Using 16s rRNA sequencing to characterize the microbiome of tropical cutaneous ulcer disease: insights into the microbial landscape and implications for diagnosis and treatment.

Cutaneous ulcers are common in yaws-endemic areas. Although often attributed to 'Treponema pallidum subsp. pertenue' and Haemophilus ducreyi, quantitative PCR has highlighted a significant proportion of these ulcers are negative for both pathogens and are considered idiopathic. This is a retrospective analysis utilising existing 16S rRNA sequencing data from two independent yaws studies that took place in Ghana and the Solomon Islands. We characterized bacterial diversity in 38 samples to identify potential causative agents for idiopathic cutaneous ulcers. We identified a diverse bacterial profile, including Arcanobacterium haemolyticum, Campylobacter concisus, Corynebacterium diphtheriae, Staphylococcus spp. and Streptococcus pyogenes, consistent with findings from previous cutaneous ulcer microbiome studies. No single bacterial species was universally present across all samples. The most prevalent bacterium, Campylobacter ureolyticus, appeared in 42% of samples, suggesting a multifactorial aetiology for cutaneous ulcers in yaws-endemic areas. This study emphasizes the need for a nuanced understanding of potential causative agents. The findings prompt further exploration into the intricate microbial interactions contributing to idiopathic yaw-like ulcers, guiding future research toward comprehensive diagnostic and therapeutic strategies.

Development of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella abortisuis isolated from pigs.

The first description of Trueperella (T.) abortisuis was presented in Japan in 2009 by Azuma and colleagues. In the current study, eight T. abortisuis strains were identified by a newly developed loop-mediated isothermal amplification (LAMP) assay based on the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) encoding gene gap. Two T. abortisuis strains were obtained from prepuce of a seven-month-old boar and pooled foetal stomach contents in the United Kingdom, while the other six T. abortisuis strains were recovered from aborted foetal material of six pigs from a single farm in Germany. The developed LAMP assay showed an analytical sensitivity of 22 pg µL-1T. abortisuis DNA. T. abortisuis DSM 19515T and field strain T. abortisuis P504054/19/1 were directly detectable in artificially contaminated vaginal swabs up to concentrations of 980 CFU and 770 CFU per swab, respectively. There was no cross reactivity with control strains representing six species of genus Trueperella and six species of the closely related genus Arcanobacterium and Schaalia (Actinomyces) hyovaginalis. Further field research is required to determine the usefulness of the designed LAMP assay for identifying T. abortisuis isolated from pigs of various origins and from test samples directly obtained at farm level.

First isolation of Arcanobacterium pinnipediorum from a grey seal pup (Halichoerus grypus) in the UK.

In the present study, a single Arcanobacterium (A.) pinnipediorum strain isolated from discharge of a jaw swelling of a grey seal pup (Halichoerus grypus) in England, UK, was identified. This strain was further characterized by phenotypical investigations, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), by Fourier transform infrared spectroscopy (FT-IR), and genotypically by sequencing the 16S rRNA gene and the genes gap encoding glyceraldehyde 3-phosphate dehydrogenase, tuf encoding elongation factor tu, and rpoB encoding the ß subunit of bacterial RNA polymerase. The present study gives a first detailed characterization of the species A. pinnipediorum from a grey seal in the UK. However, the route of infection of the grey seal with the bacterial pathogen remains unclear.

Identification of Trueperella bernardiae isolated from peking ducks (Anas platyrhynchos domesticus) by phenotypical and genotypical investigations and by a newly developed loop-mediated isothermal amplification (LAMP) assay.

Trueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.

Outbreak of Arcanobacterium haemolyticum in chronic wounds in The Netherlands.

INTRODUCTION: Aging and comorbidities such as diabetes and vascular problems contribute to the increasing occurrence of chronic wounds. From the beginning of 2016, a marked increase in Arcanobacterium haemolyticum (ARH) in chronic wound cultures was noted among patients visiting a wound expertise centre in The Netherlands. AIM: To report the outbreak investigation of ARH cultured from chronic wounds and describe the implemented infection prevention measures. METHODS: In total, 50 ARH isolates were sent to a reference laboratory for molecular typing. Samples for bacterial culture and ARH polymerase chain reaction were taken from care workers, the environment and items used for wound care. Infection prevention measures were implemented in a bundled approach, involving education, better aseptic wound care conditions and hygienic precautions. Before and after the implementation of infection prevention measures, two screening rounds of ARH testing were performed among all patients receiving home care. RESULTS: ARH isolates from wound care patients were found to be identical by core genome multi-locus sequence typing. No definite outbreak source could be determined by culture. However, three pairs of forceps, used by two nurses on multiple patients, were found to be ARH positive by polymerase chain reaction. In the two screening rounds before and after the implementation of infection prevention measures, the proportion of ARH-positive patients decreased significantly from 20% (20/99) to 3% (3/104). Subsequently, no new cases occurred. CONCLUSION: This first ARH outbreak was likely caused by re-using contaminated instruments. Through the implementation of improved infection prevention measures and re-education of all employees involved, the outbreak was controlled. With the current trend of care transition, infection control must be a major concern.

Epidemiological analysis of Arcanobacterium phocae isolated from cases of mink dermatitis of a single farm.

The present study was designed to identify nine Arcanobacterium phocae strains isolated from cases of mink dermatitis of a single farm in Finland and characterize the strains for epidemiological relationships. All nine strains and previously described A. phocae used for comparative purposes were identified and further characterized phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), by Fourier Transform Infrared Spectroscopy (FT-IR) and genotypically by detection of phocaelysin encoding gene phl with a previously developed loop-mediated isothermal amplification (LAMP) assay and by sequencing 16S rRNA gene and gene phl, the elongation factor tu encoding gene tuf and the ß subunit of bacterial RNA polymerase encoding gene rpoB. Genetic relatedness among isolates was determined using whole-genome single nucleotide polymorphism (wgSNP) analysis. The wgSNP results, partly the MALDI-TOF MS and FT-IR analyses and sequencing of the genes, revealed that the nine A. phocae strains recovered from a single farm showed close sequence similarities among each other and differed from previously investigated A. phocae strains isolated from other farms and animals in Finland and from the A. phocae type strain. This indicated a close epidemiological relationship of the A. phocae strains isolated from a single farm and that the nine A. phocae strains of the present study might have developed from a common ancestor.

Isolation and comparison of Arcanobacterium hippocoleae isolates from the genital tract of 15 mares.

The present study reports the isolation of A. hippocoleae from genital swabs of 15 apparently healthy mares (at least one had an abortion one month earlier) and describes the genotypic and phenotypic characterisation of these strains. The mares were of eight different breeds with a thoroughbred dominance and came from 11 breeding farms located in the French region of Brittany. 16S rRNA gene sequencing confirmed the species' identification by comparing it with reference strain A. hippocoleae CIP 106850T. Some degree of natural divergence within A. hippocoleae was observed by 16S rRNA sequencing (two 1,002-pb sequences), MALDI-TOF MS typing (two groups), a CAMP test (three different intensities of haemolysis from CAMP-positive results) and API® Coryne system (five profiles). The strains were all susceptible to the antimicrobials tested. A national prevalence survey would be required to estimate the frequency of A. hippocoleae carriage in mares and stallions and to verify the presence of A. hippocoleae outside the French region of Brittany, which is the only one found to be affected in the current study, probably because the isolates were recovered from a single field laboratory in this region.

Further characteristics of Arcanobacterium pinnipediorum DSM 28752T and Arcanobacterium wilhelmae DSM 102162T, two novel species of genus Arcanobacterium.

The newly described type strains Arcanobacterium pinnipediorum DSM 28752T and Arcanobacterium wilhelmae DSM 102162T, initially isolated from an anal swab of a harbor seal (Sammra et al. Int J Syst Evol Microbiol 65:4539-4543, 2015) and the genital tract of a rhinoceros (Sammra et al. Int J Syst Evol Microbiol 67:2093-2097, 2017), could be further characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Fourier transform infrared (FT-IR) spectroscopy and by sequencing the genomic targets 16S-23S rDNA intergenic spacer region (ISR) and the genes rpoB, gap, and tuf. The two strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of both species remained unclear. However, the detection of specific spectra by MALDI-TOF MS and by FT-IR spectroscopy and the presented genotypic approaches might help to identify A. pinnipediorum and A. wilhelmae in the future and might elucidate the role these two species play in infections of animals.

Identification of Arcanobacterium phocae isolated from fur animals by phenotypic properties, by MALDI-TOF MS analysis and by detection of phocaelysin encoding gene phl as probable novel target.

In the present study 12 Arcanobacterium phocae strains isolated from fur animals in Finland, including foxes, minks and Finnraccoons, could successfully be identified phenotypically, by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and phocaelysin (PHL) encoding gene phl. The PHL of all 12 A. phocae strains in the present study and reference strains A. phocae DSM 10002T and A. phocae DSM 10003 displayed, as typical members of the cholesterol dependent cytolysin-group of toxins, the variant undecapeptide sequence EATGLAWDPWW which appeared to be most closely related to arcanolysin of Arcanobacterium haemolyticum and pyolysin of Trueperella pyogenes. In addition, gene phl could be determined with a newly designed loop-mediated isothermal amplification (LAMP) assay. The detection of mass spectra by MALDI-TOF MS and the LAMP assay based on gene phl might help to reliably identify A. phocae in future and also elucidate the role this species plays in infections of fur animals.

Phenotypic and genotypic characteristics of Trueperella pyogenes isolated from ruminants.

Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in animals including humans. Data on phenotypic and genotypic properties of T. pyogenes isolated from ruminants, particularly goats and sheep, are lacking. We characterized, by phenotypic and genotypic means, T. pyogenes of caprine and ovine origin, and established their phylogenetic relationship with isolates from other ruminants. T. pyogenes isolates ( n = 50) from diagnostic specimens of bovine ( n = 25), caprine ( n = 19), and ovine ( n = 6) origin were analyzed. Overall, variable biochemical activities were observed among the T. pyogenes isolates. The fimbriae-encoding gene, fimE, and neuraminidase-encoding gene, nanH, were, respectively, more frequently detected in the large ( p = 0.0006) and small ( p = 0.0001) ruminant isolates. Moreover, genotype V ( plo/ nanH/ nanP/ fimA/ fimC) was only detected in the caprine and ovine isolates, whereas genotype IX ( plo/ nanP/ fimA/ fimC/ fimE) was solely present in the isolates of bovine origin ( p = 0.0223). The 16S rRNA gene sequences of all T. pyogenes isolates were clustered with the reference T. pyogenes strain ATCC 19411 and displayed a high degree of identity to each other. Our results highlight phenotypic and genotypic diversity among ruminant isolates of T. pyogenes and reinforce the importance of characterization of more clinical isolates to better understand the pathogenesis of this bacterium in different animal species.

Antimicrobial Resistance in Corynebacterium spp., Arcanobacterium spp., and Trueperella pyogenes.

There is currently only limited information on the antimicrobial susceptibility and resistance of Corynebacterium spp., Arcanobacterium spp., and Trueperella pyogenes from animals. The comparability of the data is hampered by the use of different antimicrobial susceptibility testing methods and interpretive criteria. To date, standard broth microdilution methods and clinical breakpoints that are approved by the Clinical and Laboratory Standards Institute and are applicable to Corynebacterium spp., Arcanobacterium spp., and T. pyogenes are available. The lack of species-specific clinical breakpoints for the different animal species reduces the explanatory power of the data. Among the isolates of the three genera, elevated MICs for different classes of antimicrobial agents (e.g., ß-lactams, macrolides, lincosamides, tetracyclines, aminoglycosides, phenicols, sulfonamides/diaminopyrimidines, and fluoroquinolones) have been described. The most comprehensive data set is available for T. pyogenes, which also includes information about genes and mutations involved in antimicrobial resistance. In T. pyogenes isolates, the macrolide-lincosamide-streptogramin B resistance genes erm(B) and erm(X) were identified. Tetracycline resistance in T. pyogenes was based on the resistance genes tet(W), tet(Z), and tet(33), whereas the aminoglycoside resistance genes aacC, aadA1, aadA2, aadA5, aadA24, and aadB have been described in T. pyogenes. So far, only single genes conferring either phenicol resistance (cmlA6), trimethoprim resistance (dfrB2a), or ß-lactam resistance (blaP1) are known to occur in T. pyogenes isolates. Various 23S rRNA mutations, including A2058T, A2058G, and G2137C, were identified in macrolide/lincosamide-resistant T. pyogenes.

Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals.

Trueperella pyogenes is an opportunistic pathogen that causes diverse pyogenic infections in livestock. The genes that encode the exotoxin pyolysin (plo) and other putative factors that promote adhesion of pathogen to host cells (fimbriae fimA, fimC, fimE, fimG, neuraminidases nanH, nanP, and collagen-binding protein cbpA) have been associated with virulence, particularly in mastitis and uterus infections of dairy cows. However, the role of these virulence markers in the pathogenicity of the agent in domestic animals infections still is incompletely understood. The genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA were investigated in 71 T. pyogenes strains recovered from cattle, sheep, goats, dogs, equines, and a pig, recovered from mastitis (n = 35), and non-mastitis (n = 36) cases (abscesses, reproductive tract diseases, pneumonia, lymphadenitis, encephalitis). The most common genes harboured by the isolates were: plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas cbpA (6/71 = 8·4%) and fimG (4/71 = 5·6%) were uncommon. The most frequent genotypes were plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%). No association was observed between the presence of genes vs clinical signs or host species. To the best of our knowledge, this is the first report on aforementioned virulence factors of pathogen detected in diseased horses and dogs. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of particular virulence factors of Trueperella pyogenes that determine different pyogenic infections among domestic animals is poorly understood. Eight putative virulence genes and genotype profiles of 71 isolates were investigated among different clinical manifestations in domestic animals. The most common genes were plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%) were the most frequent genotypes. Studies involving virulence factors are critical in the investigation of molecular epidemiology, pathogenicity, and hypothetical differences in the virulence among T. pyogenes strains from different geographical areas.

Arcanobacterium wilhelmae sp. nov., isolated from the genital tract of a rhinoceros (Rhinoceros unicornis).

A taxonomic study using a polyphasic approach was performed on an unidentified Arcanobacterium-like Gram-stain-positive bacterium isolated from the genital tract of a rhinoceros. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strains of Arcanobacterium canis (98.8 % 16S rRNA gene sequence similarity), Arcanobacterium phocisimile (97.8 %), Arcanobacterium phocae (97.7 %), Arcanobacterium haemolyticum (97.4 %), Arcanobacterium hippocoleae (96.6 %), Arcanobacterium pinnipediorum (96.4 %) and Arcarnobacterium pluranimalium (95.4 %). DNA-DNA hybridization values between strain 647T and Arcanobacterium canisDSM 25104T were very low, 13.4 % (reciprocal 15.9 %). The genomic DNA G+C content of strain 647T was 58.7 mol%. The presence of the major menaquinone MK-9(H4) supported the affiliation of this strain to the genus Arcanobacterium. The polar lipid profile consisted of the major components diphosphatidylglycerol, phosphatidylcholine and an unidentified phosphoglycolipid. The results of physiological and biochemical testing clearly distinguished the unknown bacterium from other species of the genus Arcanobacterium. Based on these tests, it is proposed that the unknown bacterium should be classified as a representative of a novel species of the genus Arcanobacterium named Arcanobacterium wilhelmaesp. nov. The type strain is 647T (=DSM 102162T=LMG 29418T).

Identification of Arcanobacterium hippocoleae by MALDI-TOF MS analysis and by various genotypical properties.

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the ß subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses.

Application of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella pyogenes isolated from various origins.

In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment.

Isolation of Trueperella pyogenes in a case of thoracic and abdominal abscess in a galago (Otolemur garnettii).

BACKGROUND: An adult male galago (Otolemur garnettii) presented for fight wounds following pairing for breeding. Treatment was symptomatic with recovery. Following resolution, the animal re-presented and died, despite additional treatment. METHODS: Necropsy, histopathology, bacterial cultures, and 16S RNA sequencing. RESULTS: A large intrathoracic/intra-abdominal abscess due to Trueperella pyogenes was found at necropsy. CONCLUSIONS: T. pyogenes should be considered in abscesses/wounds of galagos.

Phenotypic and genotypic approach to characterize Arcanobacterium pluranimalium isolated from bovine milk samples.

In the present study, three Arcanobacterium pluranimalium strains isolated from bovine milk samples of three cows of three farms (two cows with subclinical mastitis) could successfully be identified by phenotypical investigations, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis and genotypically by sequencing the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR), the ß subunit of bacterial RNA polymerase encoding gene rpoB, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, the elongation factor tu encoding gene tuf, and the pluranimaliumlysin encoding gene pla. The latter could also be identified by a loop-mediated isothermal amplification (LAMP) assay. The presented phenotypic and genotypic approaches might support the identification of A. pluranimalium in future and might help to understand the role this species plays in bovine mastitis.

Smooth and Rough Biotypes of Arcanobacterium haemolyticum Can Be Genetically Distinguished at the Arcanolysin Locus.

Arcanobacterium haemolyticum is a Gram-positive, ß-hemolytic emerging human pathogen that is classified into smooth or rough biotypes. This bacterial species is also a rare pathogen of animals. Smooth biotypes possess smooth colony edges, are moderate to strong in ß-hemolysis, and predominately cause wound infections. In contrast, rough biotypes possess rough and irregular colony edges, have weak to no ß-hemolytic activity, and predominately cause pharyngitis. Using horse erythrocytes we confirmed that smooth isolates are generally more hemolytic than rough isolates. A hemolysin from A. haemolyticum, arcanolysin (aln/ALN), was recently discovered and is a member of the cholesterol-dependent cytolysin (CDC) family. PCR amplification of aln from all 36 smooth A. haemolyticum isolates yielded the expected 2.0 kb product. While 21 rough isolates yielded the 2.0 kb product, 16 isolates had a 3.2 kb product. The extra 1.2 kb segment was 99% identical to IS911 (insertion sequence) from Corynebacterium diphtheriae. PCR amplification and sequence analysis of the upstream region of aln revealed ~40 nucleotide polymorphisms among 73 clinical isolates from Finland, Denmark, Germany and United States (Nebraska). Remarkably, multi-sequence alignments of the aln upstream region demonstrated that ~90% of the isolates phylogenetically clustered as either smooths or roughs. Differential restriction enzyme analysis of the aln upstream region also demonstrated that the aln upstream region of most (~75%) smooth isolates was cleaved with ClaI while this region in most (~86%) rough isolates was cleaved with XcmI. We conclude that the aln upstream region can be used to genetically distinguish between smooth and rough biotypes of this important emerging pathogen.

Arcanobacterium pinnipediorum sp. nov., isolated from a harbour seal.

A polyphasic taxonomic study was performed on an unidentified Arcanobacterium-like, Gram-stain-positive bacterium, strain 2710T, isolated from a harbour seal. Comparative 16S rRNA gene sequence analysis showed that this bacterial strain belonged to the genus Arcanobacterium and was related most closely to the type strains of Arcanobacterium phocae (98.4 % similarity) and Arcanobacterium phocisimile (97.5 %). 16S rRNA gene sequence similarities to the type strains of other Arcanobacterium species were between 95.3 and 96.9 %. DNA-DNA hybridization values between strain 2710T and A. phocae DSM 10002T and A. phocisimile LMG 27073T were 4.7 % (reciprocal 56 %) and 23 % (reciprocal 7.7 %), respectively. The presence of the major menaquinone MK-9(H4) and a polar lipid profile with the major compounds diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside supported the affiliation of strain 2710T to the genus Arcanobacterium. The major fatty acids were C16:0, C18:1ω9c, C18:0 and C18:2ω6,9c/anteiso-C18:0. The peptidoglycan structure was of cross-linkage type A5α (l-Lys-l-Lys-d-Glu). Physiological and biochemical tests clearly distinguished the isolate from other members of the genus Arcanobacterium. Based on these tests, it is proposed that this unknown bacterium should be classified as a novel species of the genus Arcanobacterium, with the name Arcanobacterium pinnipediorum sp. nov. The type strain is 2710T ( = DSM 28752T = LMG 28298T).

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium.

In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment.