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.

Arcanobacterium buesumense sp. nov., isolated from an anal swab of a male harbour seal (Phoca vitulina).

A polyphasic taxonomic study was performed on an unidentified previously described Arcanobacterium-like Gram-positive strain 2701T isolated from an anal swab of a dead male harbour seal. Comparative 16S rRNA sequencing showed that the bacterium belonged to the genus Arcanobacterium in the family Arcanobacteriaceae. The genome sequence of the strain was obtained by Borowiak et al. [1]. The genome had a G+C content of 49 mol% and a total length of 1.94 Mb. The presence of the major menaquinone MK-9(H4) supported the affiliation of the isolate with the genus Arcanobacterium. The polar lipid profile consisted of diphosphatidylglycerol and an unidentified phospholipid as major components and two unidentified lipids, a further unidentified phospholipid, two unidentified phosphoglycolipids as well as phosphatidylglycerol. The major fatty acids were C16 : 0, C18 : 1 and C18 : 0. Biochemical and phylogenetic analyses clearly distinguished the isolate from other members of the genus Arcanobacterium and closely related other species. Based on these results, it is proposed that the unknown Arcanobacterium sp. strain 2701T should be classified as representing a novel species with the name Arcanobacterium buesumense sp. nov. The type strain is 2701T (=DSM 112952T=LMG 32446T).

Arcanobacterium haemolyticum: A case series.

Arcanobacterium haemolyticum formerly known as Corynebacteria haemolyticum is a Gram positive bacilli. It is a fastidious, facultative anerobic, catalase negative, beta haemolytic and non motile bacterium. Gram positive bacilli are usually considered to be non-pathogenic as the majority are part of normal flora of human skin and mucous membranes. Hence, diagnosis of such infection and its treatment may be delayed by a failure of recognition. However, this bacterium has been implicated in wound, superficial and deep-seated soft tissue infections, endocarditis, osteomyelitis, meningitis, pneumonia, and also septicemia. The early diagnostic evaluation of this organism is emphasized. We report a case series which illustrates the significance of Arcanobacterium haemolyticum in skin and soft tissue infections.

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.

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.

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.

The burden of Arcanobacterium haemolyticum pharyngitis: A systematic review and management algorithm.

INTRODUCTION: Arcanobacterium haemolyticum causes pharyngotonsillitis in children and young adults. It is rarely isolated in pharyngeal swabs as testing for it is not routine. Data on complications, management, and antibiotic susceptibility testing is limited. We sought to review the available literature on the presentation and management of A. haemolyticum pharyngotonsillitis in this age group. METHODS: A systematic review of eligible studies reporting pharyngotonsillitis and related complications in children and young adults caused by A. haemolyticum was conducted. Literature from case reports, case series, and available cohorts was compiled. Data were analyzed using descriptive statistics. RESULTS: The initial database search yielded 63 articles, after applying exclusion criteria 17 studies were included. 191 patients were identified with a median age of 16.5 years. The most common presentation was throat pain reported in 93.7% of patients. Tonsillar exudates, fever at presentation and rash were present in more than half of the reviewed cases. The diagnosis was established by a positive culture on a pharyngeal swab in 98.8% of swabs collected. Complications described included peritonsillar abscesses, Lemierre's syndrome, pneumonia, and sepsis. Penicillin was the first line antibiotic in 81% of patients followed by macrolides in 19 patients (18%). CONCLUSIONS: A. haemolyticum occurs in children and young adults and may result in complications. Our review supports its susceptibility to penicillin. We suggest a diagnostic and management algorithm to guide clinicians in targeted testing and aid with decision making regarding timely and appropriate antibiotic therapy, in an effort to reduce the burden of its complications.

Bacteremia and sepsis by Arcanobacterium haemolyticum in a young immunocompetent patient / Bacteriemia y sepsis por Arcanobacterium haemolyticum en un paciente joven inmunocompetente

Abstract We report the case of a twenty-year-old immunocompetent male patient presenting to the emergency room with pharyngitis and fever. Blood cultures were drawn and Arcanobacterium haemolyticum (rough biotype) was recovered. The presence of the arcanolysin gene was investigated at the molecular level and the upstream region was amplified and sequenced in order to correlate it with the smooth or rough biotype. Although the isolate was susceptible to penicillin, vancomycin and gentamicin, empirical treatments first with amoxicillin/clavulanic acid (1g/12h) and then with ceftriaxone (1g/12h) failed and the infection evolved to sepsis. Finally, treatment with vancomycin (1 g/12 h) plus piperacillin/tazobactam (4.5g/8h) was effective. Lemierre's syndrome was ruled out. To the best of our knowledge, this is the first case of bacteremia by A. haemolyticum reported in Argentina.

Resumen Se describe el caso de un paciente varón inmunocompetente de veinte anos de edad que se presentó en la sala de emergencias con faringitis y fiebre. Se extrajeron muestras para realizar hemocultivos y se recuperó Arcanobacterium haemolyticum (biotipo rugoso). Se investigó la presencia del gen de la arcanolisina por un método molecular, y se amplificó y Faringitis; secuenció la región upstream de dicho gen para determinar su correlación con los biotipos lisos Bacteriemia; o rugosos. Aunque el aislamiento fue sensible a la penicilina, la vancomicina y la gentamicina, Sepsis; los tratamientos empíricos primero con amoxicilina/ácido clavulánico (1 g/12 h) y luego con Síndrome de Lemierre ceftriaxona (1 g/12 h) no fueron efectivos, y la infección evolucionó a sepsis. Finalmente, el tratamiento con vancomicina (1 g/12 h) más piperacilina/tazobactam (4,5g/8h) fue efectivo. Se descartó la presencia del síndrome de Lemierre. Según nuestro conocimiento, este es el primer caso de bacteriemia por A. haemolyticum reportado en Argentina.

Liver abscess caused by Arcanobacterium haemolyticum.

We report the case of a 90-year-old female who was admitted to our hospital due to a three-day history of right abdominal pain and fever of 39 °C (102 °F). The patient's blood pressure was low, with good blood perfusion and no jaundice, and her abdomen was soft and tender in the right hypochondriac and lateral region, with no guarding. Laboratory tests showed: blood glucose level of 201 mg/dl, 362 U/l AST, 237 U/l ALT, 2.5 mg/dl bilirubin, 237 U/l alkaline phosphatase and leukocytosis associated with a left shift. An abdominal ultrasound scan showed a collection of echogenic material and a shadow suggestive of air in hepatic segment 3. Meropenem and metronidazole treatment was started after taking blood cultures, which were negative. A computed tomography (CT) scan confirmed the presence of a liver abscess in segment 3, containing a high-density linear image.

Arcanobacterium bovis sp. nov., isolated from the milk of a cow with mastitis.

A polyphasic taxonomic study was performed on an unidentified Arcanobacterium-like Gram-stain-positive bacterium designated strain C605018/01/1T isolated from a milk sample collected from the udder of a cow at post mortem. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strain of Arcanobacterium pluranimalium (99.76 %); sequence similarities to all other Arcanobacterium species were below 97 %. The wet-lab DNA-DNA hybridization values among strain C605018/01/1T and A. pluranimalium DSM 13483ᵀ were low, 16.9 % (reciprocal, 49.8 %). Pertaining to the whole genome sequence with a total length of 2.02 Mb and 1654 protein counts, the novel strain C605018/01/01T displayed a G+C content of 51.6 % 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, phosphatidylinositol, phosphatidylinositol-mannoside and unidentified glycolipid and aminophospholipids. Based on these results it is proposed that strain C605018/01/1T should be classified as representing a novel species, Arcanbacterium bovis sp. nov. The type strain C605018/01/1T (CCUG 45425T=DSM 107286T=BCCM/LMG 30783T).

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.

Bacteremia and sepsis by Arcanobacterium haemolyticum in a young immunocompetent patient.

We report the case of a twenty-year-old immunocompetent male patient presenting to the emergency room with pharyngitis and fever. Blood cultures were drawn and Arcanobacterium haemolyticum (rough biotype) was recovered. The presence of the arcanolysin gene was investigated at the molecular level and the upstream region was amplified and sequenced in order to correlate it with the smooth or rough biotype. Although the isolate was susceptible to penicillin, vancomycin and gentamicin, empirical treatments first with amoxicillin/clavulanic acid (1g/12h) and then with ceftriaxone (1g/12h) failed and the infection evolved to sepsis. Finally, treatment with vancomycin (1g/12h) plus piperacillin/tazobactam (4.5g/8h) was effective. Lemierre's syndrome was ruled out. To the best of our knowledge, this is the first case of bacteremia by A. haemolyticum reported in Argentina.

Clinically Infrequent Arcanobacterium haemolyticum Bacteremia Complicated by Foot Decubitus Ulcer: An Educational Reminder for Primary Care Physicians.

An 81-year-old Japanese man with no history of diabetes mellitus was admitted to our hospital for a fever with a new ulcerative lesion on the left heel. Blood cultures on admission grew Arcanobacterium haemolyticum in aerobic bottles. He was therefore diagnosed with A. haemolyticum bacteremia and osteomyelitis complicated with foot decubitus ulcer. He was successfully treated with intravenous antibiotic therapy and debridement of the left heel. Our case and literature review show that it is important to recognize that A. haemolyticum is a systemic causative pathogen in immunocompetent patients in primary care practice.

Arcanobacterium haemolyticum Utilizes Both Phospholipase D and Arcanolysin To Mediate Its Uptake into Nonphagocytic Cells.

Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis and wound infections. A few studies have suggested that A. haemolyticum is able to induce its uptake into nonphagocytic epithelial cells, but the bacterial factors associated with host cell invasion and the host cell processes involved have yet to be studied. We investigated how two A. haemolyticum virulence factors, arcanolysin (ALN) and phospholipase D (PLD), affect the ability of the bacteria to adhere to and subsequently invade Detroit 562 pharyngeal epithelial cells. The sphingomyelinase activity of phospholipase D was necessary to increase bacterial adherence, while the absence of a functional arcanolysin had no effect on A. haemolyticum adherence but did lead to a decrease in A. haemolyticum invasion into Detroit 562 cells. Because of the known roles of cholesterol-dependent cytolysins in disrupting calcium gradients and inducing F-actin-mediated bacterial internalization, we sought to determine whether ALN and PLD played a similar role in the ability of A. haemolyticum to invade nonphagocytic cells. Elimination of extracellular calcium and inhibition of the Arp2/3 complex or F-actin polymerization also caused a decrease in the ability of A. haemolyticum to invade Detroit 562 cells. Overall, our findings suggest that A. haemolyticum utilizes phospholipase D primarily for adherence and utilizes arcanolysin primarily for invasion into Detroit 562 cells in a process dependent on extracellular calcium and F-actin polymerization. Our work marks the first insight into how the individual activities of arcanolysin and phospholipase D affect A. haemolyticum host-pathogen interactions using the biologically relevant Detroit 562 cell line.

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.

Thrombotic thrombocytopenic purpura-like syndrome associated with arcanobacterium pyogenes endocarditis in a post-transplant patient: A case report.

Systemic Arcanobacterium pyogenes is a rare bacterial infection in humans.1The diagnosis of thrombotic thrombocytopenic purpura (TTP)-like syndrome and infective endocarditis (IE) is often elusive. We report a case of TTP-like syndrome associated with A. pyogenes endocarditis in a post-allogenic transplant patient.

Arcanobacterium haemolyticum Phospholipase D Enzymatic Activity Promotes the Hemolytic Activity of the Cholesterol-Dependent Cytolysin Arcanolysin.

Arcanolysin, produced by the human pathogen Arcanobacterium haemolyticum, is a cholesterol-dependent cytolysin. To mediate the pore-formation process, arcanolysin is secreted by A. haemolyticum and then must interact with cholesterol embedded within a host membrane. However, arcanolysin must compete with membrane components, such as the phospholipid sphingomyelin, to interact with cholesterol and form pores. Cholesterol forms transient hydrogen bonds with the extracellular portion of sphingomyelin, shielding cholesterol from extracellular factors, including arcanolysin. A. haemolyticum also produces a sphingomyelin-specific phospholipase D, which removes the choline head from sphingomyelin, leaving cyclic-ceramide phosphate and eliminating the potential for cholesterol sequestration. We hypothesized that the enzymatic activity of phospholipase D decreases sphingomyelin-mediated cholesterol sequestration and increases cholesterol accessibility for arcanolysin. Using purified arcanolysin and phospholipase D, we demonstrate that the enzymatic activity of phospholipase D is necessary to promote arcanolysin-mediated hemolysis in both time- and concentration-dependent manners. Phospholipase D promotion of arcanolysin-mediated cytotoxicity was confirmed in Detroit 562 epithelial cells. Furthermore, we determined that incubating phospholipase D with erythrocytes corresponds with an increase in the amount of arcanolysin bound to host membranes. This observation suggests that phospholipase D promotes arcanolysin-mediated cytotoxicity by increasing the ability of arcanolysin to bind to a host membrane.