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.

Mycobacterium ulcerans-Bordetella trematum chronic tropical cutaneous ulcer: A four-case series, Côte d'Ivoire.

BACKGROUND: Chronic tropical cutaneous ulcers remain a neglected medical condition in West Africa, particularly Buruli ulcer, which is caused by mycolactone cytotoxin-secreting Mycobacterium ulcerans (M. ulcerans). Medical management of this highly debilitating and necrotising skin infection may be modified by colonisation and co-infection of the ulcer by opportunistic and pathogenic microorganisms, which considerably delays and increases the cost of treatment. METHODOLOGY/PRINCIPAL FINDING: We diagnosed chronic tropical cutaneous ulcers in nine patients in Côte d'Ivoire using M. ulcerans-specific PCRs and culturomics. This revealed M. ulcerans in 7/9 ulcer swabs and 5/9 control swabs as well as an additional 122 bacterial species, 32 of which were specific to ulcers, 61 specifics to the controls, and 29 which were shared, adding 40 bacterial species to those previously reported. Whole genome sequencing of four Bordetella trematum (B. trematum) isolates in four Buruli ulcer swabs and no controls indicated cytolethal distending toxins, as confirmed by cytotoxic assay. CONCLUSIONS/SIGNIFICANCE: In four cases of Buruli ulcer in Côte d'Ivoire, B. trematum was a co-pathogen which was resistant to rifampicin and clarithromycin, unmatching M. ulcerans antibiotic susceptibility profile and counteracting the current treatment of Buruli ulcer in West Africa and Australia. Thus, we report here chronic mixed M. ulcerans-B. trematum chronic tropical ulcer as a specific form of Buruli ulcer in West Africa.

Two Streptococcus pyogenes emm types and several anaerobic bacterial species are associated with idiopathic cutaneous ulcers in children after community-based mass treatment with azithromycin.

BACKGROUND: In yaws-endemic areas, two-thirds of exudative cutaneous ulcers (CU) are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD); one-third are classified as idiopathic ulcers (IU). A yaws eradication campaign on Lihir Island in Papua New Guinea utilizing mass drug administration (MDA) of azithromycin initially reduced but failed to eradicate yaws; IU rates remained constant throughout the study. Using 16S rRNA gene sequencing, we previously determined that Streptococcus pyogenes was associated with some cases of IU. Here, we applied shotgun metagenomics to the same samples we analyzed previously by 16S rRNA sequencing to verify this result, identify additional IU-associated microorganisms, and determine why S. pyogenes-associated IU might have persisted after MDA of azithromycin. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced DNA extracted from 244 CU specimens separated into four groups based upon microorganism-specific PCR results (HD+, TP+, TP+HD+, and TP-HD- or IU). S. pyogenes was enriched in IU (24.71% relative abundance [RA]) specimens compared to other ulcer sub-groups, confirming our prior results. We bioinformatically identified the emm (M protein gene) types found in the S. pyogenes IU specimens and found matches to emm156 and emm166. Only ~39% of IU specimens contained detectable S. pyogenes, suggesting that additional organisms could be associated with IU. In the sub-set of S. pyogenes-negative IU specimens, Criibacterium bergeronii, a member of the Peptostreptococcaceae, and Fusobacterium necrophorum (7.07% versus 0.00% RA and 2.18% versus 0.00% RA, respectively), were enriched compared to the S. pyogenes-positive sub-set. Although a broad range of viruses were detected in the CU specimens, none were specifically associated with IU. CONCLUSIONS/SIGNIFICANCE: Our observations confirm the association of S. pyogenes with IU in yaws-endemic areas, and suggest that additional anaerobic bacteria, but not other microorganisms, may be associated with this syndrome. Our results should aid in the design of diagnostic tests and selective therapies for CU.

Trial of Three Rounds of Mass Azithromycin Administration for Yaws Eradication.

BACKGROUND: Treponema pallidum subspecies pertenue causes yaws. Strategies to better control, eliminate, and eradicate yaws are needed. METHODS: In an open-label, cluster-randomized, community-based trial conducted in a yaws-endemic area of Papua New Guinea, we randomly assigned 38 wards (i.e., clusters) to receive one round of mass administration of azithromycin followed by two rounds of target treatment of active cases (control group) or three rounds of mass administration of azithromycin (experimental group); round 1 was administered at baseline, round 2 at 6 months, and round 3 at 12 months. The coprimary end points were the prevalence of active cases of yaws, confirmed by polymerase-chain-reaction assay, in the entire trial population and the prevalence of latent yaws, confirmed by serologic testing, in a subgroup of asymptomatic children 1 to 15 years of age; prevalences were measured at 18 months, and the between-group differences were calculated. RESULTS: Of the 38 wards, 19 were randomly assigned to the control group (30,438 persons) and 19 to the experimental group (26,238 persons). A total of 24,848 doses of azithromycin were administered in the control group (22,033 were given to the participants at round 1 and 207 and 2608 were given to the participants with yaws-like lesions and their contacts, respectively, at rounds 2 and 3 [combined]), and 59,852 doses were administered in the experimental group. At 18 months, the prevalence of active yaws had decreased from 0.46% (102 of 22,033 persons) at baseline to 0.16% (47 of 29,954 persons) in the control group and from 0.43% (87 of 20,331 persons) at baseline to 0.04% (10 of 25,987 persons) in the experimental group (relative risk adjusted for clustering, 4.08; 95% confidence interval [CI], 1.90 to 8.76). The prevalence of other infectious ulcers decreased to a similar extent in the two treatment groups. The prevalence of latent yaws at 18 months was 6.54% (95% CI, 5.00 to 8.08) among 994 children in the control group and 3.28% (95% CI, 2.14 to 4.42) among 945 children in the experimental group (relative risk adjusted for clustering and age, 2.03; 95% CI, 1.12 to 3.70). Three cases of yaws with resistance to macrolides were found in the experimental group. CONCLUSIONS: The reduction in the community prevalence of yaws was greater with three rounds of mass administration of azithromycin at 6-month intervals than with one round of mass administration of azithromycin followed by two rounds of targeted treatment. Monitoring for the emergence and spread of antimicrobial resistance is needed. (Funded by Fundació "la Caixa" and others; ClinicalTrials.gov number, NCT03490123.).

Corynebacterium rouxii, a recently described member of the C. diphtheriae group isolated from three dogs with ulcerative skin lesions.

Corynebacterium (C.) diphtheriae is one of the two etiological pathogens for human diphtheria with significant morbidity and mortality. Recently, members of its biovar Belfanti have been described as two novel species, C. belfantii and C. rouxii. The most important virulence factor and also the premise to cause diphtheria is the isolate's capacity to encode and express the diphtheria toxin (DT). In contrast to C. ulcerans, which represents a potentially zoonotic pathogen, C. diphtheriae (incl. the novel deduced species) has almost exclusively been found to comprise a human pathogen. We here report three rare cases of C. rouxii isolation from dogs suffering from disseminated poly-bacterial exsudative to purulent dermatitis and a traumatic labial defect, respectively. The isolates were identified as C. diphtheriae based on commercial biochemistry and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis. However, recently described specific spectral peaks were highly similar to spectra of C. rouxii, which was confirmed by whole genome sequencing. Further investigations of the dog isolates for the presence of DT by tox gene qPCR revealed negative results. The findings from this study point out that skin infections in companion animals can be colonized by uncommon and so believed human specific pathogens, thereby resembling the clinical signs of cutaneous diphtheria.

An Eschar-like souvenir from a journey to Colombia: Ecthyma gangrenosum as a differential diagnosis of tropical diseases in immunocompromised patients - a case report.

BACKGROUND: Ecthyma gangrenosum (EG) is a cutaneous infectious disease characterized by eschar-like skin ulcers typically caused by Pseudomonas aeruginosa. Here, we report a case of relapsing EG in a patient who had returned from a trip to Colombia, thus establishing EG as an important differential diagnosis of tropical diseases, and demonstrating that even long-term antibiotic treatment can result in only partial remission of EG. CASE PRESENTATION: A 77-year-old man with underlying chronic lymphocytic leukemia (CLL) on ibrutinib treatment was admitted because of a superinfected mosquito bite on the left ear and multiple partially necrotic skin lesions disseminated all over the entire body five days after returning from a trip to Colombia. The initial clinical suspicion of a tropical disease (leishmaniosis, systemic mycosis, or others) could not be confirmed. During the diagnostic workup, microbiological cultures of the skin biopsies and bronchoalveolar lavage revealed Pseudomonas aeruginosa, leading to a diagnosis of EG. Initial antibiotic treatment resulted in partial remission. However, the patient had to be re-admitted due to a relapse 3-4 weeks after the first episode. Finally, the patient was successfully treated with a combined approach consisting of antibiotics, recurrent surgical incisions, and administration of immunoglobulins. CONCLUSIONS: In conclusion, EG should be considered as a differential diagnosis in immunosuppressed patients presenting with eschar-like skin ulcers. A combined treatment approach seems to be the best choice to achieve clinical cure and avoid relapse.

Tropical ulcers: the first imported cases and review of the literature.

BACKGROUND: A tropical ulcer is a bacterial necrotizing disease of the skin, with an acute or chronic clinical course, caused by anaerobic bacteria, notably Fusobacteria spp. OBJECTIVES: We present six Italian tourists who acquired tropical ulcers in tropical and subtropical countries. MATERIALS & METHODS: Four males and two females acquired a skin ulcer during trips to Brazil, Malaysia, Fiji Islands, Zambia, Tanzania and India. In all patients, medical history, physical and dermatological examination, laboratory tests, bacteriological examinations and biopsy were carried out. RESULTS: All patients were in good general health. All patients stated that the ulcer was caused by a trauma. No fever was reported. Neither lymphangitis nor lymphadenopathy were detected. The ulcer was located on a forearm in one patient, on a leg in two and on an ankle in three patients. All ulcers were malodorous and painful. Laboratory tests revealed mild leucocytosis and a mild increase in erythrocyte sedimentation rate and C-reactive protein. Results of bacteriological examinations revealed the presence of Fusobacterium spp. in five patients. Other bacteria were identified in all patients. Histopathological examination showed: necrosis of the epidermis and dermis; vascular dilatation; oedema in the dermis; massive infiltration with neutrophils, lymphocytes and histiocytes; and fragmented collagen bundles. No signs of vasculitis were observed. All patients were successfully treated with oral metronidazole (1 g/day for two weeks) and, according to antibiograms, with different systemic antibiotics. CONCLUSION: To our knowledge, these are the first cases of tropical ulcers reported in Western tourists.

Ulcerative skin lesions among children in Cameroon: It is not always Yaws.

Outbreaks of yaws-like ulcerative skin lesions in children are frequently reported in tropical and sub-tropical countries. The origin of these lesions might be primarily traumatic or infectious; in the latter case, Treponema pallidum subspecies pertenue, the yaws agent, and Haemophilus ducreyi, the agent of chancroid, are two of the pathogens commonly associated with the aetiology of skin ulcers. In this work, we investigated the presence of T. p. pertenue and H. ducreyi DNA in skin ulcers in children living in yaws-endemic regions in Cameroon. Skin lesion swabs were collected from children presenting with yaws-suspected skin lesions during three outbreaks, two of which occurred in 2017 and one in 2019. DNA extracted from the swabs was used to amplify three target genes: the human ß2-microglobulin gene to confirm proper sample collection and DNA extraction, the polA gene, highly conserved among all subspecies of T. pallidum, and the hddA gene of H. ducreyi. A fourth target, the tprL gene was used to differentiate T. p. pertenue from the other agents of human treponematoses in polA-positive samples. A total of 112 samples were analysed in this study. One sample, negative for ß2-microglobulin, was excluded from further analysis. T. p. pertenue was only detected in the samples collected during the first 2017 outbreak (12/74, 16.2%). In contrast, H. ducreyi DNA could be amplified from samples from all three outbreaks (outbreak 1: 27/74, 36.5%; outbreak 2: 17/24, 70.8%; outbreak 3: 11/13, 84.6%). Our results show that H. ducreyi was more frequently associated to skin lesions in the examined children than T. p. pertenue, but also that yaws is still present in Cameroon. These findings strongly advocate for a continuous effort to determine the aetiology of ulcerative skin lesions during these recurring outbreaks, and to inform the planned mass treatment campaigns to eliminate yaws in Cameroon.

Non-surgical treatment of tetanus infection associated with breast cancer skin ulcer: a case report and literature review.

BACKGROUND: Previous studies have reported poor prognosis in cases of tetanus that develops after bacteria enters via breast cancer-related skin ulcers that are not treated with surgical debridement. Herein, we review the literature concerning this presentation and report the first case of complete remission from tetanus without surgical debridement of the skin ulcer. CASE PRESENTATION: An Asian woman aged over 60 years had a history of skin ulcer caused by breast cancer. She was diagnosed with tetanus due to trismus and opisthotonus. Based on the suspicion that the skin ulcer was the portal of entry for tetanus bacteria, we considered several debridement and thoracic surgical options for tetanus treatment. However, debridement was not performed as the surgery was considered high risk and the patient did not consent to it. The patient received treatment with anti-tetanus globulin and metronidazole; sound insulation and shielding were also performed in a dark room. Subsequently, the patient's symptoms improved, and sound insulation and deep sedation management were completed on 19th day of hospitalization. With no symptom recurrence, the patient was discharged on Day 54. To date, over 3 years after treatment, no evidence of tetanus recurrence has been observed. The case was characterized by a lack of autonomic hyperactivity. The tetanus severity was likely representative of the low amount of toxin that the patient was exposed to. CONCLUSION: This case involved moderate severity tetanus originating from a chronic skin ulcer related to breast cancer. The patient survived without undergoing extensive debridement. No evidence of tetanus relapse was observed during the follow-up period, likely due to vaccination that might have restored the patient's active immunity. Debridement is not always necessary for tetanus complicated by breast cancer skin ulcers. Furthermore, appropriate toxoid vaccination is critical for preventing the onset and recurrence of tetanus in these patients.

Streptococcus pyogenes Is Associated with Idiopathic Cutaneous Ulcers in Children on a Yaws-Endemic Island.

Exudative cutaneous ulcers (CU) in yaws-endemic areas are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD), but one-third of CU cases are idiopathic (IU). Using mass drug administration (MDA) of azithromycin, a yaws eradication campaign on Lihir Island in Papua New Guinea reduced but failed to eradicate yaws; IU rates remained constant throughout the campaign. To identify potential etiologies of IU, we obtained swabs of CU lesions (n = 279) and of the skin of asymptomatic controls (AC; n = 233) from the Lihir Island cohort and characterized their microbiomes using a metagenomics approach. CU bacterial communities were less diverse than those of the AC. Using real-time multiplex PCR with pathogen-specific primers, we separated CU specimens into HD-positive (HD+), TP+, HD+TP+, and IU groups. Each CU subgroup formed a distinct bacterial community, defined by the species detected and/or the relative abundances of species within each group. Streptococcus pyogenes was the most abundant organism in IU (22.65%) and was enriched in IU compared to other ulcer groups. Follow-up samples (n = 31) were obtained from nonhealed ulcers; the average relative abundance of S. pyogenes was 30.11% in not improved ulcers and 0.88% in improved ulcers, suggesting that S. pyogenes in the not improved ulcers may be azithromycin resistant. Catonella morbi was enriched in IU that lacked S. pyogenes As some S. pyogenes and TP strains are macrolide resistant, penicillin may be the drug of choice for CU azithromycin treatment failures. Our study will aid in the design of diagnostic tests and selective therapies for CU.IMPORTANCE Cutaneous ulcers (CU) affect approximately 100,000 children in the tropics each year. While two-thirds of CU are caused by Treponema pallidum subspecies pertenue and Haemophilus ducreyi, the cause(s) of the remaining one-third is unknown. Given the failure of mass drug administration of azithromycin to eradicate CU, the World Health Organization recently proposed an integrated disease management strategy to control CU. Success of this strategy requires determining the unknown cause(s) of CU. By using 16S rRNA gene sequencing of swabs obtained from CU and the skin of asymptomatic children, we identified another possible cause of skin ulcers, Streptococcus pyogenes Although S. pyogenes is known to cause impetigo and cellulitis, this is the first report implicating the organism as a causal agent of CU. Inclusion of S. pyogenes into the integrated disease management plan will improve diagnostic testing and treatment of this painful and debilitating disease of children and strengthen elimination efforts.

Sporothrix brasiliensis on cats with skin ulcers in Southern Brazil.

Sporotrichosis is a disease caused by thermally dimorphic fungi belonging to the Sporothrix schenckii complex. We report the occurrence of multiple cases of sporotrichosis in cats in Porto Alegre (RS), Brazil, from 2015 to 2019. A set of 21 fungal isolates were collected and the partial sequence of calmodulin (CAL) gene compared. All isolates were identified at phenotypic and molecular level as S. brasiliensis. Phylogenetic analysis showed the isolates clustered in two distinct groups, suggesting the presence of multiple genetic variants of S. brasiliensis in the region.

Yaws, Haemophilus ducreyi, and Other Bacterial Causes of Cutaneous Ulcer Disease in the South Pacific Islands.

Cutaneous ulcers in the tropics are a painful and debilitating condition that anchors people into poverty. In rural regions of the South Pacific, infectious cutaneous ulcers are caused mainly by bacteria, including Treponema pallidum pertenue (yaws), Haemophilus ducreyi, and polymicrobial ulcers. For this group of infections the term cutaneous ulcer disease (CUD) is proposed. Some infections can cause malformations on the bone that have a permanent impact on lives in endemic communities. Better characterization of CUD may help design diagnostic tools and more effective antimicrobial therapies. This review updates the knowledge of CUD and discusses optimized terminology and syndromic management.

Dual Antibacterial Activities and Biofilm Eradication of a Marine Peptide-N6NH2 and Its Analogs against Multidrug-Resistant Aeromonas veronii.

Aeromonas veronii is one of the main pathogens causing various diseases in humans and animals. It is currently difficult to eradicate drug-resistant A. veronii due to the biofilm formation by conventional antibiotic treatments. In this study, a marine peptide-N6NH2 and its analogs were generated by introducing Orn or replacing with D-amino acids, Val and Pro; their enzymic stability and antibacterial/antibiofilm ability against multi-drug resistant (MDR) A. veronii ACCC61732 were detected in vitro and in vivo, respectively. The results showed that DN6NH2 more rapidly killed A. veronii ACCC61732 and had higher stability in trypsin, simulated gastric/intestinal fluid, proteinase K, and mouse serum than the parent peptide-N6NH2. DN6NH2 and other analogs significantly improved the ability of N6NH2 to penetrate the outer membrane of A. veronii ACCC61732. DN6NH2, N6PNH2 and V112N6NH2 protected mice from catheter-associated biofilm infection with MDR A. veronii ACCC61732, superior to N6NH2 and CIP. DN6NH2 had more potent efficacy at a dose of 5 µmol/kg (100% survival) in a mouse peritonitis model than other analogs (50-66.67%) and CIP (83.33%), and it inhibited the bacterial translocation, downregulated pro-inflammatory cytokines, upregulated the anti-inflammatory cytokine, and ameliorated multiple-organ injuries (including the liver, spleen, lung, and kidney). These data suggest that the analogs of N6NH2 may be a candidate for novel antimicrobial and antibiofilm agents against MDR A. veronii infections.

THE EFFECT OF PHOTODYNAMIC THERAPY AND PLATELET-ENRICHED PLASMA ON THE HEALING OF SKIN RADIATION ULCERS INFECTED BY STAPHYLOCOCCUS AUREUS. / VPLYV FOTODYNAMIChNOÏ TERAPIÏ TA ZBAGAChENOÏ TROMBOTsYTAMY PLAZMY NA PROTsESY ZAGOIeNNIa PROMENEVYKh VYRAZOK ShKIRY, INFIKOVANYKh STAPHYLOCOCCUS AUREUS.

OBJECTIVE: To determine in experiment the quality of healing of skin radiation ulcers infected with Staphylococcusaureus (S. aureus) under the photodynamic therapy (PDT) administration and the use of platelet-rich plasma (PRP). MATERIALS AND METHODS: The experiment was performed on 95 male WAG rats of 6 months of age, which were divided into three groups. Group 1 included animals in whom a skin radiation ulcers in the thigh area was simulated, followed by application of a 0.2 ml suspension of reference strain of S. aureus to its surface on the 7th day after irradiation. Group 2 included animals with S. aureus-infected skin radiation ulcers, in whom the PET was administered aday after infection contamination. Group 3 included animals with S. aureus-infected skin radiation ulcers, in whomthe PDT was administered a day after infection in the morning, and the PRP was manifold injected in periwound areain the afternoon. The skin with underlying soft tissues from the area of radiation damage were the material for morphological examination. The hematoxylin and eosin, picrofuxin according to van Gizon, Mallory staining wereapplied to micropreparations. A morphometric study was conducted. RESULTS: In animals with skin radiation ulcers, in whom the PDT was administered upon infection with S. aureus(group 2), compared with animals with simulated infected skin radiation ulcers without treatment (group 1), Theactivation (i.e. accelerating) of the healing occurred for the period from the 14th to the 52nd day of experiment dueto the active processes of wound cleansing from necrotized tissues, less pronounced inflammatory changes in thelesion, and active of appearance and maturation of granulation tissue, less pronounced hemodynamic, ischemic andalternative disorders in the dermis, hypodermis, muscle tissue surrounding the wound cavity, activation of proliferative processes in epithelial layer localized in the marginal parts of the wound. Formation of pathological (hypertrophic or keloid) scar of the skin was the result of healing of skin radiation ulcer infected with S. aureus. In animalswith radiation ulcers infected with S. aureus, in the case of PDT and PRP (group 3) the regenerative process wasdirected not only at accelerating the rate of healing, but also on restoration of original structure of the lost parts ofthe skin compared with only PDT administration (group 2). Acceleration of the healing of the infected skin radiation ulcer in animals of groups 2 and 3 was due to similar processes. CONCLUSIONS: Photodynamic therapy activates and accelerates the healing process of skin radiation ulcers infectedwith S. aureus and leads to formation of a pathological scar (hypertrophic or keloid). Healing of the infected S. aureusradiation ulcers occurs more actively upon the photodynamic therapy administration in combination with multipleperiwound injections of the platelet-enriched plasma, compared with only photodynamic therapy administration,and finishes with an organotypic regeneration and almost complete skin recovery.