence, particularly tcdA and tcdB, showed a gradual decline over time, with sharper reductions for cdtA and cdtB. Gene presence patterns (GPP-1) for resistance, virulence, and toxin genes were primarily linked to ST2, ST42, and ST8.ConclusionThis study highlights C. difficile’s adaptability and genetic diversity. The decline in toxin genes reflects fewer toxigenic isolates, but the bacterium’s increasing preserved resistance factors and virulence genes enable its rapid evolution. ST2, ST42, and ST8 dominate globally, emphasizing the need for ongoing monitoring.

Pathogenic potential of ornithogenic <i>Escherichia coli</i> strains detected in the Earth's polar regions

Introduction. Pathogenic strains of Escherichia coli are an important object of surveillance within the One Health concept in the wild, agriculture and human society. Migratory bird colonies and high latitude avian colonies may be points of active intraspecies and interspecies contact between different animal species, accompanied by the spread of pathogens. At the same time, the phylogeography of E. coli in relation to the presence of natural foci of colibacillosis in polar regions remains virtually unstudied. The aim of this study was to assess the pathogenic potential of E. coli strains from the polar regions of the Earth, based on the analysis of the genomes of these bacteria from typical ornithogenic ecosystems of the Arctic and Antarctic. Materials and methods. The study used collections of E. coli isolated from ornithogenic biological material during expeditions to high latitude areas of the Arctic (archipelagos of Novaya Zemlya, Franz Josef Land, Svalbard) and Antarctic (Haswell Archipelago). 16 cultures associated with avian E. coli (12 polar and 4 temperate strains) were selected for genome-wide sequencing using BGI technology. The annotation of the genomes focused on the identification of genes for pathogenicity factors and antimicrobial resistance, as well as the identification of strains belonging to individual genetic lineages using the cgMLST method. Results. The annotation of the genomes allowed their assignment to different sequence types in the multilocus sequencing typing and genome-wide sequencing typing schemes. The analysis of the geographical distribution of the sequence types of polar E. coli strains determined by the cgMLST method showed their global representation in geographically distant regions of the planet. For example, cgST 133718 was observed in Antarctica (strain 17_1myr) and in the UK, and sequence 11903, to which strain 32-1 from the northernmost point of Novaya Zemlya belonged, was previously identified in the USA. All strains studied were characterized by the presence of extensive virulence. Among the pathogenicity factors identified were haemolysins A, E, F, siderophores, including the yersiniabactin gene cluster, a number of adhesion, colonization and invasion factors, as well as the thermostable enterotoxin EAST-1 and genes that characterize enteroaggregative strains of E. coli (the virulence regulator gene eilA and enteroaggregative protein (air)). One of the Arctic strains (33-1) had determinants of antibiotic resistance, in particular the extended-spectrum beta-lactamase gene TEM-1b and the Tn1721 transposon, including tetracycline resistance genes (tetA-TetR), were detected in its genome. Conclusion. The results of the study indicate the circulation of E. coli strains with strong pathogenic potential in high-latitude Arctic and Antarctic ornithogenic ecosystems. The analysis of genomic data indicates the presence of geographically widespread genetic lineages in these regions, which justifies the importance of monitoring epidemic clones of E. coli, along with monitoring for other pathogens, in bird colonies in high-latitude areas.

High Antimicrobial Susceptibility of Cloacal Enterococci and Escherichia coli from Free-Living Dalmatian and Great White Pelicans with Detection of Cefotaximase CTX-M-15 Producing Escherichia coli ST69

Background/Objectives: In 2022, an outbreak of H5N1 highly pathogenic avian influenza (HPAI) killed 60% of the largest breeding colony of Dalmatian pelicans (DPs) in the world at Mikri Prespa Lake (Greece), prompting a multidisciplinary study on HPAI and other pathogens. This study determines the antimicrobial resistance rates of cloacal enterococci and Escherichia coli in DPs. Methods: Fifty-two blood and cloacal swab samples were collected from 31 nestlings (20 DP/11 great white pelicans) hatched after the H5N1 outbreak at the Prespa colony and 21 subadult/adult DPs captured at a spring migration stopover. The swabs were inoculated in non-selective and chromogenic-selective media. Identification was performed using MALDI-TOF, and antimicrobial susceptibility was tested. The genetic content was characterized using PCR and sequencing, and the clonality of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates was characterized using Multilocus Sequence Typing. Results: Twenty-eight non-repetitive E. coli and 45 enterococci isolates were recovered in non-selective media; most of them were susceptible to all antibiotics tested (85.7% E. coli/91.1% enterococci). Three of the fifty-two samples (6%, all adults) contained ESBL-E. coli isolates (detected in chromogenic ESBL plates), all carrying the blaCTX-M-15 gene and belonging to the lineage ST69. Conclusions: Despite the susceptibility of most fecal E. coli and enterococci isolates to all antibiotics tested, the finding that E. coli of lineage ST69 carry blaCTX-M-15 is of concern. This high-risk clone needs further investigation to elucidate its primary sources and address the growing threat of antimicrobial resistance from an integrated “One Health” perspective. Furthermore, it is imperative to study the potential impacts of ESBL-E. coli on the endangered DP further.

Genetic diversity atlas of Brucella melitensis strains from Sichuan Province, China

Human brucellosis is a re-emerging disease in Sichuan Province, China. In this study, bacteriology, conventional bio-typing, multi-locus sequence typing (MLST), and multiple locus variable-number tandem repeat analysis (MLVA) were applied to preliminarily characterize the strains in terms of genetic diversity and epidemiological links. A total of 101 Brucella strains were isolated from 16 cities (autonomous prefectures) from 2014 to 2021, and all of the strains were identified as Brucella melitensis bv. 3, suggesting that surveillance should focus on ruminants. MLST analysis identified four STs, namely, ST8 (n = 93), ST39 (n = 6), ST101 (n = 1), and ST118 (n = 1). The latter were new STs, indicating that strains displayed high population diversity. Six MLVA-8, namely, 42, 43, 45, 63, 83, and 114, and eight MLVA-11, namely, 111, 115, 116, 125, 180, 291, 298, and 342, genotypes were identified, demonstrating that all of the strains were from the Eastern Mediterranean lineage, and these strains exhibited a high genotype diversity. MLVA-16 analysis revealed that there was a co-existing transmission pattern, where sporadic cases and multiple outbreak events had a common origin. The dominant STs and MLVA genotypes of strains were epidemic in Northern, China, and 36 MLVA-16 genotypes were shared among strains (n = 51, 50.4%, 51/101) from Sichuan and strains from 22 other provinces. The findings imply that infected animals were introduced from outside the province. The surveillance and control of the disease have become public health challenges. Animal quarantines should be strengthened to prevent the spread of B. melitensis species among adjacent regions.

Virulence profile of carbapenem-resistant Klebsiella pneumoniae strains by an in vivo model of Galleria mellonella.

Klebsiella pneumoniae is a significant healthcare-associated pathogen, notable for its diverse virulence and antibiotic resistance profiles. This study aimed to characterize the genotypic and phenotypic diversity of K. pneumoniae isolates and evaluate their virulence using the Galleria mellonella model. Biomass production, metabolic activity, capsule formation, and siderophore production were assessed in 27 K. pneumoniae isolates from hospital-associated infections. Lethality curves were generated using the G. mellonella model, with survival monitored hourly from 16 to 48 hours. The most common sequence types (ST) identified were the high-risk clones ST307 (N = 10), ST512 (N = 8), ST101 (N = 7), and ST661 (N = 2). These STs were associated with distinct K-locus, including KL102, KL107, KL17, and KL39. Most isolates belonged to the O2afg locus (N = 18), with the K. pneumoniae carbapenemase genotype detected in 96.3% of strains. None of the isolates were classified as hypervirulent. Phenotypically, ST661 exhibited the highest biomass production despite showing similar metabolic activity to other STs. A positive correlation was observed between biomass and siderophore production, while capsule production was inversely correlated with biomass. In the G. mellonella model, ST661 demonstrated the highest virulence, resulting in 100% mortality by 48 hours, compared to survival rates of 21.4% for ST101, 38.0% for ST307, and 31.2% for ST512. These findings underscore the pathogenic potential of ST661 isolates with enhanced biofilm production. The G. mellonella model may serve as an effective in vivo system for evaluating the virulence of emerging K. pneumoniae lineages.IMPORTANCEWe demonstrate that the Galleria mellonella model is a useful tool to analyze the virulence of carbapenem-resistant Klebsiella pneumoniae strains. Our findings highlight the pathogenicity of carbapenem-resistant K pneumoniae isolates, particularly the role of the ST661 that, despite being a rare lineage, harbors the blaVIM gene and is associated with high biofilm production and the highest mortality rates.

Naked-Eye Molecular Testing for the Detection of Xylella fastidiosa in Mallorca (Balearic Island) Almond Orchards by Colorimetric LAMP

Xylella fastidiosa (Xf) is a quarantine pathogen heavily affecting economically important crops worldwide. Different sequence types (STs) belonging to Xf subspecies are present in various areas of Spain, including the Balearic Islands, and cause the almond leaf scorch disease (ALSD) in Prunus spp. The increased demand for rapid tests for early detection of the pathogen should enforce strict containment measures. Molecular detection through isothermal amplification reactions enables simplified instrumentation and the use of raw nucleic acid extracts. Colorimetric loop-mediated isothermal amplification (cLAMP) was applied to rapidly detect Xf in naturally infected almonds on Mallorca Island (Spain), using a quick crude sap extraction without DNA purification. Following tissue homogenization, an alkaline treatment for target DNA extraction was conducted before the cLAMP test. The cLAMP assay was able to detect up to 100 CFU/mL of the Xf bacterial suspension diluted in healthy almond sap. The same crude extracts used in the cLAMP test were also tested by qPCR. An overall positive agreement of about 47% was observed between the results of the two techniques, while a decrease in cLAMP sensitivity was evident as the bacterial titer declined in infected plants over Cq > 26–27. This study shows the potential of the cLAMP application as a rapid and low-cost point-of-care diagnostic method for the timely monitoring of Xf directly in the field.

Molecular Epidemiological Characteristics of Staphylococcus pseudintermedius, Staphylococcus coagulans, and Coagulase-Negative Staphylococci Cultured from Clinical Canine Skin and Ear Samples in Queensland

Background/Objectives: Infections in dogs caused by methicillin-resistant staphylococci (MRS) present limited treatment options. This study’s objective was to investigate the molecular epidemiology of Staphylococcus spp. cultured exclusively from clinical canine skin and ear samples in Queensland, Australia, using whole-genome sequencing (WGS). Methods: Forty-two Staphylococcus spp. isolated from clinical canine skin and ear samples, from an unknown number of dogs, were sourced from two veterinary diagnostic laboratories between January 2022 and May 2023. These isolates underwent matrix-assisted laser desorption ionisation– time of flight bacterial identification, minimum inhibitory concentration testing using SensititreTM plates and WGS. Phylogenetic trees and core genome multilocus sequence typing (cgMLST) minimum spanning trees (MSTs) were constructed. Results: The isolates included methicillin-resistant and -sensitive S. pseudintermedius (MRSP: 57.1%, 24/42; and MSSP: 19.1%, 8/42), methicillin-resistant and -sensitive S. coagulans (MRSC: 14.3%, 6/42; and MSSC: 2.4%, 1/42) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS: 7.1%, 3/42). Thirty-nine isolates were included after WGS, where all MRS harboured the mecA gene. Eighteen sequence types (STs) were identified, including three novel MRSP and six novel MSSP STs. MRSP ST496-V-VII (23%; 9/39) and MRSP ST749-IV-(IVg) (12.8%; 5/39) were commonly isolated. Phylogenetic analysis of single nucleotide polymorphisms showed that MRSP, MRSC and MSSC were similar to globally isolated staphylococci from canine skin and ear infections. Using cgMLST MSTs, MRSP isolates were not closely related to global strains. Conclusions: Our findings revealed a genotypically diverse geographical distribution and phylogenetic relatedness of staphylococci cultured from clinical canine skin and ear samples across Queensland. This highlights the importance of ongoing surveillance to aid in evidence-based treatment decisions and antimicrobial stewardship.

Serological, phenotypic and molecular characterization of brucellosis in small ruminants in northern Algeria

Brucellosis is considered a common bacterial zoonotic disease of high prevalence in countries of the Middle East and the Mediterranean region with economic and public health impact. The present study aimed to investigate the current situation of brucellosis in small ruminants reared in Médéa and Sidi Bel-Abbès provinces, north Algeria. To achieve this objective, 96 sera (77 sheep and 19 goat) and 57 milk (42 sheep and 15 goat) samples were collected from suspected infected animals and serologically analyzed by using ELISA. For isolation of Brucella spp., four placentas, two fetuses and forty-four milk samples were subjected to microbiological investigation. Whole genome sequencing (WGS) was used for genomic analysis of isolated Brucella species. The results of this study showed that anti-Brucella antibodies were detected in 46 (83.6%) and 52 (54.2%) milk and serum samples, respectively. However, among 27 cases where blood samples were negative, anti-Brucella antibodies were still detected in 19 of the corresponding milk samples, resulting in an overall discordance rate of 36.5%. Ten Brucella melitensis were isolated and identified from six sheep and four goats. Of these, eight originated from milk samples. The isolated strains were assigned to sequence type ST-11 using Multilocus sequence typing (MLST). Five isolates revealing high similarity (0–2 nucleotide differences) originated from different farms, indicating a close transmission link. However, two identical caprine isolates and three other isolates showed notable genotypic variation, in comparison. The highest base difference (449–462 nucleotides) was observed for an ovine isolate originating from Sidi Bel-Abbès. The phylogenetic analysis and clustering with the West Mediterranean lineage of B. melitensis revealed high genetic similarity of the investigated isolates with B. melitensis of human origin from North Africa and travel-associated ‘European’ cases, especially from Morroco, Tunisia, Sweden and Italy. The results of this study highlight brucellosis in small ruminants as a significant public health risk and will help to develop effective control strategies in Algeria. These findings provide specific evidence of this risk, with Brucella isolation from milk and by linking theses isolates to human cases in Algeria and abroad. The use of WGS-based analysis has revealed effective in tracing patterns of transmission, and can be recommended for tracking outbreaks at a high resolution.

“Sichuanvirus”, a novel bacteriophage viral genus, able to lyse carbapenem-resistant Klebsiella pneumoniae

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is a severe threat for human health and urgently needs new therapeutic approaches. Lytic bacteriophages (phages) are promising clinically viable therapeutic options against CRKP. We attempted to isolate lytic phages against CRKP of sequence type 11 and capsular type 64 (ST11-KL64), the predominant type in China.ResultsWe recovered a lytic phage from sewage collected at a wastewater treatment station in Sichuan province, China. We obtained the genome of this phage and found that it is distinct from all known phages with the highest overall DNA similarity (12.5%, 16% coverage and 78.4% identity) with phage vB_EcoM_PHB05 (accession no. NC_052652) in ICTV. This phage represents a novel viral genus of the subfamily Stephanstirmvirinae, for which we proposed “Sichuanvirus” as the genus name. This phage has a narrow host range lyse specific for KL64 Klebsiella. This phage has no genes referring to antimicrobial resistance, virulence, and lysogen and is stable to a wide range of pH and temperatures. We also obtained three bacterial mutants resistant to the phage and performed genome sequencing for them. We therefore discovered that the interruption of a capsular polysaccharide biosynthesis-related gene wcaJ by insertion sequences mediated the resistance to this phage.ConclusionWe recovered and characterized a phage of “Sichuanvirus”, a novel viral genus of subfamily Stephanstirmvirinae, which is suitable for phage therapy. The discovery of this phage expands the arsenal against CRKP.

Understanding the clinical and molecular epidemiological characteristics of carbapenem-resistant Acinetobacter baumannii infections within intensive care units of three teaching hospitals

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) is recognized as a common clinical conditional pathogen with blaOXA−23 gene-mediated multidrug-resistance that is a significant threat to public health safety. Timely and effective infection control measures are needed to prevent their spread.MethodsWe conducted a retrospective study of CRAB patients at three teaching hospitals from 2019 to 2022. We identified bacterial isolates, collected clinical data, and performed antimicrobial susceptibility testing. Genome characteristics of isolates were investigated by whole genome sequencing. Multilocus sequence typing and phylogenetic trees were used to assess the genetic similarity of isolates. Acquired antimicrobial resistance genes and virulence factors carried in the isolated group genome were analyzed by ResFinder, PubMLST and VFDB. Sequence alignment was used to analyze genetic environment around blaOXA−23. Phylogenetic tree was constructed to analyze the genetic relationship of isolates.ResultsA total of 92 non-repetitive CRAB isolates were collected, with sputum samples accounting for the majority (94.57%, n = 87) of samples. These were distributed into ST2, with ST2 identified to have the highest prevalence of infection, accounting for 99.99% (n = 91) of all isolates. The major resistance genes identified were blaOXA−23, blaOXA−66, blaOXA−51, and blaADC. Also, 92 CRAB strains showed high levels of resistance to common clinical antibiotics, but not minocycline. Meanwhile, most of the isolates carried virulence genes such as various ompA, csuA, csuB, csuC, csuD, abaI, abaR, lpxC, lpxA, and bmfRS. Single nucleotide polymorphism (SNP) analyses further indicated that the bacterial genome was progressively polymorphic with time. We analyzed the environment of the blaOXA−23 gene and found that CRAB accumulated in the context of prominent environmental antibiotic exposure and had longer survival times in the antibiotic environment, resulting in the tendency of bacteria to develop greater antibiotic resistance.ConclusionsWe find that CRAB is prevalent within the ICU and is progressively resistant to antibiotics over time. Enhanced clinical understanding and timely management of CRAB infections will be crucial to minimize or even eliminate the spread of CRAB within the ICU setting.

Tracking clonal and plasmid transmission in colistin- and carbapenem-resistant Klebsiella pneumoniae.

The surveillance of mobile genetic elements facilitating the spread of antimicrobial resistance genes has been challenging. Here, we tracked both clonal and plasmid transmission in colistin- and carbapenem-resistant Klebsiella pneumoniae using short- and long-read sequencing technologies. We observed three clonal transmissions, all containing Incompatibility group (Inc) L plasmids and New Delhi metallo-beta-lactamase blaNDM-1, although not co-located on the same plasmid. One IncL-blaNDM-1 plasmid had been transferred between sequence type (ST) 392 and ST15, and the promiscuous IncL-blaOXA-48 plasmid was likely shared between a singleton and a clonal transmission of ST392. Plasmids within clonal outbreaks and between clusters and STs had 0-2 single nucleotide polymorphism (SNP) differences, showing high stability upon transfer to same or different STs. The simplest explanation, without a comprehensive analysis with long-read sequencing, would be the spread of a single common IncL-blaNDM-1 plasmid. However, here, we report blaNDM-1 in five different plasmids, emphasizing the need to investigate plasmid-mediated transmission for effective containment of outbreaks.IMPORTANCEAntimicrobial resistance occupies a central stage in global public health emergencies. Recently, efforts to track the genetic elements that facilitate the spread of resistance genes in plasmids outbreaks, utilizing short-read sequencing technologies, have been described. However, incomplete plasmid reconstruction from short-read sequencing data hinders full knowledge about plasmid structure, which makes the exploration very challenging. In this study, we used both short- and long-read sequencing in clinical Klebsiella pneumoniae from University Hospital Centre Zagreb, Croatia, which was resistant to both last-resort antibiotics colistin and carbapenem. Our results show complex transmission networks and sharing of plasmids, emphasizing multiple transmissions of plasmids harboring carbapenem and/or colistin resistance genes between and within K. pneumoniae clones. Only full-length sequencing plus a novel way of determining plasmid clusters resulted in the complete picture, showing how future active monitoring of plasmids as a vital tool for infection prevention and control could be implemented.

Chronic Carriage of Leptospira interrogans Genotype Associated With the Australis Serogroup by Naturally-Infected Hedgehogs (Erinaceus europaeus) at a Wildlife Health Centre in Northwestern France.

Leptospirosis is a widespread zoonosis caused by bacteria in the genus Leptospira. Basic epidemiological information is crucial to mitigating disease risk but is lacking for leptospirosis; notably, the hosts responsible for maintaining Leptospira remain largely unknown. Frequently observed near human habitations, hedgehogs (Erinaceus europaeus) are taken to wildlife rescue centres when found sick or injured. Thus, they may pose a risk to human and animal health if they carry pathogenic Leptospira. This study aimed to describe Leptospira carriage in a hedgehog population and the potential clinical impacts of the infection. We investigated Leptospira carriage frequency and diversity in urine samples from 69 hedgehogs at a wildlife rescue centre, between April and June 2022. We used quantitative PCR, typing of the 16S rRNA and lfb1 genes, variable number tandem repeat and multispacer sequence typing to characterise Leptospira DNA. An analysis of urinary biochemical parameters was conducted to assess renal function. We detected Leptospira DNA in 25 (35%) of the urine samples, of which 21 were successfully typed. The latter analysis revealed a limited degree of genetic diversity. L. interrogans (n = 19) predominated, and the only genotype detected was related to the Australis serogroup (n = 17). We also noted the presence of L. borgpetersenii (n = 1) and L. kirschneri (n = 1). There was no relationship between infection status and urinalysis parameters. These results suggest hedgehogs may act as long-term shedders of Leptospira in natural ecosystems.

Highly viable gastrointestinal Chlamydia trachomatis in women abstaining from receptive anal intercourse

Chlamydia trachomatis (CT) may employ persistence to evade antimicrobial clearance, possibly residing in the gastrointestinal tract. This study assessed the reliability of droplet digital PCR (ddPCR) in CT detection, its functionality in viability assessment, and predictions on CT transmission dynamics by combining viability PCR (vPCR) and clinical data from 52 infected women. The ddPCR showed 94% positive and 100% negative agreement with Abbott Alinity STI-M for endocervical samples, and 92% positive and 87% negative agreement in rectal samples. Viability was higher in endocervical samples (89.1%) than in rectal samples (69.4%). Samples from participants not engaging in anal intercourse, and with non-concordant multi-locus sequence typing between rectum and endocervix, had on average the highest viability in rectum, indicating a persistent population residing in the gastrointestinal tract. This study demonstrates the effectiveness of ddPCR in detecting CT, especially in samples with high inhibition or low bacterial load, suggesting its superiority over quantitative real-time PCR. These findings support that rectal CT infection can occur independently of anal intercourse, possibly through vaginorectal contamination or oral routes. High rectal CT viability, independent of endocervical infection, indicates potential gastrointestinal establishment. Understanding CT dynamics in various infection sites can provide insights into the epidemiology and pathogenesis of CT.

Accelerated High-resolution T1- and T2-weighted Breast MRI with Deep Learning Super-resolution Reconstruction.

To assess the performance of an industry-developed deep learning (DL) algorithm to reconstruct low-resolution Cartesian T1-weighted dynamic contrast-enhanced (T1w) and T2-weighted turbo-spin-echo (T2w) sequences and compare them to standard sequences. Female patients with indications for breast MRI were included in this prospective study. The study protocol at 1.5 Tesla MRI included T1w and T2w. Both sequences were acquired in standard resolution (T1S and T2S) and in low-resolution with following DL reconstructions (T1DL and T2DL). For DL reconstruction, two convolutional networks were used: (1) Adaptive-CS-Net for denoising with compressed sensing, and (2) Precise-Image-Net for resolution upscaling of previously downscaled images. Overall image quality was assessed using 5-point-Likert scale (from 1=non-diagnostic to 5=excellent). Apparent signal-to-noise (aSNR) and contrast-to-noise (aCNR) ratios were calculated. Breast Imaging Reporting and Data System (BI-RADS) agreement between different sequence types was assessed. A total of 47 patients were included (mean age, 58±11 years). Acquisition time for T1DL and T2DL were reduced by 51% (44 vs. 90s per dynamic phase) and 46% (102 vs. 192s), respectively. T1DL and T2DL showed higher overall image quality (e.g., 4 [IQR, 4-4] for T1S vs. 5 [IQR, 5-5] for T1DL, P<0.001). Both, T1DL and T2DL revealed higher aSNR and aCNR than T1S and T2S (e.g., aSNR: 32.35±10.23 for T2S vs. 27.88±6.86 for T2DL, P=0.014). Cohen k agreement by BI-RADS assessment was excellent (0.962, P<0.001). DL for denoising and resolution upscaling reduces acquisition time and improves image quality for T1w and T2w breast MRI.

Phylogeny and virulence determinant detection of Fusobacterium necrophorum strains isolated at the UK Anaerobe Reference Unit between 1982 and 2019.

Explore the presence, or absence, of virulence genes and the phylogeny of a multi-decade UK collection of clinical and reference Fusobacterium necrophorum isolates. Three hundred and eighty-five F. necrophorum strains (1982-2019) were recovered from storage (-80°C). Illumina whole genome sequencing (WGS) was undertaken with 374/385 genomes available for examination after quality checking. Sequences were analysed, using BioNumerics (bioMérieux; v 8.1), for the presence of known virulence genes. Strain phylogeny was investigated using a bespoke Fusobacterium spp. whole genome multi-locus sequence typing (wgMLST) method and single nucleotide polymorphism (SNP) analysis. F. necrophorum ssp. necrophorum and F. necrophorum ssp. funduliforme phylogeny showed clear separation of the two subspecies and clustering of F. necrophorum ssp. funduliforme into three distinct clades. Congruence between SNP and wgMLST analysis was high (99.3%) and indistinguishable clusters were observed with wgMLST (n=3) and SNP (n=1) analysis of isolates derived from different students attending the same education setting. There was no grouping of strains by disease state or decade of isolation. No association was demonstrated with specific virulence gene detection although conspicuous virulence gene patterns were seen amongst the different subspecies and clades. There was no evidence that the pathogenesis of F. necrophorum infection was associated with the presence of the virulence determinants investigated. Host-pathogen interactions should, therefore, be a focus of future research. Person-to-person transmission is a feature of this important pathogen.

First Report of Pseudomonas amygdali Causing Leaf Spot on Hibiscus rosa-sinensis in a greenhouse in New York State, U.S.A.

Hibiscus rosa-sinensis, commonly known as the "Chinese hibiscus", is a widely cultivated shrub with ornamental and medicinal applications (Jadhav et al., 2009). However, it is known to be susceptible to a range of pathogens including bacteria (Chase, 1986). In March 2019, Hibiscus rosa-sinensis plants in production at a greenhouse in New York, but originally from Florida, developed widespread, conspicuous leaf spots and associated chlorosis for a month after arrival. "Hibiscus 35-1" was isolated from a suspension of symptomatic plant tissue pieces soaked in sterile deionized water and streaked on potato dextrose agar (PDA) (González-Tobón et al., 2023), resulting in numerous white bacterial colonies with a uniform appearance. Previously we reported the complete species identification for this bacterial isolate using whole genome sequencing via Oxford Nanopore and Illumina platforms (BioProject: PRJNA765326) as a Pseudomonas amygdali with an ANI ≥ 95% ± 0.5%. (González-Tobón et al., 2023). The hypersensitive response (HR) to P. amygdali 35-1 was first evaluated in three tobacco (Nicotiana tabacum) and three tomato (Solanum lycopersicum) plants. An overnight culture was washed, resuspended, adjusted to an OD600 of 0.2-0.3, and used to inoculate two leaves per plant via syringe infiltration (Chakravarthy et al., 2017). HR was observed on all leaves at the inoculation site as early as 48hpi for tobacco and 24hpi for tomato. No symptoms were observed for mock inoculations. The pathogenicity of P. amygdali 35-1 in hibiscus was then tested using six 'Social Butterfly' plants in 4.0 in pots inoculated by hand spraying. Inoculum was made from a 72 h culture on solid King's B medium resuspended in sterile deionized water with 2 drops/L of Tween 80 and sprayed at a concentration of 3.6 x 108 bacteria/mL, as determined by dilution plating. Inoculated plants and six matching controls were sealed inside plastic bags and placed in a randomized complete block in a growth chamber at 30 ºC with a 12h photoperiod for 40 days. No changes were observed on any of the control plants but spotting and chlorosis similar to the original symptoms gradually developed on five of the inoculated plants. After 40 days, seven 2-mm tissue squares were excised from the lesion margins or from the asymptomatic tissue of controls, surface-sterilized with 0.75% NaCl, and plated on King's B. No bacteria were recovered from the control plants but white colonies resembling those of the original inoculation were recovered from several diseased tissue pieces. Genomic DNA was extracted from this bacterium using the New England Biolabs Monarch Genomic DNA Extraction Kit. Polymerase chain reaction (PCR) tests were performed to amplify five genes (16S, cts, gapA, gyrB, and rpoD) typically used for multilocus sequence typing (MLST) (Sarkar & Guttman, 2004). The amplicons were Sanger sequenced and BLAST analyses confirmed 100% match to P. amygdali 35-1 (GenBank: CP084212). To our knowledge, our lab is the first to report P. amygdali causing disease on hibiscus in New York. Similar symptoms on hibiscus plants in Florida were reported in the past as caused by other Pseudomonas species of the P. syringae G group, mainly P. syringae and P. cichorii (Chase, 1986; Gardan et al., 1999: Girard et al., 2021; Jones et al., 1986). Given the lack of consistent reporting on bacterial leaf spot pathogens of hibiscus, as well as the economic importance of this ornamental, further research on the disease is needed.

Genomic characterization of Escherichia coli with a polyketide synthase (pks) island isolated from ulcerative colitis patients

The E. coli strains harboring the polyketide synthase (pks) island encode the genotoxin colibactin, a secondary metabolite reported to have severe implications for human health and for the progression of colorectal cancer. The present study involves whole-genome-wide comparison and phylogenetic analysis of pks harboring E. coli isolates to gain insight into the distribution and evolution of these organisms. Fifteen E. coli strains isolated from patients with ulcerative colitis (UC) were sequenced, 13 of which harbored pks islands. In addition, 2,654 genomes from the public database were also screened for pks harboring E. coli genomes, 158 of which were pks-positive (pks+) isolates. Whole-genome-wide comparison and phylogenetic analysis revealed that 171 (158 + 13) pks+ isolates belonged to phylogroup B2, and most of the isolates belong to sequence types ST73 and ST95. One isolate from a UC patient was of the sequence type ST8303. The maximum likelihood tree based on the core genome of pks+ isolates revealed horizontal gene transfer across sequence types and serotypes. Virulome and resistome analyses revealed the0020preponderance of virulence genes and a reduced number of antimicrobial genes in pks+ isolates. This study significantly contributes to understanding the evolution of pks islands in E. coli.

Genomic Analysis of Virulent, Multidrug Resistant Klebsiella pneumoniae and Klebsiella oxytoca from Bloodstream Infections, South Africa.

A total of 46 suspected Klebsiella species (spp.) were collected from blood cultures within the uMgungundlovu District in the KwaZulu-Natal Province. Antibiotic susceptibility was determined against a panel of 19 antibiotics using the disk diffusion test. A subset of 14 MDR K. pneumoniae (n=10) and K. oxytoca (n=4) isolates were selected based on their antibiograms and subjected to whole genome sequencing (WGS). The sequence types (STs), resistome, virulome, mobilome, capsule loci (KLs) were analysed using relevant WGS and bioinformatics tools. Of the 10 K. pneumoniae sequence types (ST) identified, the most common were ST25 (n=3), ST101 (n=3), and 4 K. oxytoca belonged to ST450 (n=3). The two high-risk K. pneumoniae clones ST15, and ST17 were identified. O and K capsule types were identified, with predominance of KL2, KL17, KL29, O1/O2v2, O1/O2v1, and OL104 respectively. The majority of isolates displayed multidrug resistance predominantly carrying β-lactamase genes, including blaCTX-M-15, blaTEM-1B, blaSHV, and blaOXA-1, and blaOXY including the carbapenemase blaOXA-181 in two (14.3%) study isolates. Other resistance genes included: aac(6')-lb-cr, aac(3), aac, aph, aad, dfr, tet(A), and tet(D), mph(A), sul1, sul2, oqx, qnr, acrR, ramR, parC, gyrA, arr-3, cat, fosA, qacE genes conferring resistance to aminoglycosides, trimethoprim, tetracycline, macrolide, sulfonamides, fluoroquinolones, rifampicin phenicols, fosfomycin, and quaternary ammonium compound disinfectant. Virulence factors related to hypervirulence: encoding aerobactin (iuc, iutA), salmochelin (iro), yersiniabactin (ybt), enterobactin (ent), type 1 and 3 (mrk and fim), and capsule synthesis (rcsA and rcsB) were identified. IncF, IncR, and Col plasmid replicon types and class I integrons were detected, with IncFIB(K) predominance. The blaCTX-M-15 and blaTEM-1 genes were bracketed by Tn3 transposons, ISEc9, recombinase and IS91 insertion sequences. The convergence of multidrug resistance and hypervirulence genes in Klebsiella strains is a potential clinical concern. Carbapenemase, ESBL screening and genomic surveillance are urgently required in hospital environments.