Clinical characteristics and molecular analysis of USA300 and ST 764 methicillin-resistant Staphylococcus aureus isolates from outpatients in Japan by PCR-Based open reading frame typing.

INTRODUCTION: USA300 is the most common community-acquired methicillin-resistant Staphylococcus aureus (MRSA) strain. Sequence type (ST) 764 MRSA is a new local variant of the ST 5 lineage. The objective of this study was to determine the clinical characteristics of USA300 and ST 764 infections among outpatients in Japan. METHODS: We obtained MRSA isolates from 132 outpatients who visited our hospital from January 2016 to December 2017 and compared USA300 infection group to ST 764 infection group. Molecular analysis, including that of various toxins and other virulence factors, of the MRSA isolates were performed. In particular, we investigated the relationships among PCR-based open reading frame typing (POT) scores, MRSA clones, and virulence factors. RESULTS: Twenty-seven USA300 isolates (20.5%) and 16 ST 764 isolates (12.1%) were identified. Although USA300 and ST 764 had lower rates of risk factors, their infection rates were higher. USA300-infected patients had higher rates of deep skin and soft tissue infections compared with the non-USA300 CA-MRSA-infected patients. Notably, the USA300 and ST 764 isolates had unique POT scores. CONCLUSIONS: Our results indicated that USA300 MRSA was spreading in an area 120 km west of Tokyo, Japan. We observed multiple cases of ST 764 MRSA infection, raising concerns about the antimicrobial resistance of ST 764, as it limits the choices of antibiotics to treat infection. The POT score can predict the presence of toxins and virulence factors, as well as the clone identity of MRSA with high accuracy.

Isolation of Salmonella enterica serovar Agona strains and their similarities to strains derived from a clone caused a serovar shift in broilers.

Salmonella enterica serovar Agona strains isolated from human cases were compared to strains that were derived from a clone caused a serovar shift in broilers. Pulsed field gel electrophoresis (PFGE) analysis with XbaI or BlnI digestion showed that three of seven strains from human case strains and most of the 81 strains from broilers were clustered in single complex in a minimum spanning tree (MST) reconstructed from the PFGE data. All the strains from human cases and 22 randomly selected strains from broilers were also analyzed by whole genome sequencing (WGS). Analysis of single nucleotide polymorphism (SNP) in the S. Agona core genes showed that four strains from human cases and all the strains from broilers were clustered in a maximum likelihood phylogenetic tree (ML tree) and an MST. These results indicated that the strains derived from the clone caused the serovar shift had already spread to humans. PFGE analysis with XbaI showed that four strains from broilers did not cluster with the other strains in an MST, though all those strains clustered in an ML tree and an MST reconstructed from SNP data. Moreover, three strains from broilers did not cluster in an MST reconstructed from PFGE with BlnI digestion, though those strains clustered in an ML tree and an MST reconstructed from SNP data. Therefore, it was suggested that S. Agona strains derived from a particular clone could not be traced by PFGE analysis but can be investigated by WGS analysis.

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.

Isolation of Alpha-Toxin-Deficient Clostridium perfringens Type F from Sewage Influents and Effluents.

Clostridium perfringens is classified into types A to G, and all types produce alpha-toxins; however, C. perfringens type F that is negative for phospholipase C (PLC) activity of alpha-toxin has been isolated from the environment and cases of humans afflicted by food poisoning. This study aimed to elucidate the distribution of PLC-negative C. perfringens type F in sewage influents and effluents. Influents and effluents of two wastewater treatment plants were collected monthly between July 2016 and January 2020 and between August 2018 and January 2020, respectively. Isolation rates of PLC-negative C. perfringens type F from sewage influents and effluents were 38% (33/86) and 22% (8/36), and the numbers of isolates were 43 and 13, respectively. The locus of the enterotoxin gene of all isolates was determined to be in a plasmid with an IS1151 sequence, and multilocus sequence typing revealed that all 17 representative isolates were assigned as sequence type 186. Sequencing of the plc gene of these representative isolates showed that nonsense mutation (p.W98*) causing alpha-toxin deficiency should be responsible for a loss of PLC enzymatic activity. These results suggest that alpha toxin-deficient C. perfringens type F is distributed in living and water environments since sewage influents contain community wastewater, and effluents contaminate the environment. Detection of C. perfringens type F, independent of PLC activity, should be carried out on human and environmental samples. IMPORTANCE Understanding the diversity of biochemical characteristics that may affect the identification of bacteria is essential. C. perfringens is a ubiquitous bacterium found in the environment, humans, and animals and is responsible for infectious disease in the intestine. Although the alpha-toxin of C. perfringens may be used for its detection, variants of the alpha-toxin lacking its activity have been isolated from soil and humans experiencing symptoms of diarrhea. It is valuable to disclose the prevalence of the alpha-toxin variant in the sewage of wastewater treatment plants, as it may reflect the hygienic condition of the community, as it would be a pollution source for the environment. This study shows the persistent existence and genetic characteristics of the alpha-toxin variant in sewage and reveals a lacking mechanism of the alpha-toxin activity and proposes the detection method of C. perfringens, independent of the alpha-toxin activity.

Characterization of Salmonella Isolates from Wastewater Treatment Plant Influents to Estimate Unreported Cases and Infection Sources of Salmonellosis.

Salmonella enterica is a major cause of gastroenteritis usually caused by animal-based contaminated foods. Since the current passive surveillance is not sufficient to detect all infections and infection sources, we determined the prevalence of Salmonella isolated from sewage influent of wastewater treatment plants (WWTPs) and compared the characteristics of human and food isolates to identify the infection sources. Sewage influent samples were collected monthly from two WWTPs located in the Yamanashi Prefecture, Japan, for three years. Serotypes, antimicrobial resistances, isolation periods, isolated areas, and pulsed-field gel electrophoresis patterns of six isolates belonging to five serotypes were consistent with those of the isolates from patients. Real-time PCR for Salmonella indicated that sewage influents reflect cases of patients infected with Salmonella, including unreported cases. Serovars Schwarzengrund and Anatum were predominant in sewage, but not in humans, and their characteristics were closely related or identical to those isolated from poultry heart and liver, respectively. These results suggest that sewage influent contains Salmonella isolates from humans and that some originated from unreported human cases infected by poultry-associated products. Therefore, it is necessary to take countermeasures against Salmonella infection based on the unreported cases, which would be disclosed by analysis of sewage influent.

The Circulation of Type F Clostridium perfringens among Humans, Sewage, and Ruditapes philippinarum (Asari Clams).

Clostridium perfringens is an important pathogen that is responsible for gastroenteritis; the causative agent for the symptoms is C. perfringens enterotoxin (CPE), which is mainly produced by type F C. perfringens. Since shellfishes may gather C. perfringens in the water environment, this study estimated the potential circulation of type F C. perfringens among humans, sewage, and Ruditapes philippinarum (asari clams) as a result of sewage pollution. A comparison of the characteristics among the isolates from 86 sewage influents, 36 effluents, 76 asari clams, and 37 humans was conducted. Serotyping, cpe genotyping, and toxin genotyping showed that C. perfringens with a plasmid IS1151 sequence downstream of cpe was predominant among sewage influents, effluents, humans, and asari clams. Multilocus sequence typing suggested that some isolates from a human, sewage influents, effluents, and asari clams were linked to each other. These results demonstrated that asari clams are the necessary infection sources of C. perfringens responsible for carriers and foodborne diseases, and that these pathogens from humans infected by asari clams can pollute the water environment. It is useful to assess bacteria such as C. perfringens isolates from sewage to estimate the trend of those from the community.

Genetic characteristics of emerging Salmonella enterica serovar Agona strains isolated from humans in the prior period to occurrence of the serovar shift in broilers.

Our previous studies found that a dominant serovar of Salmonella enterica isolates from three farms raising broilers in 2014 and 2015 was serovar Agona and the number of Infantis isolates decreased (the serovar shift). In this study, 52 S. Agona strains which isolated between 1993 and 2008, were compared to the serovar shift clone by molecular epidemiology and phylogenetic analyses, using pulsed field gel electrophoresis and whole genome sequence analyses. Of the 52 strains, one strain isolated from a human case in 1995 was genetically identical to the serovar shift clone, even though it was isolated prior to the serovar shift. These results suggested that the S. Agona serovar shift clone had existed in a source other than chicken penetrated chicken population.

Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actin-like protein, MreB, in Shigella flexneri.

S-Benzylisothiourea compound A22 induces coccoid forms in Escherichia coli by inhibiting the function of the actin-like cytoskeletal protein, MreB. The minimum inhibitory concentration of A22 and the minimum concentration to induce coccoid forms for various pathogenic bacteria were determined. At 10 microg/ml, A22 induced coccoid forms in Shigella flexneri but did not inhibit the growth. No alteration of coccoid forms in the Gram-positive bacteria and anaerobic bacteria tested were observed following treatment with A22. To study the relationship between pathogenicity and alterations in bacterial shape, the infectious capacity of A22-induced coccoid S. flexneri was examined using CHO-K1 cells. Invasion of the coccoid cells was significantly reduced, however, no changes in adherence were observed. Using a mutant defective in the type III secretion apparatus, which delivers effectors to the host, we examined the secretion of effectors by A22-induced coccoid S. flexneri. The amount of secreted effectors in the coccoid cells was clearly decreased compared to rod-shaped cells. These results showed that the maintenance of rod-shaped cells by MreB in bacteria was essential for the secretion of effectors via the type III secretion system. Therefore, our results suggest that A22 is a useful lead compound for a novel anti-infectious agent without bactericidal activity and MreB is a candidate target site for development of new anti-infectious agents.