Helicobacter suis-Associated Gastritis Mimicking Conventional H. pylori-Associated Atrophic Gastritis.

A 45-year-old Japanese man underwent esophagogastroduodenoscopy, which revealed spotty redness at the gastric fornix, mucosal swelling, diffuse redness in the corpus, and mucosal atrophy in the gastric angle and antrum. Histological examination showed rod-shaped bacteria that appeared larger than Helicobacter pylori. The patient tested positive for rapid urease test, and serum anti-H. pylori IgG antibody test results were negative. Further examination of the bacteria revealed that H. suis antibody test was positive, and the presence of H. suis was confirmed using H. suis-specific real-time PCR. H. suis was successfully eradicated after triple therapy with vonoprazan, amoxicillin, and clarithromycin. This case reinforces the notion that non-H. pylori Helicobacter species such as H. suis and H. heilmannii may be involved in the pathogenesis of active gastritis in patients who test negative for H. pylori antibodies.

Comparison Approach for Identifying Missed Invasive Fungal Infections in Formalin-Fixed, Paraffin-Embedded Autopsy Specimens.

Invasive fungal infection (IFI) has a high mortality rate in patients who undergo hematopoietic stem cell transplantation, and it is often confirmed by postmortem dissection. When IFI is initially confirmed after an autopsy, the tissue culture and frozen section are challenging to secure, and in many cases, formalin-fixed, paraffin-embedded (FFPE) samples represent the only modality for identifying fungi. Histopathological diagnosis is a useful method in combination with molecular biological methods that can achieve more precise identification with reproducibility. Meanwhile, polymerase chain reaction (PCR) using fungal-specific primers helps identify fungi from FFPE tissues. Autopsy FFPE specimens have a disadvantage regarding the quality of DNA extracted compared with that of specimens obtained via biopsy or surgery. In the case of mucormycosis diagnosed postmortem histologically, we examined currently available molecular biological methods such as PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) to identify fungi. It is reasonable that PCR with some modification is valuable for identifying fungi in autopsy FFPE specimens. However, PCR does not always correctly identify fungi in autopsy FFPE tissues, and other approaches such as ISH or IHC are worth considering for clarifying the broad classification (such as the genus- or species-level classification).

Structome analysis of Escherichia coli cells by serial ultrathin sectioning reveals the precise cell profiles and the ribosome density.

Structome analysis, the quantitative three-dimensional structural analysis of whole cells at the electron microscopic level, of Exophiala dermatitidis (black yeast), Saccharomyces cerevisiae, Mycobacterium tuberculosis (MTB) and Myojin spiral bacteria (MSB) have already been reported. Here, the results of the structome analysis of Escherichia coli cells based on transmission electron microscope observation of serial ultrathin sections was reported, and compared with the data obtained from phase contrast microscopy and scanning electron microscopy. On average, the cells had 0.89 µm in diameter, 2.47 µm in length and 1.16 fl (µm3) in cell volume in the structome analysis. Furthermore, E. coli cells had 26 100 ribosomes per whole cell with density of 2840 per 0.1 fl cytoplasm. The total ribosome number per cell was 15 times larger than that of MTB and about one-eighth of those of the yeast cells above. On the other hand, the ribosome density of E. coli cells are more than 13 times, 4 times, 2.5-times and 1.5-times higher than MSB, MTB, E. dermatitidis and S. cerevisiae, respectively. Finally, our ribosome enumeration data were compared between the structome-analyzed species and the relationship between the ribosome density and the growth rate among these species was discussed.

In vitro resistance of Aspergillus fumigatus to azole farm fungicide.

Azole resistance in Aspergillus fumigatus is mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which encodes the target of azole fungicides. Moreover, overexpression of CYP51B or multidrug resistance (MDR) gene is supposedly related to the mechanism of azole resistance in A. fumigatus. In this study, we tried to induce resistance to tetraconazole, an azole fungicide, in strains of A. fumigatus from a farm and then investigated mutation and expression of their CYP51A, CYP51B, and multidrug resistance (MDR) genes. Three tetraconazole resistant strains were induced and their minimum inhibitory concentration (MIC) for tetraconazole was 145 mg/L. However, the MICs of itraconazole (ITZ), posaconazole (POS), and voriconazole (VRZ) obtained by an E-test of the three tetraconazole resistant strains were 0.064-0.19 mg/L for ITZ, 0.023-0.32 mg/L for POS, and 0.047-0.064 mg/L for VRZ. No gene mutations were detected in the CYP 51A sequence amplified in these strains. RT-PCR of cyp51A and cyp51B indicated that the tetraconazole resistant strains more highly expressed these genes than the susceptible strain in tetraconazole containing medium.

[Invasive Aspergillosis Accompanied by a Subcutaneous Nodular Lesion].

We report herein on a case of invasive aspergillosis accompanied by a subcutaneous nodular lesion. A 74-years-old male with myelodysplastic syndrome was hospitalized due to high fever and a painful subcutaneous nodule on the left thigh. Chest radiography and CT scans showed multiple nodular lesions of both lungs, and bacterial pneumonia was initially suspected. He was treated with meropenem, but the symptoms did not subside. Three days after admission, we found that ß-D-glucan levels were elevated at 52.6 pg/mL. He was treated with liposomal amphotericin B (L-AMB) for invasive fungal pneumonia, and the symptoms regressed thereafter. Excisional biopsy of the nodular lesion showed a cluster of septated and branching hyphae. Serum Aspergillus antigen tests and sputum fungal culture were negative, and the fungal species could not be identified. Thus, we performed in situ hybridization (ISH) and polymerase chain reaction (PCR) with the excised subcutaneous specimens, and as a result Aspergillus fumigatus infection was diagnosed. Invasive aspergillosis with a subcutaneous lesion is a rare case, and we found that treatment with L-AMB was effective. ISH, PCR and measurement of serum trough concentration of AMPH-B are useful in diagnosis and treatment.

Helicobacter suis-Infected Nodular Gastritis and a Review of Diagnostic Sensitivity for Helicobacter heilmannii-Like Organisms.

Helicobacter heilmannii-like organisms (HHLOs) are associated with mucosa-associated lymphoid tissue lymphoma and peptic ulcer. However, the sensitivity of diagnostic tests for HHLOs, such as rapid urease test (RUT), urea breath test (UBT) and blood antibody, is not high. Tightly coiled spiral microorganisms were found in the gastric mucosal biopsy specimen of a 48-year-old asymptomatic woman. Her findings were positive for RUT and UBT, but negative for blood antibody and stool antigen against H. pylori. A 7-day course of esomeprazole, amoxicillin and clarithromycin was administered, resulting in the successful eradication of the HHLOs. Analysis of the 16S rRNA and urease genes suggested a diagnosis of the HHLO H. suis. The sensitivity results of RUT, UBT, culture, blood antibody, immunohistochemistry and stool antigen were 40.0, 14.8, 0, 23.1, 40.0 and 0%, respectively. We report asymptomatic nodular gastritis due to an HHLO. Histological techniques, most likely with smears, are expected to be the most effective method for diagnosing infections by HHLOs, and genetic diagnosis by polymerase chain reaction can be very useful to identify the species of HHLOs.

Mouse Models for Assessing the Protective Efficacy of Lactobacillus gasseri SBT2055 against Helicobacter suis Infection Associated with the Development of Gastric Mucosa-Associated Lymphoid Tissue Lymphoma.

BACKGROUND: Helicobacter suis strain TKY infection has been strongly associated with the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma in a C57BL/6J mouse model. MATERIALS AND METHODS: 1. C57BL/6J mice were intragastrically administered Lactobacillus strains once daily with 10(8)-10(9) colony-forming units (CFU), starting 2 days before intragastric infection with H. suis TKY (approximately 1 × 10(4) copies of 16S rRNA genes) or H. pylori Sydney strain 1 (SS1; 3 × 10(8) CFU) and continuing for 14 days after infection. 2. C57BL/6J mice were given powdered feed mixed with lyophilized L. gasseri SBT2055 (LG2055) cells (5 × 10(8) CFU/g), starting 2 weeks before intragastric infection with H. suis TKY and continuing 12 months after infection. RESULTS: 1. Among the 5 Lactobacillus strains that we examined, only LG2055 exhibited significantly preventive efficacy against both H. suis TKY and H. pylori SS1 at day 15 after infection. 2. Dietary supplementation with LG2055 protected mice from the formation of round protrusive lesions in the gastric fundus 12 months after infection with H. suis TKY, whereas such lesions had developed in the gastric fundus of nonsupplemented mice 12 months after infection. In addition, the formation of lymphoid follicles in gastric mucus layers was suppressed by dietary LG2055 at 3 months after infection. CONCLUSIONS: LG2055 administration is effective for suppressing the progression of gastric MALT lymphoma by reducing H. suis colonization.

Does farm fungicide use induce azole resistance in Aspergillus fumigatus?

Azole resistance of Aspergillus fumigatus isolates has been reported worldwide and it would appear to be mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which is the target enzyme for azoles. The mutation has been confirmed in isolates from patients who received long-term itraconazole (ITZ) therapy and from agricultural fields where high levels of azole fungicides were employed. However, the relationship between farm environments and azole-resistant A. fumigatus has not been fully studied. In this investigation, 50 isolates of A. fumigatus were obtained from a farm where tetraconazole has been sprayed twice a year for more than 15 years. The mean minimum inhibitory concentration (MIC) of isolates was 0.74 (0.19-1.5) mg/L against ITZ, which was below the medical resistance level of ITZ. The sequence of CYP51A from isolates indicated no gene mutations in isolates from the farm. Antifungal susceptibility of isolates to tetraconazole showed that spraying with tetraconazole did not induce resistance to tetraconazole or ITZ in A. fumigatus.

Histopathological evaluation of the efficacy of antifungals for experimental Trichosporon bloodstream infection.

The efficacy of polyene macrolides to treat experimental Trichosporon bloodstream infection was evaluated by histopathological examination and viable cell counts in the kidneys of infected mice. Viable cell counts on the 5th day after infection confirmed that liposomal amphotericin B (L-AMB) is a more effective treatment than fluconazole (FLC) for mice infected with an azole-resistant strain of Trichosporon. Histological examination revealed that the administration of L-AMB induced a transformation from acute purulent inflammation caused by both azole-susceptible and -resistant strain infections to a chronic and subsiding form, whereas FLC failed to convert the acute inflammation induced by the azole-resistant strain to a subsiding form. Our results demonstrate that polyene macrolides can be used as an alternative therapy for infection of azole-resistant strains of Trichosporon and that histopathological evaluation is useful for elucidating the pathophysiology of an experimental Trichosporon infection.

Development of a peptide nucleic acid probe to Trichosporon species and identification of trichosporonosis by use of in situ hybridization in formalin-fixed and paraffin-embedded (FFPE) sections.

In order to identify Trichosporon species in formalin-fixed and paraffin-embedded sections from which visual discrimination of non-glabrata Candida species is mostly ineffective but critical for the choice of antifungals, we tested the usefulness of a newly designed peptide nucleic acid probe (PNA) for in situ hybridization (ISH). Results confirmed the usefulness of ISH with our PNA probe in identifying Trichosporon species from Candida albicans.

Evolutionary paths of streptococcal and staphylococcal superantigens.

BACKGROUND: Streptococcus pyogenes (GAS) harbors several superantigens (SAgs) in the prophage region of its genome, although speG and smez are not located in this region. The diversity of SAgs is thought to arise during horizontal transfer, but their evolutionary pathways have not yet been determined. We recently completed sequencing the entire genome of S. dysgalactiae subsp. equisimilis (SDSE), the closest relative of GAS. Although speG is the only SAg gene of SDSE, speG was present in only 50% of clinical SDSE strains and smez in none. In this study, we analyzed the evolutionary paths of streptococcal and staphylococcal SAgs. RESULTS: We compared the sequences of the 12-60 kb speG regions of nine SDSE strains, five speG(+) and four speG(-). We found that the synteny of this region was highly conserved, whether or not the speG gene was present. Synteny analyses based on genome-wide comparisons of GAS and SDSE indicated that speG is the direct descendant of a common ancestor of streptococcal SAgs, whereas smez was deleted from SDSE after SDSE and GAS split from a common ancestor. Cumulative nucleotide skew analysis of SDSE genomes suggested that speG was located outside segments of steeper slopes than the stable region in the genome, whereas the region flanking smez was unstable, as expected from the results of GAS. We also detected a previously undescribed staphylococcal SAg gene, selW, and a staphylococcal SAg -like gene, ssl, in the core genomes of all Staphylococcus aureus strains sequenced. Amino acid substitution analyses, based on dN/dS window analysis of the products encoded by speG, selW and ssl suggested that all three genes have been subjected to strong positive selection. Evolutionary analysis based on the Bayesian Markov chain Monte Carlo method showed that each clade included at least one direct descendant. CONCLUSIONS: Our findings reveal a plausible model for the comprehensive evolutionary pathway of streptococcal and staphylococcal SAgs.

A CD46 transgenic mouse model for studying the histopathology of arthritis caused by subcutaneous infection with Streptococcus dysgalactiae subspecies equisimilis.

Ankle arthritis was induced by a single subcutaneous (s.c.) infection of 1×10(7) c.f.u. of the Streptococcus dysgalactiae subspecies equisimilis strain RE378, which was isolated from a patient suffering from multiple organ failure due to septicaemia, into both hind footpads of human CD46-expressing transgenic (Tg) mice. In contrast, in non-Tg mice, the incipient foot lesions (swelling and redness) resolved before arthritis developed. The number of viable bacteria in tissue samples and the arthritis frequency on days 3 and 28 after infection were higher in CD46 Tg mice than in non-Tg mice. The histopathological findings in the hind ankle sections of CD46 Tg mice showed the stimulation of osteoclast formation associated with inflammation of the synovial membrane and the development of aggressive granulation tissue (pannus). In addition, increased expression levels of interleukin (IL)-6, receptor activator of NF-κB ligand, IL-1ß and tumour necrosis factor alpha were detected in the foot bones of CD46 Tg mice but not in those of non-Tg mice. These observations suggest that the s.c. infection with S. dysgalactiae subsp. equisimilis induced arthritis in the ankle joints of CD46 Tg mice as a consequence of the prolonged inflammation associated with focal bone loss.

Novel postentry inhibitor of human immunodeficiency virus type 1 replication screened by yeast membrane-associated two-hybrid system.

Human immunodeficiency virus (HIV) Gag protein targets to the plasma membrane and assembles into viral particles. In the next round of infection, the mature Gag capsids disassemble during viral entry. Thus, Gag plays a central role in the HIV life cycle. Using a yeast membrane-associated two-hybrid assay based on the SOS-RAS signaling system, we developed a system to measure the Gag-Gag interaction and isolated 6 candidates for Gag assembly inhibitors from a chemical library composed of 20,000 small molecules. When tested in the human MT-4 cell line and primary peripheral blood mononuclear cells, one of the candidates, 2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine (BMMP), displayed an inhibitory effect on HIV replication, although a considerably high dose was required. Unexpectedly, neither particle production nor maturation was inhibited by BMMP. Confocal microscopy confirmed that BMMP did not block Gag plasma membrane targeting. Single-round infection assays with envelope-pseudotyped and luciferase-expressing viruses revealed that BMMP inhibited HIV replication postentry but not simian immunodeficiency virus (SIV) or murine leukemia virus infection. Studies with HIV/SIV Gag chimeras indicated that the Gag capsid (CA) domain was responsible for the BMMP-mediated HIV postentry block. In vitro studies indicated that BMMP accelerated disassembly of HIV cores and, conversely, inhibited assembly of purified CA protein in a dose-dependent manner. Collectively, our data suggest that BMMP primarily targets the HIV CA domain and disrupts viral infection postentry, possibly through inducing premature disassembly of HIV cores. We suggest that BMMP is a potential lead compound to develop antiretroviral drugs bearing novel mechanisms of action.

Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS).

BACKGROUND: Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes invasive streptococcal infections, including streptococcal toxic shock syndrome (STSS), as does Lancefield group A Streptococcus pyogenes (GAS). We sequenced the entire genome of SDSE strain GGS_124 isolated from a patient with STSS. RESULTS: We found that GGS_124 consisted of a circular genome of 2,106,340 bp. Comparative analyses among bacterial genomes indicated that GGS_124 was most closely related to GAS. GGS_124 and GAS, but not other streptococci, shared a number of virulence factor genes, including genes encoding streptolysin O, NADase, and streptokinase A, distantly related to SIC (DRS), suggesting the importance of these factors in the development of invasive disease. GGS_124 contained 3 prophages, with one containing a virulence factor gene for streptodornase. All 3 prophages were significantly similar to GAS prophages that carry virulence factor genes, indicating that these prophages had transferred these genes between pathogens. SDSE was found to contain a gene encoding a superantigen, streptococcal exotoxin type G, but lacked several genes present in GAS that encode virulence factors, such as other superantigens, cysteine protease speB, and hyaluronan synthase operon hasABC. Similar to GGS_124, the SDSE strains contained larger numbers of clustered, regularly interspaced, short palindromic repeats (CRISPR) spacers than did GAS, suggesting that horizontal gene transfer via streptococcal phages between SDSE and GAS is somewhat restricted, although they share phage species. CONCLUSION: Genome wide comparisons of SDSE with GAS indicate that SDSE is closely and quantitatively related to GAS. SDSE, however, lacks several virulence factors of GAS, including superantigens, SPE-B and the hasABC operon. CRISPR spacers may limit the horizontal transfer of phage encoded GAS virulence genes into SDSE. These findings may provide clues for dissecting the pathological roles of the virulence factors in SDSE and GAS that cause STSS.

Dermal mast cells reduce progressive tissue necrosis caused by subcutaneous infection with Streptococcus pyogenes in mice.

A single subcutaneous (s.c.) infection with 1×10(7) c.f.u. GAS472, a group A streptococcus (GAS) serotype M1 strain isolated from the blood of a patient suffering from streptococcal toxic shock syndrome, led to severe damage of striated muscle layers in the feet of mast cell (MC)-deficient WBB6F(1)-Kit(W)/Kit(W-v) (W/W(v)) mice 72 h after infection. In contrast, no damage was recognized in striated muscle layers in the feet of the control WBB6F(1)-Kit(+/+) (+/+) mice 72 h after infection. In addition, adoptively transferred MCs reduced progressive tissue necrosis of the feet of W/W(v) mice after infection. However, there was no significant difference in the mortality rates between the W/W(v) and +/+ mice, or between the human CD46-expressing transgenic (Tg) mouse bone marrow-derived cultured MC-reconstituted W/W(v) and non-Tg mouse bone marrow-derived cultured MC-reconstituted W/W(v) mice after infection. Consequently, although MCs can help to reduce the severity of necrosis of the feet caused by s.c. infection with GAS472, such reduction of tissue necrosis scarcely improves the mortality rates of these mice. Moreover, human CD46 does not play a crucial role in the MC-mediated innate immune defence against GAS infection.

Identification of Fusarium species in formalin-fixed and paraffin-embedded sections by in situ hybridization using peptide nucleic acid probes.

Fusarium has recently emerged as an opportunistic pathogen of humans, but the histological differentiation of Fusarium from Aspergillus and Scedosporium is particularly difficult because these fungi may induce similar clinical features and exhibit filamentous development in host tissues. Thus, there is a need to establish rapid and reliable methods that are applicable to pathological diagnoses. The aim of this study was to evaluate and establish in situ hybridization (ISH) using peptide nucleic acid (PNA) probes targeting the 28S rRNA to identify Fusarium species in tissue sections. This technique was validated using both formalin-fixed and paraffin-embedded pulmonary tissues from mice infected with seven different species of fungi and cell blocks from fungal cultures of 30 strains. As a result, strong positive signals were observed within fungal organisms present in tissues of the lung from mice infected with Fusarium solani. Furthermore, this probe reacted strongly with both F. solani and Fusarium oxysporum in sections from cell blocks. Although some cross-reactivity occurred with the Pseudallescheria boydii in sections from cell blocks, the signal intensity was low and most hyphae were not reactive. In conclusion, it was confirmed that ISH with PNA probes is accurate and is a valuable tool for identifying Fusarium spp. among organisms that have identical morphological features in formalin-fixed and paraffin-embedded sections.

CD46 transgenic mouse model of necrotizing fasciitis caused by Streptococcus pyogenes infection.

We developed a human CD46-expressing transgenic (Tg) mouse model of subcutaneous (s.c.) infection into both hind footpads with clinically isolated 11 group A streptococcus (GAS) serotype M1 strains. When the severity levels of foot lesions at 72 h and the mortality rates by 336 h were compared after s.c. infection with 1x10(7) CFU of each GAS strain, the GAS472 strain, isolated from the blood of a patient suffering from streptococcal toxic shock syndrome (STSS), induced the highest severity levels and mortality rates. GAS472 led to a 100% mortality rate in CD46 Tg mice after only 168 h postinfection through the supervention of severe necrotizing fasciitis (NF) of the feet. In contrast, GAS472 led to a 10% mortality rate in non-Tg mice through the supervention of partial necrotizing cutaneous lesions of the feet. The footpad skin sections of CD46 Tg mice showed hemorrhaging and necrotic striated muscle layers in the dermis, along with the exfoliation of epidermis with intracellular edema until 48 h after s.c. infection with GAS472. Thereafter, the bacteria proliferated, reaching a 90-fold or 7-fold increase in the livers of CD46 Tg mice or non-Tg mice, respectively, for 24 h between 48 and 72 h after s.c. infection with GAS472. As a result, the infected CD46 Tg mice appeared to suffer severe liver injuries. These findings suggest that human CD46 enhanced the progression of NF in the feet and the exponential growth of bacteria in deep tissues, leading to death.

Capsular type and antibiotic resistance in Streptococcus agalactiae isolates from patients, ranging from newborns to the elderly, with invasive infections.

Streptococcus agalactiae isolates (n = 189) from patients with invasive infections were analyzed for capsular type by PCR, for antimicrobial susceptibility, and for the presence of resistance genes. In contrast to the predominance of capsular type III in children, types Ib and V were most common among adults. All 45 levofloxacin-resistant strains had two amino acid substitutions, Ser(81)Leu in the gyrA gene and Ser(79)Phe in the parC gene, and showed similar pulsed-field gel electrophoresis patterns.

The lipolytic enzymes activities of Malassezia species.

Malassezia yeasts are part of the cutaneous microflora commonly found on animals and human and may sometimes cause various opportunistic skin diseases. As most of Malassezia species show lipid-dependency, lipolytic enzymes such as lipase and phospholipase are necessary for them to obtain useful lipids from the environment. Consequently, these enzymes are thought to play an important role in the growth and pathogenicity of Malassezia. Here we analyze and compare extracellular lipase and phospholipase activities of several Malassezia species cultivated under common growth conditions. M. globosa showed the highest lipase activity of all of the Malassezia species included in our studies. The lipid-independent M. pachydermatis also showed high lipase and phospholipase activity. These results indicate that this Malassezia species are capable of utilizing lipids well in contrast to the other lipid-dependent species of the genus. Our data suggest that lipase may be a pathogenic factor in the skin disease associated with Malassezia and provide an explanation as to why M. globosa is an important pathogenic species in several human skin diseases despite its slow rate of growth.