Genetic Characterization of Streptococcus pyogenes emm89 Strains Isolated in Japan From 2011 to 2019.

Invasive infection caused by Streptococcus pyogenes emm89 strains has been increasing in several countries linked to a recently emergent clade of emm89 strains, designated clade 3. In Japan, the features of emm89 S. pyogenes strains, such as clade classification, remains unknown. In this study, we collected emm89 strains isolated from both streptococcal toxic shock syndrome (STSS) (89 STSS isolates) and noninvasive infections (72 non-STSS isolates) in Japan from 2011 to 2019, and conducted whole-genome sequencing and comparative analysis, which resulted in classification of a large majority into clade 3 regardless of disease severity. In addition, invasive disease-associated factors were found among emm89 strains, including mutations of control of virulence sensor, and absence of the hylP1 gene encoding hyaluronidase. These findings provide new insights into genetic features of emm89 strains.

Application of proteotyping Strain Solution™ ver. 2 software and theoretically calculated mass database in MALDI-TOF MS typing of Salmonella serotype.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.

Shiga Toxin Subtypes and Virulence Genes in Escherichia coli Isolated from Cattle.

Subtypes of stx1 and stx2 in 45 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle were investigated by PCR. Only subtype stx1a was detected among all the stx1-positive strains. The major stx2 subtype was stx2a followed by stx2d, stx2c, stx2b, and stx2g in decreasing order of frequency. stx2c was found in strains of serotypes O157 and O174. stx2d was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were eae-negative, and 3 contained stx2d as the only stx. stx2g was found in 2 strains in association with stx2a, estA1, and astA. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic E. coli (STEC/ETEC) were found to harbor one or both of stx1a and stx2a (stx1a/stx2a) and estA1. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H- strains were eae-positive. Other putative adhesin genes, such as saa, iha, espP, and lpfAO113 were detected in more than 12 serotypes.

Comprehensive Study of Pathogenic Genes Distributed in Escherichia coli Isolated from Cattle.

Pathogenic genes such as stx1, stx2, STh gene, STp gene, LT gene, invE, eae, aggR, afaD, astA, cdt and cnf were investigated in Escherichia coli isolated from cattle during Nov. 2012 and Aug. 2013. Plural pathogenic genes were concurrently detected by multiplex PCR, and screen-positive genes were confirmed and sub-classified by PCR. Among 100 cattle investigated, 180 E. coli strains with diarrheic genes (DEC) were detected in 79 cattle, and 45 of them, isolated from 32 cattle, were Shiga toxin-producing E. coli (STEC). More than 30% of cattle carried astA, cdt, cnf and stx2 in descending order. STh gene, LT gene, invE, aggR and afaD were not detected in this study. Both stx1 and stx2 were concurrently detected from 6 of 45 STEC strains and stx2 alone was detected from 19. Seventeen STEC strains carried STp gene, astA, or cdt along with stx1 or stx2. Additionally, 135 remaining DEC were classified into 18 enterotoxigenic E. coli with STp gene, 25 enteropathogenic E. coli with eae, and 92 other DEC with astA, cdt and cnf. Both O and H serotypes were identified in 48 strains, including O157 : H7, O1H7 and so on. O157 : H7 were identified in 3 strains that carried stx2 and eae together, as found in human pathogenic strains isolated from patients with gastroenteritis and hemolytic-uremic syndrome.

[Simultaneous screening method for Bordetella species by conventional PCR assay].

A simultaneous screening method using conventional PCR was developed for the detection and discrimination of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii. A formulated multiprex method employing 4 kinds of paired primers on amplification of 4 corresponding different insertion sequences (IS481, IS1001, IS1002 and hIS1001) enabled rapid screening and identification. The detection limits of each DNA extracted from 3 kinds of Bordetella species were 5fg/microL for each. Obscure existences of B. pertussis and B. holmesii at low levels were confirmed with the LAMP method. This multiplex assay was applied to the clinical specimens obtained from patients with pertussis-like symptoms at sentinel clinics under the epidemiological surveillance of infectious diseases of Hyogo prefecture in FY2012. Among 42 nasopharyngeal swabs, B. pertussis was detected from 12 samples including 8 samples collected at outbreak in nursery school. The use of this method for the surveillance of infectious agents enabled us to search for 3 kinds of Bordetella species at once with low costs.

[Screening method of pesticides in meat using cleanup with GPC and mini-column].

A multiresidue screening method using GC/MS and LC/MS was investigated for analysis of 202 pesticides (including metabolites) in meat. Many target pesticides with a wide range of polarity were successfully extracted with ethyl acetate-cyclohexane (1 : 1) from meat samples rich in lipid components. Matrix components such as cholesterol, monoglycerides and fatty acids were effectively eliminated by means of GPC and 2 kinds of mini-columns (PSA and Silica). These procedures enabled analysis in the SCAN mode by GC/MS and LC/MS without any matrix interference. Among the pesticides tested, 17 had low recoveries (<50%), but 185 pesticides showed acceptable recoveries of 50-140% when spiked at 0.1 microg/g into muscle of beef, chicken and pork. The limits of quantitation were 0.01 microg/g, except for linuron, triforine and isoxaflutole (<0.02 microg/g). This proposed method is expected to be useful for screening analysis of residual pesticides in meat.

[Study of pesticide residues in agricultural products for the "Positive List" system].

During a 3-year monitoring survey (April 2002-March 2005) of pesticide residues in agricultural products, 592 samples (324 domestic; 268 imported) collected in Hyogo prefecture, Japan were analyzed. The number of pesticides tested increased from 232 in FY 2002 to 323 in FY 2004. The purpose of the study was to clarify the residue status by accumulating information about pesticides detected frequently, to allow effective and efficient regulation under the new "Positive List" legislation to be implemented in FY 2006. Overall, 47% of domestic and 61% of imported samples contained detectable residues and ca. 60% of positive samples contained multiple residues. The limit of quantitation was set at 0.01 microg/g and the limit of detection was 0.001-0.003 microg/ g. Most of the residues were present at low concentrations: 80% of the detections in samples excluding imported citrus fruits were < 0.05 microg/g. More than 5 different pesticides (> 0.01 microg/g) were detected simultaneously in 13 samples. The sum of the ratios of residues to MRLs was calculated as one of the indexes to represent the risk of multiple residues, and they exceeded 100% in 3 imported frozen vegetables; baby kidney bean, spinach, Welsh onion. Samples in violation of the Food Sanitation Law were not found in our survey, but 1.9% of the samples might be in conflict with the new "Positive List" legislation.

Rapid simultaneous determination of o-phenylphenol, diphenyl, thiabendazole, imazalil and its major metabolite in citrus fruits by liquid chromatography-mass spectrometry using atmospheric pressure photoionization.

A simple and rapid simultaneous analytical method of four post-harvest fungicides, o-phenylphenol (OPP), diphenyl (DP), thiabendazole (TBZ), imazalil (IMZ) and its major metabolite R14821 (IMZ-M) in citrus fruits was developed. These compounds were extracted under basic conditions with diethyl ether. The organic layer was washed with water and evaporated at moderate pressure adding methanol. These compounds were determined by liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure photoionization (APPI). The recoveries of the five compounds added to citrus fruits at 1 microg/g ranged from 67 to 100%, with relative standard deviations (R.S.D.) ranging from 2 to 8%. The detection limits (S/N = 3) were 0.01 microg/g and 0.05 microg/g (DP).

Determination of ten sulfonyl urea herbicides in unpolished rice by solid-phase extraction cleanup and LC-diode array detection.

A liquid chromatographic (LC) method with diode array detection (DAD) was developed for screening of 10 sulfonyl urea herbicide residues in unpolished rice. The investigated herbicides were azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, ethoxysulfuron, flazasulfuron, imazosulfuron, metsulfuron-methyl, pyrazosulfuron-ethyl and tribenuron-methyl. Acetonitrile-water (2:1) extracts of rice samples were cleaned up with solid-phase extraction cartridges (octadecylsilane-bonded silica (ODS) and graphitized carbon black (GCB)). Three fractions of the GCB eluate were taken for analysis using 3 separate injections in order to avoid interference in LC-DAD analysis and to reduce analyte coelution problems. Recoveries from rice samples fortified with the 10 herbicides at 0.05 and 0.2 microgram/g ranged from 46.6 to 119.6%, and coefficients of variation were 3.1-12.6%. The quantitation limits were 0.01-0.02 microgram/g.

Pesticide residues in agricultural products monitored in Hyogo Prefecture, Japan, FYs 1995-1999.

During a 5-year monitoring survey (April 1995-March 2000) of pesticide residues in agricultural products, 765 samples (478 domestic; 287 imported) collected in Hyogo Prefecture, Japan, were analyzed. The number of pesticides tested increased from 107 in fiscal year (FY) 1995 to 204 in FY 1999. The purpose of the study was to promote consumer safety by excluding the food illegally containing pesticide residues from markets. Overall, 51 % of domestic and 32% of imported samples contained no detectable residues. Multiple residues were detected in 152 (32%) of domestic and 146 (51%) of imported samples. The limit of quantitation was set at 0.01 microg/g and the limit of detection was 0.001 microg/g. Most of the residues were present at low concentrations; 70% of detections in domestic samples were <0.05 microg/g, and 97% were <0.5 microg/g. Although 86% of antifungal agent residues in imported citrus fruits were > or = 0.1 microg/g, 59% of the other residues in imported samples were <0.05 microg/g, and 96% were <0.5 microg/g. Violations of maximum residue limits (MRL) were observed in 3 samples: diazinon in chrysanthemums, dieldrin in cucumbers, and bitertanol in bananas. Of the detectable residues above 0.01 microg/g, 55% in domestic and 38% in imported samples were <10% of the MRL. Of all the samples, 2.4% contained more than 5 different pesticides; tomatoes, strawberries, apples, and citrus fruits tended to have more multiple residues.