Isolated Cutaneous Granuloma Caused by <Emphasis Type="Italic">Candida glabrata</Emphasis>: A Rare Case Report and Literature Review

The incidence of candidiasis due to non-albicans Candida species (especially Candida glabrata) has significantly increased in recent decades. Candida glabrata often invades immunocompromised hosts and causes systemic or mucosal infections, whereas cutaneous infections are rarely reported. We present a rare case of cutaneous infection caused by C. glabrata and review all similar cases available in the PubMed database. A patient was admitted to the hospital with a 2-month history of a plaque on the face. Histopathological examination displayed typical infectious granulomas in the deep dermis, and the pathogen was finally confirmed as C. glabrata using a series of microbial examinations (fungal culture, biochemical test, and PCR-directed sequencing). The patient was completely cured after 4 months of treatment with oral itraconazole combined with topical terbinafine. We reviewed similar reports of cutaneous infection caused by C. glabrata. All the data suggested that an accurate diagnosis of cutaneous candidiasis depends mainly on histological and fungal examinations, especially molecular biological assays. Antifungal agents based on microbial susceptibility tests are the first-line treatment choice for C. glabrata infection, but the prognosis might be more dependent on the basic condition of the host.     

Melanization of <Emphasis Type="Italic">Fusarium keratoplasticum</Emphasis> (<Emphasis Type="Italic">F. solani</Emphasis> Species Complex) During Disseminated Fusariosis in a Patient with Acute Leukemia

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.     

In Vitro Antifungal Susceptibility of <Emphasis Type="Italic">Neoscytalidium dimidiatum</Emphasis> Clinical Isolates from Malaysia

Neoscytalidium dimidiatum is an opportunistic fungus causing cutaneous infections mostly, which are difficult to treat due to antifungal resistance. In Malaysia, N. dimidiatum is associated with skin and nail infections, especially in the elderly. These infections may be mistaken for dermatophyte infections due to similar clinical appearance. In this study, Neoscytalidium isolates from cutaneous specimens, identified using morphological and molecular methods (28 Neoscytalidium dimidiatum and 1 Neoscytalidium sp.), were evaluated for susceptibility towards antifungal agents using the CLSI broth microdilution (M38-A2) and Etest methods. Amphotericin B, voriconazole, miconazole and clotrimazole showed high in vitro activity against all isolates with MIC ranging from 0.0313 to 1 µg/mL. Susceptibility towards fluconazole and itraconazole was noted in up to 10% of isolates, while ketoconazole was inactive against all isolates. Clinical breakpoints for antifungal drugs are not yet available for most filamentous fungi, including Neoscytalidium species. However, the results indicate that clinical isolates of N. dimidiatum in Malaysia were sensitive towards miconazole, clotrimazole, voriconazole and amphotericin B, in vitro.     

<Emphasis Type="Italic">Myroides injenensis</Emphasis> sp. nov., a new member isolated from human urine

A Gram-negative, yellow-pigmented, rod-shaped bacteria, designated M09-0166^Tand M09-1053 were isolated from human urine samples. 16S rRNA gene sequence analysis revealed that the isolates belong to the Myroides cluster and were closely related to Myroides phaeus DSM 23313^T (96.3 %), Myroides odoratimimus KCTC 23053^T (96.1 %), Myroides profundi KCTC 23066^T (96.0 %), Myroides odoratus KCTC 23054^T (95.4 %) and Myroides pelagicus KCTC 12661^T (95.2 %). The major mena quinone was identified as MK-6. The major polar lipids were identified as phosphatidylethanolamine, amino lipids, and several unknown lipids, and the major fatty acids as iso-C_15:0 and iso-C_17:0 3-OH. Phenotypic and chemotaxonomic data supported the affiliation of the isolates with the genus Myroides and clearly indicated that two isolates represent novel species, for which the name Myroides injenensis sp. nov. (type strain, M09-0166^T = KCTC 23367^T = JCM 17451^T) is proposed.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation: an efficient tool for targeted gene disruption in <Emphasis Type="Italic">Talaromyces marneffei</Emphasis>

Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 10⁶ yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments.     

<Emphasis Type="Italic">Exophiala dermatitidis</Emphasis> Revealing Cystic Fibrosis in Adult Patients with Chronic Pulmonary Disease

Cystic fibrosis (CF) is a genetic inherited disease due to mutations in the gene cystic fibrosis transmembrane conductance regulator (CFTR). Because of the huge diversity of CFTR mutations, the CF phenotypes are highly heterogeneous, varying from typical to mild form of CF, also called atypical CF. These atypical features are more frequently diagnosed at adolescence or adulthood, and among clinical signs and symptoms leading to suspect a mild form of CF, colonization or infection of the respiratory tract due to well-known CF pathogens should be a warning signal. Exophiala dermatitidis is a melanized dimorphic fungus commonly detected in respiratory specimens from CF patients, but only very rarely from respiratory specimens from non-CF patients. We described here two cases of chronic colonization of the airways by E. dermatitidis, with recurrent pneumonia and hemoptysis in one patient, which led clinicians to diagnose mild forms of CF in these elderly patients who were 68- and 87-year-old. These cases of late CF diagnosis suggest that airway colonization or respiratory infections due to E. dermatitidis in patients with bronchiectasis should led to search for a mild form of CF, regardless of the age and associated symptoms. On a broader level, in patients with chronic respiratory disease and recurrent pulmonary infections, an allergic bronchopulmonary mycosis or an airway colonization by CF-related fungi like E. dermatitidis or some Aspergillus, Scedosporium or Rasamsonia species, should be considered as potential markers of atypical CF and should led clinicians to conduct investigations for CF diagnosis.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

A Case Report of Penile Infection Caused by Fluconazole- and Terbinafine-Resistant <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida albicans is the most common pathogen that causes balanoposthitis. It often causes recurrence of symptoms probably due to its antifungal resistance. A significant number of balanitis Candida albicans isolates are resistant to azole and terbinafine antifungal agents in vitro. However, balanoposthitis caused by fluconazole- and terbinafine-resistant Candida albicans has rarely been reported. Here, we describe a case of a recurrent penile infection caused by fluconazole- and terbinafine-resistant Candida albicans, as well as the treatments administered to this patient. The isolate from the patient was tested for drug susceptibility in vitro. It was sensitive to itraconazole, voriconazole, clotrimazole and amphotericin B, but not to terbinafine and fluconazole. Thus, oral itraconazole was administrated to this patient with resistant Candida albicans penile infection. The symptoms were improved, and mycological examination result was negative. Follow-up treatment of this patient for 3 months showed no recurrence.     

The <Emphasis Type="Italic">Brucella melitensis</Emphasis> M5-90 phosphoglucomutase (PGM) mutant is attenuated and confers protection against wild-type challenge in BALB/c mice

Brucellae are Gram-negative intracellular bacterial pathogens that infect humans and animals, bringing great economic burdens to developing countries. Live attenuated Brucella vaccines (strain M5-90 or others) are the most efficient means for prevention and control of animal brucellosis. However, these vaccines have several drawbacks, including residual virulence in animals, and difficulties in differentiating natural infection from vaccine immunization, which limit their application. A vaccine that can differentiate infection from immunization will have extensive applications. A Brucella melitensis (B. melitensis) strain M5-90 pgm mutant (M5-90Δpgm) was constructed to overcome these drawbacks. M5-90Δpgm showed significantly reduced survival in embryonic trophoblast cells and in mice, and induced high protective immunity in BALB/c mice. Moreover, M5-90Δpgm elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon (IFN-γ) and interleukin-2 (IL-2). In addition, M5-90Δpgm induced the secretion of IFN-γ in immunized sheep. Serum samples from sheep inoculated with M5-90Δpgm were negative by the Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT). Furthermore, the PGM antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90Δpgm is an ideal live attenuated vaccine candidate against B. melitensis 16 M and deserves further evaluation for vaccine development.     

Identification of essential genes of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> for its growth in airway mucus

Pseudomonas aeruginosa has been identified as an important causative agent of airway infection, mainly in cystic fibrosis. This disease is characterized by defective mucociliary clearance induced in part by mucus hyper-production. Mucin is a major component of airway mucus and is heavily O-glycosylated, with a protein backbone. Airway infection is known to be established with bacterial adhesion to mucin. However, the genes involved in mucin degradation or utilization remain elusive. In this study, we sought to provide a genetic basis of P. aeruginosa airway growth by identifying those genes. First, using RNASeq analyses, we compared genome-wide expression profiles of PAO1, a prototype P. aeruginosa laboratory strain, grown in M9-mucin (M9M) and M9-glucose (M9G) media. Additionally, a PAO1 transposon (Tn) insertion mutants library was screened for mutants defective in growth in M9M medium. One mutant with a Tn insertion in the xcpU gene (PA3100) was determined to exhibit faulty growth in M9M medium. This gene contributes to the type II secretion system, suggesting that P. aeruginosa uses this secretion system to produce a number of proteins to break down and assimilate the mucin molecule. Furthermore, we screened the PAO1 genome for genes with protease activity. Of 13 mutants, one with mutation in PA3247 gene exhibited defective growth in M9M, suggesting that the PA3247-encoded protease plays a role in mucin utilization. Further mechanistic dissection of this particular process will reveal new drug targets, the inhibition of which could control recalcitrant P. aeruginosa infections.     

Characterization of a novel lytic bacteriophage from an industrial <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

Bacterial–bacteriophage interactions are a well-studied and ecologically-important aspect of microbiology. Many commercial fermentation processes are susceptible to bacteriophage infections due to the use of high-density, clonal cell populations. Lytic infections of bacterial cells in these fermentations are especially problematic due to their negative impacts on product quality, asset utilization, and fouling of downstream equipment. Here, we report the isolation and characterization of a novel lytic bacteriophage, referred to as bacteriophage DTL that is capable of rapid lytic infections of an Escherichia coli K12 strain used for commercial production of 1,3-propanediol (PDO). The bacteriophage genome was sequenced and annotated, which identified 67 potential open-reading frames (ORF). The tail fiber ORF, the largest in the genome, was most closely related to bacteriophage RTP, a T1-like bacteriophage reported from a commercial E. coli fermentation process in Germany. To eliminate virulence, both a fully functional Streptococcus thermophilus CRISPR3 plasmid and a customized S. thermophilus CRISPR3 plasmid with disabled spacer acquisition elements and seven spacers targeting the bacteriophage DTL genome were constructed. Both plasmids were separately integrated into a PDO production strain, which was subsequently infected with bacteriophage DTL. The native S. thermophilus CRISPR3 operon was shown to decrease phage susceptibility by approximately 96%, while the customized CRISPR3 operon provided complete resistance to bacteriophage DTL. The results indicate that the heterologous bacteriophage-resistance system described herein is useful in eliminating lytic infections of bacteriophage DTL, which was prevalent in environment surrounding the manufacturing facility.     

Serological diagnosis of <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis> infection by using the mimic epitopes

Nowadays, there is lack of effective serological detection method for Mycoplasma pneumoniae (M. pneumoniae) infection in clinic. In this study, the mimic epitopes of M. pneumoniae were screened to evaluate the role in the serodiagnosis of M. pneumoniae infection. The M. pneumoniae-positive serum was used as the target for biopanning to phage display random 7-peptide library. The positive phage clones were selected and the DNA were sequenced and analyzed by BLAST. The representative phages were identified using dot immunoblotting and ELISA. The exogenous heptapeptides were synthesized and their reactions with M. pneumonia-positive serum were tested by indirect ELISA. Two heptapeptides, namely heptapeptide 1: TVNFKLY and heptapeptide 2: LPQRLRT, were screened out from the randomly selected 40 phages after the four bio-panning rounds. They had high homologies to some M. pneumoniae antigens. Besides, the representative bacteriophage containing heptapeptide 1 or 2 could react with the M. pneumonia- positive serum. The sensitivities of heptapeptide 1 and heptapeptide 2 for the diagnosis of M. pneumoniae infection were 90.1 and 80.0%, respectively, and the specificities were 94.3 and 97.1%, respectively. Therefore the two heptapeptides were the mimic epitopes of M. pneumoniae and might have potential serological diagnosis value for M. pneumoniae infection.     

Strain-specific response to ampicillin in <Emphasis Type="Italic">Wolbachia-</Emphasis>infected mosquito cell lines

Wolbachia pipientis (Rickettsiales; Anaplasmataceae) is an obligate intracellular alpha proteobacterium that occurs in arthropods and filarial worms. Some strains of Wolbachia can be maintained as persistent infections in insect cell lines. C/wStr1 cells from the mosquito Aedes albopictus maintain a robust infection with Wolbachia strain wStr, originally isolated from the planthopper, Laodelphax striatellus. To explore possible functions of penicillin-binding proteins expressed from the wStr genome, C/wStr1 cells were exposed to ampicillin. Absolute levels of Wolbachia increased 3.5-fold in ampicillin-treated cells and fivefold in naive cells newly infected with wStr. Because cell numbers were depressed by ampicillin treatment, Wolbachia yield on a per-cell basis increased by 15-fold. The absence of a similar effect on wAlbB in Aa23 host cells suggests that the Wolbachia strain, the presence/absence of genes encoding penicillin-binding proteins, or the interaction between wAlbB and its host cells may modulate the effects of ampicillin.     

Genetically diverse mice are novel and valuable models of age-associated susceptibility to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background

Tuberculosis, the disease due to Mycobacterium tuberculosis, is an important cause of morbidity and mortality in the elderly. Use of mouse models may accelerate insight into the disease and tests of therapies since mice age thirty times faster than humans. However, the majority of TB research relies on inbred mouse strains, and these results might not extrapolate well to the genetically diverse human population. We report here the first tests of M. tuberculosis infection in genetically heterogeneous aging mice, testing if old mice benefit from rapamycin.

Findings

We find that genetically diverse aging mice are much more susceptible than young mice to M. tuberculosis, as are aging human beings. We also find that rapamycin boosts immune responses during primary infection but fails to increase survival.

Conclusions

Genetically diverse mouse models provide a valuable resource to study how age influences responses and susceptibility to pathogens and to test interventions. Additionally, surrogate markers such as immune measures may not predict whether interventions improve survival.

     

Bile salt hydrolase-mediated inhibitory effect of <Emphasis Type="Italic">Bacteroides ovatus</Emphasis> on growth of <Emphasis Type="Italic">Clostridium difficile</Emphasis>

Clostridium difficile infection (CDI) is one of the most common nosocomial infections. Dysbiosis of the gut microbiota due to consumption of antibiotics is a major contributor to CDI. Recently, fecal microbiota transplantation (FMT) has been applied to treat CDI. However, FMT has important limitations including uncontrolled exposure to pathogens and standardization issues. Therefore, it is necessary to evaluate alternative treatment methods, such as bacteriotherapy, as well as the mechanism through which beneficial bacteria inhibit the growth of C. difficile. Here, we report bile acid-mediated inhibition of C. difficile by Bacteroides strains which can produce bile salt hydrolase (BSH). Bacteroides strains are not commonly used to treat CDI; however, as they comprise a large proportion of the intestinal microbiota, they can contribute to bile acid-mediated inhibition of C. difficile. The inhibitory effect on C. difficile growth increased with increasing bile acid concentration in the presence of Bacteroides ovatus SNUG 40239. Furthermore, this inhibitory effect on C. difficile growth was significantly attenuated when bile acid availability was reduced by cholestyramine, a bile acid sequestrant. The findings of this study are important due to the discovery of a new bacterial strain that in the presence of available bile acids inhibits growth of C. difficile. These results will facilitate development of novel bacteriotherapy strategies to control CDI.     

Fatal <Emphasis Type="Italic">Talaromyces marneffei</Emphasis> Infection in a Patient with Autoimmune Hepatitis

Talaromyces marneffei, previously known as Penicillium marneffei, is the most important pathogenic thermally dimorphic fungus causing systemic mycosis in Southeast Asia. Traditionally, T. marneffei infection in human was mainly associated with acquired immunodeficiency syndrome caused by HIV infection. In recent years, there has been an increasing number of T. marneffei infections reported in non-HIV-infected patients with other immunocompromised conditions, including autoantibodies against interferon-gamma, systemic lupus erythematosis, solid organ transplantation, Job’s syndrome, hematological malignancies, and use of novel targeted therapies. In this article, we describe the first case of fatal T. marneffei infection in a patient with underlying autoimmune hepatitis, presented as fever without localizing features. The diagnosis of talaromycosis was confirmed with the identification of the fungi isolated from the blood culture specimen by conventional methods and using matrix-assisted laser desorption–ionization time-of-flight mass spectrometer. This case shows the importance of a high index of suspicion, particularly for such a highly fatal but potentially treatable fungal infection.     

Functional characterization of the powdery mildew susceptibility gene <Emphasis Type="Italic">SmMLO1</Emphasis> in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Eggplant (Solanum melongena L.) is one of the most important vegetables among the Solanaceae and can be a host to fungal species causing powdery mildew (PM) disease. Specific homologs of the plant Mildew Locus O (MLO) gene family are PM susceptibility factors, as their loss of function results in a recessive form of resistance known as mlo resistance. In a previous work, we isolated the eggplant MLO homolog SmMLO1. SmMLO1 is closely related to MLO susceptibility genes characterized in other plant species. However, it displays a peculiar non-synonymous substitution that leads to a T → M amino acid change at protein position 422, in correspondence of the MLO calmodulin-binding domain. In this study, we performed the functional characterization of SmMLO1. Transgenic overexpression of SmMLO1 in a tomato mlo mutant compromised resistance to the tomato PM pathogen Oidium neolycopersici, thus indicating that SmMLO1 is a PM susceptibility factor in eggplant. PM susceptibility was also restored by the transgenic expression of a synthetic gene, named s-SmMLO1, encoding a protein identical to SmMLO1, except for the presence of T at position 422. This indicates that the T → M polymorphism does not affect the protein role as PM susceptibility factor. Overall, the results of this work are of interest for the functional characterization of MLO proteins and the introduction of PM resistance in eggplant using reverse genetics.     

Epidemiology and resistance features of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> isolates from the ward environment and patients in the burn ICU of a Chinese hospital

Acinetobacter baumannii is an important opportunistic pathogen that causes severe nosocomial infections, especially in intensive care units (ICUs). Over the past decades, an everincreasing number of hospital outbreaks caused by A. baumannii have been reported worldwide. However, little attention has been directed toward the relationship between A. baumannii isolates from the ward environment and patients in the burn ICU. In this study, 88 A. baumannii isolates (26 from the ward environment and 62 from patients) were collected from the burn ICU of the Southwest Hospital in Chongqing, China, from July through December 2013. Antimicrobial susceptibility testing results showed that drug resistance was more severe in isolates from patients than from the ward environment, with all of the patient isolates being fully resistant to 10 out of 19 antimicrobials tested. Isolations from both the ward environment and patients possessed the β-lactamase genes bla_OXA-51, bla_OXA-23, bla_AmpC, bla_VIM, and bla_PER. Using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), these isolates could be clustered into 4 major PFGE types and 4 main sequence types (ST368, ST369, ST195, and ST191) among which, ST368 was the dominant genotype. Epidemiologic and molecular typing data also revealed that a small-scale outbreak of A. baumannii infection was underway in the burn ICU of our hospital during the sampling period. These results suggest that dissemination of β-lactamase genes in the burn ICU might be closely associated with the high-level resistance of A. baumannii, and the ICU environment places these patients at a high risk for nosocomial infection. Cross-contamination should be an important concern in clinical activities to reduce hospitalacquired infections caused by A. baumannii.