An interlaboratory study on the detection method for Escherichia albertii in food using real time PCR assay and selective agars.

Escherichia albertii is an emerging enteropathogen. Although E. albertii-specific detection and isolation methods have been developed, their efficiency on food samples have not yet been systematically studied. To establish a series of effective methods for detecting E. albertii in food, an interlaboratory study was conducted in 11 laboratories using enrichment with modified E. coli broth supplemented with cefixime and tellurite (CT-mEC), real-time PCR assay, and plating on four kinds of selective agars. This study focused on the detection efficiency of an E. albertii-specific real-time PCR assay (EA-rtPCR) and plating on deoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), DHL supplemented with rhamnose and xylose (RX-DHL), and MAC supplemented with rhamnose and xylose (RX-MAC). Chicken and bean sprout samples were inoculated with E. albertii either at 17.7 CFU/25 g (low inoculation level) or 88.5 CFU/25 g (high inoculation level), and uninoculated samples were used as controls. The sensitivity of EA-rtPCR was 1.000 for chicken and bean sprout samples inoculated with E. albertii at low and high inoculation levels. The Ct values of bean sprout samples were higher than those of the chicken samples. Analysis of microbial distribution by 16S rRNA gene amplicon sequencing in enriched cultures of bean sprout samples showed that approximately >96 % of the population comprised unidentified genus of family Enterobacteriaceae and genus Acinetobacter in samples which E. albertii was not isolated. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a high inoculation level of E. albertii was 1.000 and 0.848-0.970, respectively. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a low inoculation level of E. albertii was 0.939-1.000 and 0.515-0.727, respectively. The E. albertii-positive rate in all colonies isolated in this study was 89-90 % in RX-DHL and RX-MAC, and 64 and 44 % in DHL and MAC, respectively. Therefore, the sensitivity of RX-supplemented agar was higher than that of the agars without these sugars. Using a combination of enrichment in CT-mEC and E. albertii isolation on selective agars supplemented with RX, E. albertii at an inoculation level of over 17.5 CFU/25 g of food was detected with a sensitivity of 1.000 and 0.667-0.727 in chicken and bean sprouts, respectively. Therefore, screening for E. albertii-specific genes using EA-rtPCR followed by isolation with RX-DHL or RX-MAC is an efficient method for E. albertii detection in food.

Agminated pigmented matricoma: a case of a unique tumor with a multifocal appearance composed of neoplastic matrical cells with a significant component of melanocyte.

Matricoma is benign follicular neoplasm with the same constituent cells as pilomatricoma but with a different silhouette. Matricoma consists mostly of solid aggregations of matrical cells, as opposed to pilomatricoma, which is often a cystic lesion. We herein describe a case of agminated pigmented matricoma. A 27-year-old Japanese man visited our hospital complaining of three separate, pigmented, firm and sessile nodules on the back. Histopathologic examination revealed mostly solid aggregations of matrical and supramatrical cells located mainly within the dermis and the upper part of the subcutis. Shadow cells were present mostly within the aggregations. At higher magnifications, occasional indication of inner sheath differentiation, in addition to differentiation toward hair in the form of shadow cells, could be discerned in the center of the aggregations. The matrical and supramatrical cells within the aggregations were relatively uniform in size and shape, and contained several mitotic figures. Heavily pigmented melanocytes were present within the aggregations of matrical and supramatrical cells, and heavily pigmented melanophages were found in adjacent stroma. Pigmentation is a rare event in benign and malignant adnexal tumors, and to our knowledge, no similar case has been reported.

Prednisolone Treatment Is Effective for an Idiopathic Penile Abscess: A Case Report and Review.

BACKGROUND Penile abscesses were traditionally regarded as an infectious disease; however, idiopathic cases in which prednisolone was effective have been reported. CASE REPORT A 64-year-old man was admitted to the hospital with symptoms of penile induration and dysuria. He was diagnosed with a penile abscess, which was punctured and then relapsed. An incision and drainage were performed on the abscess, and the pus and tissue samples were cultured and examined histologically. There was no evidence of malignancy or bacterial infection, and he was diagnosed with an idiopathic penile abscess. As pus continuously drained from the incision, prednisolone 40 mg was initiated, which resulted in a decreased amount of pus and eventual wound closure. Over 15 months, prednisolone was gradually tapered to 5 mg, and the abscess continued to decrease in size. CONCLUSIONS Idiopathic penile abscesses are rare but often lead to penectomy. Prednisolone is a new treatment method for such patients. This is the third case of an idiopathic penile abscess that was successfully treated with prednisolone. The causative agent of the idiopathic penile abscess was suggested to be pyoderma gangrenosum; however, this case did not exhibit the typical characteristics of pyoderma gangrenosum. Therefore, further investigation was needed. A differential diagnosis of an infectious abscess is required before initiating steroid treatment. Open drainage is useful, but the size of the incision should be minimized for the purpose of preserving penile function. The prednisolone dose should be started at 20 to 40 mg and reduced gradually to avoid relapse.

Evaluating Methods for Detecting Escherichia albertii in Chicken Meat.

ABSTRACT: Escherichia albertii is an emerging foodborne pathogen. The source of the E. albertii infection in most foodborne outbreaks is unknown because E. albertii is difficult to isolate from suspected food or water. E. albertii has a broad host range among birds and can be isolated from chicken meat. In this study, PCR assay, enrichment, and isolation conditions for detecting E. albertii in chicken meat were evaluated. The growth of 47 E. albertii strains isolated in Japan between 1994 and 2018 and a type strain was evaluated in modified EC broth (mEC) and mEC supplemented with novobiocin (NmEC) and on media containing carbohydrates. The enzyme used for the nested PCR, the enrichment conditions, the most-probable-number (MPN) method, and agar media were also evaluated with chicken meat. To distinguish E. albertii from presumptive non-E. albertii bacteria, desoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), and these agars supplemented with rhamnose and xylose (RX-DHL and RX-MAC, respectively) were used. All E. albertii strains grew in mEC and NmEC at both 36 and 42°C and did not utilize rhamnose, sucrose, or xylose. Both the first and nested PCRs with TaKaRa Ex Taq, which was 10 to 100 times more active than the other enzymes, produced positive results in enrichment culture of 25 g of chicken meat inoculated with >20 CFU of E. albertii and incubated in mEC and NmEC at 42°C for 22 ± 2 h. Thus, the first PCR was sensitive enough to detect E. albertii in chicken meat. The MPN values in mEC and NmEC were 0.5- and 2.3-fold higher than the original inoculated bacterial levels, respectively. E. albertii in chicken meat was more efficiently isolated with enrichment in NmEC (70.1 to 100%) and plating onto RX-DHL (85.4%) and RX-MAC (100%) compared with enrichment in mEC (53.5 to 83.3%) and plating onto DHL (70.1%) and MAC (92.4%). Thus, optimized conditions for the surveillance of E. albertii contamination in food and investigations of E. albertii outbreaks, including the infectious dose, were clarified.

Oculocutaneous albinism type IV: A boy of Moroccan descent with a novel mutation in SLC45A2.

Oculocutaneous albinism type IV (OCA4 [MIM606574]) caused by mutations of the SLC45A2 gene is an autosomal recessive disorder of pigmentation characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eye. We had the opportunity to examine a Belgian boy of Moroccan descent with clinically severe OCA and screened the mutation in his SLC45A2 gene. Sequencing of exon 1, of which the PCR product showed aberrant patterns in the SSCP gel, revealed that the patient was a homozygote for p.H38R mutation. We demonstrated that the p.H38R-mutant protein was functionally incapable of melanin synthesis using melanocyte cultures (under white cells; uw) established from a mouse model of OCA4. This is the second report of the occurrence of OCA4 in a member of an African ethnic group.

O-antigen biosynthesis gene clusters of Escherichia albertii: their diversity and similarity to Escherichia coli gene clusters and the development of an O-genotyping method.

Escherichia albertii is a recently recognized human enteropathogen that is closely related to Escherichia coli. In many Gram-negative bacteria, including E. coli, O-antigen variation has long been used for the serotyping of strains. In E. albertii, while eight O-serotypes unique to this species have been identified, some strains have been shown to exhibit genetic or serological similarity to known E. coli/Shigella O-serotypes. However, the diversity of O-serotypes and O-antigen biosynthesis gene clusters (O-AGCs) of E. albertii remains to be systematically investigated. Here, we analysed the O-AGCs of 65 E. albertii strains and identified 40 E. albertii O-genotypes (EAOgs) (named EAOg1-EAOg40). Analyses of the 40 EAOgs revealed that as many as 20 EAOgs exhibited significant genetic and serological similarity to the O-AGCs of known E. coli/Shigella O-serotypes, and provided evidence for the inter-species horizontal gene transfer of O-AGCs between E. albertii and E. coli. Based on the sequence variation in the wzx gene among the 40 EAOgs, we developed a multiplex PCR-based O-genotyping system for E. albertii (EAO-genotyping PCR) and verified its usefulness by genotyping 278 E. albertii strains from various sources. Although 225 (80.9 %) of the 278 strains could be genotyped, 51 were not assigned to any of the 40 EAOgs, indicating that further analyses are required to better understand the diversity of O-AGCs in E. albertii and improve the EAO-genotyping PCR method. A phylogenetic view of E. albertii strains sequenced so far is also presented with the distribution of the 40 EAOgs, which provided multiple examples for the intra-species horizontal transfer of O-AGCs in E. albertii.

Non-biogroup 1 or 2 Strains of the Emerging Zoonotic Pathogen Escherichia albertii, Their Proposed Assignment to Biogroup 3, and Their Commonly Detected Characteristics.

Escherichia albertii, a zoonotic enteropathogen, is responsible for outbreaks of disease in humans. Identifying strains of E. albertii by phenotypic characterization tests is difficult because of its poorly defined properties. Screening its phenotypic characteristics is, nevertheless, a necessary prerequisite for further genetic analysis of its properties, and species-specific polymerase chain reaction (PCR) analysis can be used to type the pathogen. While two E. albertii biogroups (1 and 2) have been described, strains with characteristics divergent from both biogroups have been reported worldwide. The aim of the present study was to evaluate the characteristics of non-biogroup 1 or 2 strains, and discern the characteristics common to all of the E. albertii strains from this study. Altogether, 107/414 field isolates were selected for examination based on pulsed-field gel electrophoresis analysis. The 107 strains were isolated from 92 sources, including humans and pigeon feces, other wild birds, and retail chicken livers. All strains were then examined using various culture-based, biochemical (API 50CHE tests, API Zym test, and others) and molecular (virulence gene screening, multi-locus sequence analysis) testing methods. Our results revealed that all field strains (n = 107) showed non-biogroup 1 or 2 characteristics, with multiple sequence differences. Variations in indole production and the lysine decarboxylase activity profiles among the isolates made identification of E. albertii very difficult. Therefore, we propose that non-biogroup 1 or 2 of E. albertii should be assigned to biogroup 3 to make screening of them easier in public health and clinical laboratory settings. Clearly, having group criteria for indole-negative/lysine-positive, indole-positive/lysine-negative, and indole-positive/lysine-positive E. albertii biogroups 1, 2, and 3 strains, respectively, should provide for more accurate identification of E. albertii isolates. Based on our findings, we recommend that isolates displaying phenotype mobility-negativity (sulfide-indole-motility medium, 37°C), hydrogen sulfide production-negativity (triple sugar iron medium), acid production-negativity from xylose, negative ß-glucuronidase activity properties, and showing indole production and lysine decarboxylase activity profiles in accordance with one of the three biogroups, should be further assessed using an E. albertii-specific PCR assay.

Competition between trans-translation and termination or elongation of translation.

The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3'-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3'-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

A randomized double-blind trial of intravenous immunoglobulin for bullous pemphigoid.

BACKGROUND: Patients with steroid-resistant bullous pemphigoid (BP) require an appropriate treatment option. OBJECTIVE: A multicenter, randomized, placebo-controlled, double-blind trial was conducted to investigate the therapeutic effect of high-dose intravenous immunoglobulin (IVIG; 400mg/kg/day for 5days) in BP patients who showed no symptomatic improvement with prednisolone (≥0.4mg/kg/day) administered. METHODS: We evaluated the efficacy using the disease activity score on day15 (DAS15) as a primary endpoint, and changes in the DAS over time, the anti-BP180 antibody titer, and safety for a period of 57days as secondary endpoints. RESULTS: We enrolled 56 patients in this study. The DAS15 was 12.5 points lower in the IVIG group than in the placebo group (p=0.089). The mean DAS of the IVIG group was constantly lower than that of the placebo group throughout the course of observation, and a post hoc analysis of covariance revealed a significant difference (p=0.041). Furthermore, when analyzed only in severe cases (DAS≥40), the DAS15 differed significantly (p=0.046). The anti-BP180 antibody titers showed no difference between the two groups. CONCLUSION: IVIG provides a beneficial therapeutic outcome for patients with BP who are resistant to steroid therapy.

A minimum structure of aminoglycosides that causes an initiation shift of trans-translation.

Trans-translation is an unusual translation in which transfer-messenger RNA plays a dual function--as a tRNA and an mRNA--to relieve the stalled translation on the ribosome. It has been shown that paromomycin, a typical member of a 4,5-disubstituted class of aminoglycosides, causes a shift of the translation-resuming point on the tmRNA by -1 during trans-translation. To address the molecular basis of this novel effect, we examined the effects of various aminoglycosides that can bind around the A site of the small subunit of the ribosome on trans-translation in vitro. Tobramycin and gentamicin, belonging to the 4,6-disubstituted class of aminoglycosides having rings I and II similar to those in the 4,5-disubstituted class, possess similar effects. Neamine, which has only rings I and II, a common structure shared by 4,5- and 4,6-disubstituted classes of aminoglycosides, was sufficient to cause an initiation shift of trans-translation. In contrast, streptomycin or hygromycin B, lacking ring I, did not cause an initiation shift. The effect of each aminoglycoside on trans-translation coincides with that on conformational change in the A site of the small subunit of the ribosome revealed by recent structural studies: paromomycin, tobramycin and geneticin which is categorized into the gentamicin subclass, but not streptomycin and hygromycin B, flip out two conserved adenine bases at 1492 and 1493 from the A site helix. The pattern of initiation shifts by paromomycin fluctuates with variation of mutations introduced into a region upstream of the initiation point.

Nosocomial outbreak of ceftazidime-resistant Serratia marcescens strains that produce a chromosomal AmpC variant with N235K substitution.

Serratia marcescens is a Gram-negative bacterium that is often associated with nosocomial infections. Here we analyzed the resistance mechanism of the ceftazidime-resistant S. marcescens nosocomial strains. The five S. marcescens urinary tract infection-associated isolates were positive for chromosomal ampC and bla(TEM-1). Four of the five strains, ES11, ES31, ES42, and ES46, were single clone and ceftazidime resistant. The fifth strain, ES71, was susceptible to ceftazidime. Analysis of the deduced amino acid sequence revealed a Glu-235-Lys substitution in the third amino acid of the third motif of AmpC from both ES46 and ES71, and a site-directed mutagenesis experiment confirmed that this substitution is involved in the ceftazidime resistance phenotype. However, the resistance phenotypes of strains ES46 and ES71 to ceftazidime were quite different from one another, indicating that another mechanism, in addition to the AmpC mutation, is also involved in the determination of the resistance phenotype of these strains. Basal AmpC activity was more than two times higher in strain ES46 than in ES71, which could result in the differing resistance phenotypes of these two strains. The clinical significance and prevalence of extended-spectrum cephalosporin-resistant S. marcescens strains harboring the mutated chromosomal ampC gene are unclear in Japan and remain to be elucidated.

Large axillary defect after lymph node dissection directly closed by suturing the pectoralis major to the latissimus dorsi.

SUMMARY: In 2009, the Union for International Cancer Control defined lymph node (LN) metastasis ≥6 cm in diameter as stage 4 in squamous cell carcinoma of the skin. Lesions from such LNs become ulcerated and infected and bleed without treatment. A 67-year-old man suffered from skin cancer on his right back and a 7-cm-diameter LN metastasis. After axillary LN dissection, a large skin and soft tissue defect was apparent. To rectify the defect, we simply sutured the pectoralis major muscle to the latissimus dorsi muscle and covered the suture with a split-skin mesh graft. After the surgery, the range of motion of the upper limb on the side where surgery was performed remained in good condition.

Determination of enterohemorrhagic Escherichia coli serotype O165:HNM infection in a hemolytic uremic syndrome patient with adenovirus seroype 41.

A 4-year-old girl who was positive for adenovirus according to a rapid immunochromatographic test conducted at a hospital, progressed to hemorrhagic diarrhea and hemolytic uremic syndrome (HUS). The presence of adenovirus serotype 41 (AdV-41) was confirmed by TaqMan real-time PCR and sequence analysis. However, most enteric viral infections cause mild to moderate diarrhea. In the present case, enterohemorrhagic Escherichia coli (EHEC) O165:HNM was isolated concomitantly with AdV-41. In addition, O165 antibody was specifically detected in patient sera. The EHEC isolate was positive for the virulence genes stx1, stx2a, eae type ε, ehxA, and norV. Therefore, we concluded that EHEC O165:HNM was the precise pathogen leading to HUS in this patient.

Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients.

Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.