Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

Comparison of disease and economic burden between MRSA infection and MRSA colonization in a university hospital: a retrospective data integration study.

BACKGROUND: Although there is a growing concern and policy regarding infections or colonization caused by resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), the prognosis of MRSA infections compared to that of methicillin-susceptible Staphylococcus aureus (MSSA) infections remains controversial. Moreover, there have not been any studies comparing both the burden of disease and its impact on the healthcare economy between MRSA infection and colonization while adjusting for confounding factors. These comparisons are crucial for developing effective infection control measures and healthcare policies. We aimed to compare the disease and economic burden between MRSA and MSSA infections and between MRSA infection and colonization. METHODS: We retrospectively investigated data of 496 in-patients with MRSA or MSSA infections and of 1178 in-patients with MRSA infections or MRSA colonization from a university hospital in Japan from 2016 to 2021. We compared in-hospital mortality, length of stay, and hospital charges between in-patients with MRSA and MSSA infections and those with MRSA infections and MRSA colonization using multiple regressions. We combined surveillance data, including all microbiological test results, data on patients with infections, treatment histories, and clinical outcomes, to create the datasets. RESULTS: There was no statistically significant difference in in-hospital mortality rates between matched MRSA vs. MSSA infections and MRSA infection vs. colonization. On the contrary, the adjusted effects of the MRSA infection compared to those of MSSA infection on length of stay and hospital charges were 1.21-fold (95% confidence interval [CI] 1.03-1.42, P = 0.019) and 1.70-fold (95% CI 1.39-2.07, P < 0.00001), respectively. The adjusted effects of the MRSA infection compared to those of MRSA colonization on length of stay and hospital charges were 1.41-fold (95% CI 1.25-1.58, P < 0.00001) and 1.53-fold (95% CI 1.33-1.75, P < 0.00001), respectively. Regarding confounding factors, hemodialysis or hemofiltration was consistently identified and adjusted for in the multiple regression analyses comparing MRSA and MSSA infections, as well as MRSA infection and MRSA colonization. CONCLUSIONS: MRSA infection was associated with longer length of stay and higher hospital charges than both MSSA infection and MRSA colonization. Furthermore, hemodialysis or hemofiltration was identified as a common underlying factor contributing to increased length of stay and hospital charges.

Detection of methicillin-resistant Staphylococcus aureus using a specific anti-PBP2a chicken IgY antibody.

Immunodetection of methicillin-resistant Staphylococcus aureus (MRSA) by conventional methods employing mammalian immunoglobulins has unknown detection limits, and often yields false-positive results because of the presence of S. aureus protein A, which binds the Fc region of mammalian IgG. In this study, a new PBP2a-specific chicken IgY antibody was developed in inbred and conventional chickens, and used for the detection of MRSA using whole cell lysate samples. Our results showed that this chicken IgY antibody minimized the side effects of protein A. Moreover, enzyme-linked immunosorbent assay and immunochromatography systems were used with a monoclonal and polyclonal anti-PBP2a IgY antibody, clearly differentiating MRSA from methicillin-sensitive S. aureus and other methicillin-sensitive Staphylococcus spp. The detection limit of the immunochromatography was 10(8) colony-forming units; therefore, 1 colony on an agar plate was adequate to distinguish MRSA from non-MRSA. The specificity and sensitivity of this assay were almost similar to that of a commercially available latex agglutination test; however, the procedure used in this study was less complicated. The entire detection procedure, including sample preparation, takes only 20 min and does not require special equipment. Therefore, the use of this IgY antibody as a new tool for the detection of MRSA is highly recommended.

Recurrent vascular catheter-related bacteremia caused by Delftia acidovorans with different antimicrobial susceptibility profiles.

An 11-year-old girl with metastatic neuroblastoma developed recurrent bacteremia during sustained neutropenia after autologous peripheral blood transplantation. All febrile episodes of bacteremia were caused by single Delftia acidovorans strain revealed by ERIC-PCR. This strain became resistant to broad-spectrum penicillins and cephalosporins through antibiotic treatments. Removal of the indwelling vascular catheter resulted in resolution of the infection. So far as we know, this is the first report of vascular catheter-related D. acidovorans bacteremia in Japan.

Evaluation of selection media for the detection of borderline MRSA.

Recently, hospital-associated as well as community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains showing a low level of resistance to oxacillin have emerged worldwide, and as a result, a highly sensitive method to detect MRSA has become more important. To prevent MRSA being overlooked, some selection agar media have recently been developed. We evaluated six commercially available selection agar media in regard to the detection of 35 borderline MRSA (BOMRSA) strains which were mecA-positive but showed low resistance to oxacillin. The MIC values of oxacillin differed between the broth dilution method and the agar dilution method, and 11 of the 35 BOMRSA strains were judged as sensitive by the broth dilution method and 14 of the 35 strains were judged as sensitive by the agar dilution method. Thirty-two of the 35 strains were also judged as sensitive by an oxacillin disk diffusion test. Moreover, there was no consistent pattern of resistance to the tested beta-lactams among the BOMRSA strains. Some commercially available selection media designed for the detection of MRSA contain a beta-lactam antibiotic; oxacillin, ceftizoxime, or cefoxitin, and we evaluated media containing each of these agents. The detection sensitivities of cefoxitin-based agar media, such as CHROMagar MRSA and MRSA ID, for BOMRSA were 100% at 24-h culture. On the other hand, the media containing oxacillin or ceftizoxime gave lower results at 24 h, suggesting that, possibly, BOMRSA strains may not to be able to grow on these media. These results suggest that cefoxitin-based agar media should be recommended for the first-round screening of BOMRSA.

Usefulness of phage open-reading frame typing method in an epidemiological study of an outbreak of methicillin-resistant Staphylococcus aureus infections.

A novel genotyping method for methicillin-resistant Staphylococcus aureus (MRSA), the phage open-reading frames typing (POT) method, was evaluated using 92 MRSA isolates collected from blood cultures between 1991 and 2003 at Nagoya University Hospital. These strains were divided into 64 distinct POT types, classified into 21 genotypes by pulsed-field gel electrophoresis (PFGE) using SmaI, and analyzed with the DICE coefficient of 80% in dendrogram analysis, with 48 subtypes analyzed with the DICE coefficient of 100%. The discriminatory indices of these three methods were 0.988, 0.719, and 0.953, respectively. The first and second prevalent PFGE subtypes A1 and A2, which comprised 16 and 13 isolates recovered serially during the study period, were both divided into 11 distinct POT types. Six isolates belonging to PFGE subtype A1 were indistinguishable with POT. The six isolates were probably involved in an outbreak. Phenotypic analysis suggested that these isolates were the siblings of the New York/Japan clone which are prevalent in many Japanese hospitals. In conclusion, in the strain population studied, POT is a more rapid and discriminatory method than PFGE, and is a useful epidemiological tool for evaluating the available clinical information.

Seroprevalence of Fusobacterium varium in ulcerative colitis patients in Japan.

The etiology of ulcerative colitis (UC) is unknown, while an exacerbating factor of this disease is associated with infectious agents. Recently, Fusobacterium varium has been found in the mucosa of a significant number of patients with UC. The aim of this study was to estimate the prevalence of F. varium infection based on serology, evaluate the relationship between F. varium seropositivity and UC, and determine the clinical characteristics of infected UC individuals. Seropositive patients were determined by immunoblotting with F. varium ATCC 8501 antigen. We also identified cross-reactive protein spots by peptide mass mapping analysis. These protein spots showed putative caseinolytic protease protein, putative translation elongation factor G, and putative enolase. Immunoblotting with F. varium antigen revealed signals with sera from 45 (40.2%) of the 112 UC patients and 20 (15.6%) of the 128 healthy controls, respectively (P<0.01). In terms of disease activity, seropositive UC patients were more likely to have clinically severe disease than seronegative UC patients. Disease location in seropositive patients was more extensive than the seronegative patients. In conclusion, F. varium is a predominant infection in the UC population and is a potential pathogen of UC.

Nationwide epidemiological study revealed the dissemination of meticillin-resistant Staphylococcus aureus carrying a specific set of virulence-associated genes in Japanese hospitals.

To study comprehensive toxin profiles and the chromosomal diversity of current Japanese hospital-associated meticillin-resistant Staphylococcus aureus (HA-MRSA) strains, we conducted PCR-based identification of 28 toxin genes, and staphylococcal cassette chromosome mec (SCCmec) typing and PFGE analysis of 208 MRSA strains isolated from 100 hospitals throughout Japan. Of the tested HA-MRSA strains, 80.3 % were tst-positive. The most frequent toxin gene profile was characterized by the carriage of 13 genes, tst, sec, seg, sei, sel, sem, sen, seo, lukED, hla, hlb, hld and hlg-2. Ninety of the 208 strains had this profile, which was named pattern A. Among the 118 non-pattern A strains, 100 had similar toxin gene profiles, the concordance rates to pattern A of which were more than 80 %. Consequently, 91.3 % of the examined HA-MRSA strains carried similar toxin profiles, although PFGE patterns showed a wide variation. These strains belonged to SCCmec type II, agr II and coagulase type II. We concluded that, unlike MRSA from many other countries, most of the Japanese HA-MRSA strains belonged to, or were related to, a specific group carrying the set of 13 toxin genes, irrespective of chromosomal diversity. In addition, among the 13 toxin genes, the coexistence rates of tst, sec and sel, and those of seg, sei, sem, sen and seo, were higher than for the other toxin genes. High coexistence rates of tst, sec and sel genes suggested the presence of the pathogenicity island SaPIn1 in these strains.

Post-colonoscopic Listeria septicemia in ulcerative colitis during immunosuppressive therapy.

A 78-year-old man who had been diagnosed with ulcerative colitis was admitted because of uncontrolled severe, frequent, bloody diarrhea. He was treated with immunosuppressive therapy that included corticosteroid and azathioprine. Colonoscopy was used to assess disease activity. This revealed that the mucosa of his digestive tract from the rectum to the ileum was damaged. He developed a high-grade fever soon after colonoscopy. Blood culture demonstrated Listeria monocytogenes. Treatment was changed to intravenous ampicillin for 20 days. His general body symptoms, including the bloody diarrhea, improved after treatment. We assume that the colonoscopy induced Listeria monocytogenes septicemia through bacterial translocation in this patient.

Analysis of twin-arginine translocation pathway homologue in Staphylococcus aureus.

Staphylococcus aureus releases a large number of exoproteins, including membrane-active proteins and toxins with superantigenic activity involved in pathogenicity. However, the export pathways of exoproteins in S. aureus have not been reported. We analyzed the function of the staphylococcal twin-arginine translocation (Tat) pathway homologue, the presence of which was recently discovered according to the genome database. The amino-acid sequences of the Tat homologues of S. aureus do not have a high similarity with those of Escherichia coli and other bacteria. Constructed tatC-deficient mutants from distinct parent strains showed the same patterns of exoproteins compared with those of parent strains on two-dementional gel electrophoresis, and the amounts of secreted staphylococcal enterotoxins and toxic shock syndrome toxin-1, of which signal peptides have some features often seen in signal sequences of Tat-dependent proteins, did not change with Western blotting analyses. Therefore, it seems that the Tat pathway does not play a major role in the secretion system of S. aureus, but other export pathways may play an important role in toxin secretion. This is the first experimental report showing the influence of the Tat pathway on the secretion of S. aureus.

Cytomegalovirus infection in severe ulcerative colitis patients undergoing continuous intravenous cyclosporine treatment in Japan.

AIM: To investigate active cytomegalovirus (CMV) infection following the cyclosporine A (CyA) treatment of steroid-refractory ulcerative colitis (UC). METHODS: Twenty-three patients with severe UC not responding to steroid therapy (male 14, and female 9) enrolled at Nagoya University Hospital from 1999 to 2005. They received continuous intravenous infusion of CyA (average 4 mg/kg per day) for 1 mo. Serum and colonic biopsy samples were collected before CyA treatment and 4 d, 10 d, 20 d, and 30 d after treatment. Patients were evaluated for CMV by using serology (IgM antibody by ELISA), quantitative real-time PCR for CMV DNA, and histopathological assessment of hematoxylin and eosin (HE)-stained colonic biopsies. CMV infection was indicated by positive results in any test. RESULTS: No patients had active CMV infection before CyA treatment. Eighteen of 23 UC patients treated with CyA were infected with active CMV (IgM antibody in 16/23 patients, 69.6%; CMV DNA in 18/23 patients, 78.2%; and inclusion bodies in 4/23 patients, 17.3%). There was no difference in the active CMV-infection rate between males and females. Active CMV infection was observed after approximately 8 d of CyA treatment, leading to an exacerbation of colitis. Fifteen of these 18 patients with active CMV infection (83.3%) required surgical treatment because of severe deteriorating colitis. Treatment with ganciclovir rendered surgery avoidable in three patients. CONCLUSION: Our results suggest that active CMV infection in severe UC patients treated with CyA is associated with poor outcome. Further, ganciclovir is useful for treatment of CMV-associated UC after immuno-suppressive therapy.

Use of a combination of brushing technique and the loop-mediated isothermal amplification method as a novel, rapid, and safe system for detection of Helicobacter pylori.

Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori, detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non-H. pylori bacterial species, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Helicobacter hepaticus, and 51 H. pylori strains. Results showed that H. pylori-specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 10(2) CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection.

Characterization of group C and G streptococcal strains that cause streptococcal toxic shock syndrome.

Twelve strains (the largest number ever reported) of group C and G(1) streptococci (GCS and GGS, respectively) that caused streptococcal toxic shock syndrome (STSS) were collected and characterized. Eleven strains were identified as Streptococcus dysgalactiae subsp. equisimilis, and one strain was identified as Streptococcus equi subsp. zooepidemicus. We found that it was the first reported case of STSS caused by S. equi subsp. zooepidemicus. Cluster analysis according to the 16S rRNA gene (rDNA) sequences revealed that the S. dysgalactiae strains belonged to clusters I and II, both of which were closely related. The emm types and the restriction patterns of chromosomal DNA measured by pulsed-field gel electrophoresis were highly variable in these strains except BL2719 and N1434. The 16S rDNA sequences and other characteristics of these two strains were indistinguishable, suggesting the clonal dissemination of this particular S. dysgalactiae strain in Japan. As the involvement of superantigens in the pathogenesis of group A streptococcus-related STSS has been suggested, we tried to detect known streptococcal superantigens in GCS and GGS strains. However, only the spegg gene was detected in seven S. dysgalactiae strains, with none of the other superantigen genes being detected in any of the strains. However, the sagA gene was detected in all of the strains except Tokyo1291. In the present study no apparent factor(s) responsible for the pathogenesis of STSS was identified, although close genetic relationships of GCS and GGS strains involved in this disease were suggested.