Prevalence and genetic diversity of azole-resistant Malassezia pachydermatis isolates from canine otitis and dermatitis: A 2-year study.

Despite previous reports on the emergence of Malassezia pachydermatis strains with decreased susceptibility to azoles, there is limited information on the actual prevalence and genetic diversity of azole-resistant isolates of this yeast species. We assessed the prevalence of azole resistance in M. pachydermatis isolates from cases of dog otitis or skin disease attended in a veterinary teaching hospital during a 2-year period and analyzed the ERG11 (encoding a lanosterol 14-α demethylase, the primary target of azoles) and whole genome sequence diversity of a group of isolates that displayed reduced azole susceptibility. Susceptibility testing of 89 M. pachydermatis isolates from 54 clinical episodes (1-6 isolates/episode) revealed low minimum inhibitory concentrations (MICs) to most azoles and other antifungals, but 11 isolates from six different episodes (i.e., 12.4% of isolates and 11.1% of episodes) had decreased susceptibility to multiple azoles (fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, and/or voriconazole). ERG11 sequencing of these 11 azole-resistant isolates identified eight DNA sequence profiles, most of which contained amino acid substitutions also found in some azole-susceptible isolates. Analysis of whole genome sequencing (WGS) results revealed that the azole-resistant isolates from the same episode of otitis, or even different episodes affecting the same animal, were more genetically related to each other than to isolates from other dogs. In conclusion, our results confirmed the remarkable ERG11 sequence variability in M. pachydermatis isolates of animal origin observed in previous studies and demonstrated the value of WGS for disentangling the epidemiology of this yeast species.

We analyzed the prevalence and diversity of azole-resistant Malassezia pachydermatis isolates in a veterinary hospital. A low prevalence of multi-azole resistance (c.10% of isolates and cases) was found. Whole genome and ERG11 sequencing of resistant isolates revealed remarkable genetic diversity.

[Ear tuberculosis concomitant with pulmonary localization in an HIV-immunosuppressed patient, in Bamako, Mali]. / Tuberculose auriculaire concomitante à une localisation pulmonaire chez un patient immunodéprimé par le VIH, à Bamako, Mali.

Introduction/Rationale: Tuberculosis remains a major public health issue. It is an opportunistic pathology, very common in HIV-immunocompromised persons, classifying it at the WHO stage 4. Ear tuberculosis remains a rare and under-diagnosed clinical form. We report here a case of ear tuberculosis concomitant with pulmonary localization in an HIV-immunosuppressed person on triple antiretroviral therapy aged 32 years hospitalized in Bamako (Mali) to discuss the diagnostic and therapeutic difficulties posed by this rare localization. Description of the case: The patient had a chronic productive cough, otalgia and right chronic purulent otorrhea. The search for acid-resistant bacilli was positive for direct examination in gastric casing fluid and swabbing of the ear pus, confirming the diagnosis of tuberculosis. Anti-tuberculosis treatment instituted for 6 months associated with adjuvants resulted in complete healing of the patient. Discussion/conclusion: Although rare, ear localization must be actively sought. Etiological treatment must be instituted upon confirmation of the diagnosis to avoid complications and sequelae.

Pilot study of dogs with suppurative and non-suppurative Malassezia otitis: A case series.

BACKGROUND: Rarely, Malassezia otitis presents as a painful, erosive otitis with an otic discharge containing Malassezia and neutrophils on cytology. There are no published reports of this type of suppurative Malassezia otitis (SMO). The role of Malassezia hypersensitivity in otitis is still unknown, and no association has been demonstrated with SMO. We compared Malassezia IgE levels, intradermal test and histology changes in SMO dogs with the more conventional Malassezia otitis (MO) presentation. RESULTS: Three dogs (case 1, case 2 and case 3) were diagnosed with SMO, one dog (case 4) was diagnosed with unilateral MO and unilateral SMO, and one dog (case 5) was diagnosed with MO. Only one case (case 4) with SMO/MO had a positive Intradermal Allergy Test (IDAT) and elevated IgE levels for Malassezia. Histopathology findings from SMO revealed: interface dermatitis (case 1 and 3), lymphocytic dermatitis (case 2) and chronic hyperplastic eosinophilic and lymphoplasmacytic dermatitis (case 4). Histopathology findings from MO showed perivascular dermatitis (case 4 and 5). All the cases were treated successfully. CONCLUSIONS: SMO presents with a distinct clinical phenotype in comparison with conventional MO. No consistent aetiology could be isolated. In these clinical cases it is possible that previous treatments could have influenced the results. More research is needed to understand the possible aetiologies and the pathogenesis of SMO.

Cerumen microbial community shifts between healthy and otitis affected dogs.

Otitis externa is a common multifactorial disease in dogs, characterized by broad and complex modifications of the ear microbiota. The goal of our study was to describe the ear cerumen microbiota of healthy dogs, within the same animal and between different animals, and to compare the cerumen microbiota of otitis affected dogs with that of healthy animals. The present study included 26 healthy dogs, 16 animals affected by bilateral otitis externa and 4 animals affected by monolateral otitis externa. For each animal cerumen samples from the right and left ear were separately collected with sterile swabs, and processed for DNA extraction and PCR amplification of the 16S rRNA gene. Amplicon libraries were sequenced using an Ion Torrent Personal Genome Machine (PGM), and taxonomical assignment and clustering were performed using QIIME 2 software. Our results indicate that the bacterial community of the cerumen in healthy dogs was characterized by extensive variability, with the most abundant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria. The analysis of both alpha and beta diversity between pairs of left and right ear samples from the same dog within the group of affected animals displayed higher differences than between paired samples across healthy dogs. Moreover we observed reduced bacterial richness in the affected group as compared with controls and increased variability in population structure within otitis affected animals, often associated with the proliferation of a single bacterial taxon over the others. Moreover, Staphylococcus and Pseudomonas resulted to be the bacterial genera responsible for most distances between the two groups, in association with differences in the bacterial community structure. The cerumen microbiota in healthy dogs exhibits a complex bacterial population which undergoes significant modifications in otitis affected animals.

Recurrent polyp formation with Candida tropicalis infection and otitis in a dog.

To describe a case of a recurrent Candida tropicalis otitis externa, media and interna in a dog with an ear polyp. A 9-year-old Irish Setter was presented with 2 episodes of otitis sinistra, left-sided vestibular syndrome and Horner syndrome 7 months apart. At the first episode a benign ear polyp was extracted and Candida tropicalis cultured from the left middle ear. The neurological signs disappeared within 7 days, the Candida infection was more difficult to treat. Seven months later, a polyp was found in the ear again and cytology was consistent with Candida tropicalis. A unilateral left total ear canal ablation with lateral bulla osteotomy was performed and a middle ear culture confirmed Candida tropicalis. Treatment led to resolution of clinical signs. Candida tropicalis, an emerging pathogen, should be considered in cases of recurrent yeast otitis and may be difficult to treat.

Extended spectrum beta lactamase producing bacteria among outpatients with ear infection at FelegeHiwot Referral Hospital, North West Ethiopia.

BACKGROUND: Antibiotic resistant bacteria particularly extended-spectrum beta lactamase (ESBL) producing are of major concern for management of outpatients. They can spread rapidly and are associated with poor patient outcome. However, there is scarcity of information on ear infection with ESBL producing bacteria in Ethiopia. Therefore, this study investigates the prevalence of ear infection with ESBL producing bacteria among outpatients attending Felegehiwot Referral Hospital, Northwest Ethiopia. METHODS: A hospital based cross-sectional study was conducted from May, 2018 to January, 2019. Demographic and clinical data were collected with face to face interview and were complemented with patient card review. Ear discharge specimens were collected from study participants using swab technique. All ear swab specimens were cultured using standard microbiological techniques. The ESBL producing bacteria were detected by double disc synergy test and interpreted based on Clinical and laboratory Standards Institute Guidelines. Chi-square and fisher's exact tests were calculated to check association between variables. RESULTS: A total of 236 patients (male = 138 and female = 98) with ear infection took part in the study. The median age of the participants was 20years. Overall, 10 (4.23%, 95%CI; 2.3-7.6%) of patients had ear infection with ESBL producing bacteria. Other chronic illnesses (p = 0.003), history of hospital visit and treatment (p = 0.006) and history of antibiotic use without physician's prescription (p<0.001) had significant association with prevalence of ESBL producing bacteria in ear infection. The proportion of ear infection with ESBL producing P.mirabilis, P.aeruginosa and K.pneumoniae were 4 (1.7%), 3 (1.3%) and 2 (0.8%), respectively. All ESBL producing isolates were MDR (100%). Overall, 58 (43%) species were MDR. P.aeruginosa was the leading MDR isolate 29 (53.7%).For all bacterial isolates of ear infection, ampicillin(93.3%) and amoxicillin-clavulanic acid (58.5%) revealed high level of resistance whereas low resistance rates were observed for ciprofloxacin (5.2%), third generation cephalosporin (11.9-20%) and aztreonam (16.3%). CONCLUSIONS: Ear infection due to ESBL producing bacteria coupled with high levels of MDR is becoming a growing concern for outpatients in the study area. Regular detection of these bacteria and wise use of antibiotics are needed to stop the spread of this form of resistance.

The canine skin and ear microbiome: A comprehensive survey of pathogens implicated in canine skin and ear infections using a novel next-generation-sequencing-based assay.

This study analyzed the complex bacterial and fungal microbiota of healthy and clinically affected canine ear and skin samples. A total of 589 canine samples were included: 257 ear swab samples (128 healthy vs. 129 clinically affected) and 332 skin swab samples (172 healthy vs. 160 clinically affected) were analyzed using next-generation sequencing (NGS) to determine both relative and absolute abundances of bacteria and fungi present in the samples. This study highlighted the canine microbiota of clinically affected cases was characterized by an overall loss of microbial diversity, high microbial biomass, with overgrowth of certain members of the microbiota. The observed phenotype of these samples was best described by the combination of both relative and absolute microbial abundances. Compared to healthy samples, 78.3% of the clinically affected ear samples had microbial overgrowth; 69.8% bacterial overgrowth, 16.3% fungal overgrowth, and 7.0% had both bacterial and fungal overgrowth. The most important microbial taxa enriched in clinically affected ears were Malassezia pachydermatis, Staphylococcus pseudintermedius, Staphylococcus schleiferi, and a few anaerobic bacteria such as Finegoldia magna, Peptostreptococcus canis, and Porphyromonas cangingivalis. The anaerobic microbes identified here were previously not commonly recognized as pathogens in canine ear infections. Similar observations were found for skin samples, but yeasts and anaerobes were less abundant when compared to clinically affected cases. Results highlighted herein, signify the potential of NGS-based methods for the accurate quantification and identification of bacterial and fungal populations in diagnosing canine skin and ear infections, and highlight the limitations of traditional culture-based testing.

Whole-genome analyses reveal gene content differences between nontypeable Haemophilus influenzae isolates from chronic obstructive pulmonary disease compared to other clinical phenotypes.

Nontypeable Haemophilus influenzae (NTHi) colonizes human upper respiratory airways and plays a key role in the course and pathogenesis of acute exacerbations of chronic obstructive pulmonary disease (COPD). Currently, it is not possible to distinguish COPD isolates of NTHi from other clinical isolates of NTHi using conventional genotyping methods. Here, we analysed the core and accessory genome of 568 NTHi isolates, including 40 newly sequenced isolates, to look for genetic distinctions between NTHi isolates from COPD with respect to other illnesses, including otitis media, meningitis and pneumonia. Phylogenies based on polymorphic sites in the core-genome did not show discrimination between NTHi strains collected from different clinical phenotypes. However, pan-genome-wide association studies identified 79 unique NTHi accessory genes that were significantly associated with COPD. Furthermore, many of the COPD-related NTHi genes have known or predicted roles in virulence, transmembrane transport of metal ions and nutrients, cellular respiration and maintenance of redox homeostasis. This indicates that specific genes may be required by NTHi for its survival or virulence in the COPD lung. These results advance our understanding of the pathogenesis of NTHi infection in COPD lungs.

Tympanostomy Tube Controversies and Issues: State-of-the-Art Review.

OBJECTIVE: To review current pragmatic issues and controversies related to tympanostomy tubes in children, in the context of current best research evidence plus expert opinion to provide nuance, address uncertainties, and fill evidence gaps. METHODS: Each issue or controversy is followed by the relevant current best evidence, expert insight and opinion, and recommendations for action. The role of expert opinion and experience in forming conclusions is inversely related to the quality, consistency, and adequacy of published evidence. Conclusions are combined with opportunities for shared decision-making with caregivers to recommend pragmatic actions for clinicians in everyday settings. RESULTS: The issues and controversies discussed include (1) appropriate tube indications, (2) rationale for not recommending tubes for recurrent acute otitis media without persistent middle ear effusion, (3) role of tubes in at-risk children with otitis media with effusion, (4) role of new, automated tube insertion devices, (5) appropriateness and feasibility of in-office tube insertion in awake children, (6) managing methicillin-resistant Staphylococcus aureus acute tube otorrhea, and (7) managing recurrent or persistent tube otorrhea. CONCLUSIONS: Despite a substantial, and constantly growing, volume of high-level evidence on managing children with tympanostomy tubes, there will always be gaps, uncertainties, and controversies that benefit from clinician experience and expert opinion. In that regard, the issues discussed in this review article will hopefully aid clinicians in everyday, pragmatic management decisions.

Role of Biofilms in Post-Tympanostomy Tube Otorrhea.

OBJECTIVE: Nearly half of children who undergo tympanostomy tube (TT) insertion may experience otorrhea following surgery. We sought to review the evidence for the role of bacterial biofilms in post-tympanostomy tube otorrhea (PTTO) and the accumulated experience regarding the preventive measures for biofilm formation/adhesion on TTs. METHODS: English literature search for relevant MeSH keywords was conducted in the following databases: MEDLINE (via PubMed), Ovid Medline, Google Scholar, and Clinical Evidence (BMJ Publishing) between January 1, 1995, and December 31, 2019. Subsequently, articles were reviewed and included if biofilm was evident in PTTO. RESULTS: There is an increased evidence supporting the role of biofilms in PTTO. Studies on TT design and material suggest that nitinol and/or silicone TTs had a lower risk for PTTO and that biofilms appeared in specific areas, such as the perpendicular junction of the T-tubes and the round rims of the Paparella-type tubes. Biofilm-component DNAB-II protein family was present in half of children with PTTO, and targeting this protein may lead to biofilm collapse and serve as a potential strategy for PTTO treatment. Novel approaches for the prevention of biofilm-associated PTTO include changing the inherent tube composition; tube coating with antibiotics, polymers, plant extracts, or other biofilm-resistant materials; impregnation with antimicrobial compounds; and surface alterations by ion-bombardment or surface ionization, which are still under laboratory investigation. CONCLUSIONS: Currently, there is no type of TT on which bacteria will not adhere. The challenges of treating PTTO indicate the need for further research in optimization of TT design, composition, and coating.

Oxymetazoline, Mupirocin, Clotrimazole-Safe, Effective, Off-Label Agents for Tympanostomy Tube Care.

OBJECTIVES: Only a few medications have a United States Food and Drug Administration indications for prevention and/or treatment of infections in patients with tympanic perforations or tympanostomy tubes. We examined 3 off-label agents that have become important in tympanostomy tube care hoping to demonstrate the effectiveness and safety of each in experimental assays and human application. METHODS: Computerized literature review. RESULTS: (1) Oxymetazoline nasal spray applied at the time of surgery is equivalent to fluoroquinolone ear drops in the prevention of early postsurgical otorrhea and tympanostomy tube occlusion at the first postoperative visit. (2) Topical mupirocin 2% ointment is effective alone or in combination with culture-directed systemic therapy for the treatment of tympanostomy tube otorrhea caused by community-acquired, methicillin-resistant Staphylococcus aureus. (3) Topical clotrimazole 1% cream is highly active against the common yeast and fungi that cause otomycosis. A single application after microscopic debridement will cure fungal tympanostomy tube otorrhea in most cases. None of these 3 agents is ototoxic in animal histological or physiological studies, and each has proved safe in long-term clinical use. CONCLUSIONS: Oxymetazoline nasal spray, mupirocin ointment, and clotrimazole cream are safe and effective as off-label medications for tympanostomy tube care in children.

The Role of Mastoidectomy in Draining Ventilation Tubes.

OBJECTIVES: To discuss the indication for performing a mastoidectomy with catheter placement in patients with chronic tympanostomy tube otorrhea. METHODS: The Medical Literature Analysis and Retrieval System Online was searched via PubMed for relevant articles using serous mastoiditis, mastoidectomy, chronic otorrhea, tube otorrhea, tympanostomy tubes, and biofilm as keywords. RESULTS: Further understanding of the pathophysiology of otorrhea and the development of ototopical fluoroquinolones have made a draining tympanostomy tube more manageable. Nevertheless, chronic otorrhea refractory to an otolaryngologist's traditional treatment algorithm still occurs and may benefit from a mastoidectomy with antibiotic irrigation using a catheter in certain cases. We theorize that resolution of otorrhea results from this technique by decreasing the burden of diseased mucosa and providing a larger concentration or dose of antibiotic to the middle ear cleft through the antrum. High-resolution images of the technique and catheter placement are included in this review. CONCLUSIONS: Despite being an uncommon management strategy, the literature suggests an indication for performing a mastoidectomy in a small percentage of patients with a chronically draining tympanostomy tube.

A mouse ear skin model to study the dynamics of innate immune responses against Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus is a human pathogen that is a common cause of nosocomial infections and infections on indwelling medical devices, mainly due to its ability to shift between the planktonic and the biofilm/sessile lifestyle. Biofilm infections present a serious problem in human medicine as they often lead to bacterial persistence and thus to chronic infections. The immune responses elicited by biofilms have been described as specific and ineffective. In the few experiments performed in vivo, the importance of neutrophils and macrophages as a first line of defence against biofilm infections was clearly established. However, the bilateral interactions between biofilms and myeloid cells remain poorly studied and analysis of the dynamic processes at the cellular level in tissues inoculated with biofilm bacteria is still an unexplored field. It is urgent, therefore, to develop biologically sound experimental approaches in vivo designed to extract specific immune signatures from the planktonic and biofilm forms of bacteria. RESULTS: We propose an in vivo transgenic mouse model, used in conjunction with intravital confocal microscopy to study the dynamics of host inflammatory responses to bacteria. Culture conditions were created to prepare calibrated inocula of fluorescent planktonic and biofilm forms of bacteria. A confocal imaging acquisition and analysis protocol was then drawn up to study the recruitment of innate immune cells in the skin of LysM-EGFP transgenic mice. Using the mouse ear pinna model, we showed that inflammatory responses to S. aureus can be quantified over time and that the dynamics of innate immune cells after injection of either the planktonic or biofilm form can be characterized. First results showed that the ability of phagocytic cells to infiltrate the injection site and their motility is not the same in planktonic and biofilm forms of bacteria despite the cells being considerably recruited in both cases. CONCLUSION: We developed a mouse model of infection to compare the dynamics of the inflammatory responses to planktonic and biofilm bacteria at the tissue and cellular levels. The mouse ear pinna model is a powerful imaging system to analyse the mechanisms of biofilm tolerance to immune attacks.

Otomastoiditis por Scedosporium apiospermum. Alucinaciones como efecto adverso del voriconazol / Otomastoiditis due to Scedosporium apiospermum. Hallucinations as an adverse reaction to voriconazol

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Otopathogenic Staphylococcus aureus Invades Human Middle Ear Epithelial Cells Primarily through Cholesterol Dependent Pathway.

Chronic suppurative otitis media (CSOM) is one of the most common infectious diseases of the middle ear especially affecting children, leading to delay in language development and communication. Although Staphylococcus aureus is the most common pathogen associated with CSOM, its interaction with middle ear epithelial cells is not well known. In the present study, we observed that otopathogenic S. aureus has the ability to invade human middle ear epithelial cells (HMEECs) in a dose and time dependent manner. Scanning electron microscopy demonstrated time dependent increase in the number of S. aureus on the surface of HMEECs. We observed that otopathogenic S. aureus primarily employs a cholesterol dependent pathway to colonize HMEECs. In agreement with these findings, confocal microscopy showed that S. aureus colocalized with lipid rafts in HMEECs. The results of the present study provide new insights into the pathogenesis of S. aureus induced CSOM. The availability of in vitro cell culture model will pave the way to develop novel effective treatment modalities for CSOM beyond antibiotic therapy.

Biofilm production of Pseudomonas spp. isolates from canine otitis in three different enrichment broths.

BACKGROUND: Pseudomonas spp. are commonly isolated from dogs with clinical otitis and have been shown to produce biofilm. There is a paucity of studies demonstrating biofilm growth in veterinary medicine. HYPOTHESIS/OBJECTIVES: To compare biofilm production of Pseudomonas spp. isolated from dogs with otitis using three different enrichment broths at two different time points. Speciation was performed. ANIMALS: One hundred isolates from 98 dogs with clinical otitis were assessed for biofilm production. METHODS AND MATERIALS: One hundred isolates were assessed for biofilm production using a microtitre plate assay. Biofilm production in Luria-Bertani Broth (LBB), Mueller-Hinton Broth (MHB) and Tryptic Soy Broth (TSB) were assessed after 18 and 24 h of incubation. RESULTS: At 18 h, biofilm production was demonstrated in 87% of LBB, 91% of TSB and 93% of MHB grown isolates. By 24 h, this was 92% of LBB, 96% of TSB and 99% of MHB isolates. Biofilm production was significantly increased after 24 h incubation compared to 18 h. A significant difference was noted in biofilm production between LBB and MHB (P = 0.0349), but not between LBB and TSB (P = 0.3727) or MHB and TSB (P = 0.3687) at 24 h incubation. Two isolates were speciated as P. fluorescens and 98 as P. aeruginosa. CONCLUSION AND CLINICAL IMPORTANCE: Not all enrichment broths were equivalent to one another and 24 h incubation was superior to 18 h. Biofilm production was high in this population of Pseudomonas spp. isolates.

Insights into the Unique Nature of the East Asian Clade of the Emerging Pathogenic Yeast Candida auris.

The emerging yeast Candida auris can be highly drug resistant, causing invasive infections, and large outbreaks. C. auris went from an unknown pathogen a decade ago to being reported in over thirty countries on six continents. C. auris consists of four discrete clades, based on where the first isolates of the clade were reported, South Asian (clade I), East Asian (clade II), African (clade III), and South American (clade IV). These clades have unique genetic and biochemical characteristics that are important to understand and inform the global response to C. auris Clade II has been underrepresented in the literature despite being the first one discovered. In this issue of the Journal of Clinical Microbiology, Y. J. Kwon et al. (J Clin Microbiol 57:e01624-18, 2019, https://doi.org/10.1128/JCM.01624-18) describe the largest collection of clinical isolates from Clade II, which is also the longest-running span of clinical cases, 20 years, from any single region to date. Clade II appears to have a propensity for the ear that is uncharacteristic of the other clades, which typically cause invasive infections and large-scale outbreaks. This study provides new information on an understudied lineage of C. auris and has important implications for future surveillance.

In vitro activity of amphotericin B-azole combinations against Malassezia pachydermatis strains.

Combination therapy has become popular in clinical practice, but limited data on the effects of combinations of antifungal agents is still available for most fungal pathogens. We studied the in vitro response of 30 genetically diverse clinical strains of the basidiomycetous lipophilic yeast Malassezia pachydermatis obtained from cases of canine otitis to several amphotericin B (AMB)-azole combinations. Broth microdilution checkerboard tests revealed that AMB antagonized the effects of itraconazole (ITC) and voriconazole (VRC) in 50% and 6.7% of the strains, respectively, but did not interact with fluconazole or posaconazole (fractional inhibitory concentration index (FICI) values were <4 in all cases). Subsequent Etest-based assays performed for a subset of strains did not confirm the antagonism between AMB and ITC or AMB and VRC. In summary, the results of this study suggest that antagonistic combination effects between AMB and azoles might occur when tested against M. pachydermatis. Nevertheless, as observed for other fungi, different in vitro analyses yielded contrasting results, and the response to AMB-azole combinations was compound- and strain-dependant.