Comparative Genomic Analysis of Third-Generation-Cephalosporin-Resistant Escherichia coli Harboring the blaCMY-2-Positive IncI1 Group, IncB/O/K/Z, and IncC Plasmids Isolated from Healthy Broilers in Japan.

The off-label use of third-generation cephalosporins (3GCs) during in ovo vaccination or vaccination of newly hatched chicks has been a common practice worldwide. CMY-2-producing Escherichia coli strains have been disseminated in broiler chicken production. The objective of this study was to determine the epidemiological linkage of blaCMY-2-positive plasmids among broilers both within and outside Japan, because the grandparent stock and parent stock were imported into Japan. We examined the whole-genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002 to 2014. The predominant 3GC resistance gene was blaCMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-Iγ (n = 21; 24.1%), IncI (n = 12; 13.8%), IncB/O/K/Z (n = 28; 32.2%), and IncC (n = 22; 25.3%). Those plasmids were subjected to gene clustering, network analyses, and plasmid multilocus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single-nucleotide variant (SNV)-based phylogenetic analysis. MLST and SNV-based phylogenetic analysis revealed high diversity of E. coli isolates. The sequence type 429 (ST429) cluster harboring blaCMY-2-positive IncB/O/K/Z was closely related to isolates from broilers in Germany harboring blaCMY-2-positive IncB/O/K/Z. pST55-IncI, pST12-IncI1-Iγ, and pST3-IncC were prevalent in western Japan. pST12-IncI1-Iγ and pST3-IncC were closely related to plasmids detected in E. coli isolates from chickens in North America, whereas 26 IncB/O/K/Z types were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria inside and outside Japan.

Potential Impact of Rapid Blood Culture Testing for Gram-Positive Bacteremia in Japan with the Verigene Gram-Positive Blood Culture Test.

Background. Early detection of Gram-positive bacteremia and timely appropriate antimicrobial therapy are required for decreasing patient mortality. The purpose of our study was to evaluate the performance of the Verigene Gram-positive blood culture assay (BC-GP) in two special healthcare settings and determine the potential impact of rapid blood culture testing for Gram-positive bacteremia within the Japanese healthcare delivery system. Furthermore, the study included simulated blood cultures, which included a library of well-characterized methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) isolates reflecting different geographical regions in Japan. Methods. A total 347 BC-GP assays were performed on clinical and simulated blood cultures. BC-GP results were compared to results obtained by reference methods for genus/species identification and detection of resistance genes using molecular and MALDI-TOF MS methodologies. Results. For identification and detection of resistance genes at two clinical sites and simulated blood cultures, overall concordance of BC-GP with reference methods was 327/347 (94%). The time for identification and antimicrobial resistance detection by BC-GP was significantly shorter compared to routine testing especially at the cardiology hospital, which does not offer clinical microbiology services on weekends and holidays. Conclusion. BC-GP generated accurate identification and detection of resistance markers compared with routine laboratory methods for Gram-positive organisms in specialized clinical settings providing more rapid results than current routine testing.

Genomic characterization of third-generation cephalosporin-resistant Escherichia coli strains isolated from diseased dogs and cats: Report from Japanese Veterinary Antimicrobial Resistance Monitoring.

This study investigates the genomic characteristics of canine and feline cefotaxime (CTX, a third-generation cephalosporin)-resistant Escherichia coli using the JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring System, a nationwide monitoring. In this study, whole-genome sequencing (WGS) was performed on 51 canine and 45 feline CTX-resistant E. coli isolates, with certain isolates subjected to pulsed-field gel electrophoresis with S1 nuclease for plasmid-chromosome separation. The most common blaCTX-M genes were blaCTX-M-27 (dogs: 11/51 [21.6 %]; cat: 10/45 [22.2 %]), followed by blaCTX-M-14 (dogs: 10/51 [19.6 %]; cats: 10/45 [22.2 %]), and blaCTX-M-15 (dogs: 9/51 [17.6 %]; cats: 5/45 [11.1 %]). Besides ß-lactamase genes, all isolates harbored mdf(A), a multidrug efflux pump, with resistance genes for aminoglycosides, sulfonamides, trimethoprims, macrolides and tetracyclines. None of the isolates had carbapenemase genes, such as blaOXA-48, blaNDM, and blaIMP, whereas most of the isolates showed double mutations in gyrA and parC, which affected quinolone resistance. For the isolates separately analyzed for plasmid and chromosomal DNA via WGS, the majority of CTX-M genes were present on the plasmids. Some plasmids also harbored the same combination of resistance genes and plasmid replicon type, although they differed from isolates derived from different areas of Japan. The predominant plasmids were blaCTX-M-27,aadA5, aph(6)-Id, aph(3")-Ib, sul1, sul2, tet(A), dfrA17, and mph(A) on IncF. The predominant combination of ST131, O25:H4, and B2 isolates comprised the largest cluster in the minimum spanning tree and the ST131 E. coli harboring blaCTX-M-27 from human in Japan was closely related to these isolates. The results indicated that CTX-resistant canine and feline E. coli harbored multiple plasmids carrying the same combination of resistance genes and emphasizes the need to prevent the spread. DATA AVAILABILITY: All raw short-read sequence data have been deposited in the DNA Data Bank of Japan. (DRR Run No, DRR335726-335821).

Prevalence and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Pigs in Japan.

We investigated the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in pig slaughterhouses from 2018 to 2022 in Japan and the isolates were examined for antimicrobial susceptibility and genetic characteristics by whole-genome analysis. Although the positive LA-MRSA rates on farms (29.6%) and samples (9.9%) in 2022 in Japan remained lower than those observed in European countries exhibiting extremely high rates of confirmed human LA-MRSA infections, these rates showed a gradually increasing trend over five years. The ST398/t034 strain was predominant, followed by ST5/t002, and differences were identified between ST398 and ST5 in terms of antimicrobial susceptibility and the resistance genes carried. Notably, LA-MRSA possessed resistance genes toward many antimicrobial classes, with 91.4% of the ST398 strains harboring zinc resistance genes. These findings indicate that the co-selection pressure associated with multidrug and zinc resistance may have contributed markedly to LA-MRSA persistence. SNP analysis revealed that ST398 and ST5 of swine origin were classified into a different cluster of MRSA from humans, showing the same ST in Japan and lacking the immune evasion genes (scn, sak, or chp). Although swine-origin LA-MRSA is currently unlikely to spread to humans and become a problem in current clinical practice, preventing its dissemination requires using antimicrobials prudently, limiting zinc utilization to the minimum required nutrient, and practicing fundamental hygiene measures.

Detection of IMP metallo-ß-lactamase in carbapenem-nonsusceptible Enterobacteriaceae and non-glucose-fermenting Gram-negative rods by immunochromatography assay.

Metallo-ß-lactamases (MBLs) are transmissible carbapenemases of increasing prevalence in Gram-negative bacteria among health care facilities worldwide. Control of the further spread of these carbapenem-resistant bacteria relies on clinical microbiological laboratories correctly identifying and classifying the MBLs. In this study, we evaluated a simple and rapid method for detecting IMP, the most prevalent MBL in Japan. We used an immunochromatography (IC) assay for 181 carbapenem-nonsusceptible (CNS) (nonsusceptible to imipenem or meropenem) strains comprising 74 IMP-producing and 33 non-IMP-producing strains of non-glucose-fermenting Gram-negative rods (NFGNR), as well as 64 IMP-producing and 10 non-IMP-producing Enterobacteriaceae strains. The IC assay results were compared to those from the double-disk synergy test (DDST), the MBL Etest, and the modified Hodge test (MHT) (only for Enterobacteriaceae). The IMP type was confirmed by specific PCR and direct sequencing. The IC assay detected all of the IMP-type MBLs, including IMP-1, -2, -6, -7, -10, -11, -19, -20, and -22 and IMP-40, -41, and -42 (new types), with 100% specificity and sensitivity against all strains tested. Although the sensitivity and specificity values for the DDST and MHT were equivalent to those for the IC assay, the MBL Etest was positive for only 87% of NFGNR and 31% of Enterobacteriaceae due to the low MIC of imipenem, causing an indeterminate evaluation. These results indicated that the IC assay might be a useful alternative to PCR for IMP MBL detection screening.

Evaluation of a simple protein extraction method for species identification of clinically relevant staphylococci by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

In clinical microbiology, bacterial identification is labor-intensive and time-consuming. A solution for this problem is the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In this study, we evaluated a modified protein extraction method of identification performed on target plates (on-plate extraction method) with MALDI-TOF (Bruker Microflex LT with Biotyper version 3.0) and compared it to 2 previously described methods: the direct colony method and a standard protein extraction method (standard extraction method). We evaluated the species of 273 clinical strains and 14 reference strains of staphylococci. All isolates were characterized using the superoxide dismutase A sequence as a reference. For the species identification, the on-plate, standard extraction, and direct colony methods identified 257 isolates (89.5%), 232 isolates (80.8%), and 173 isolates (60.2%), respectively, with statistically significant differences among the three methods (P < 0.05). In conclusion, the on-plate extraction method is at least as good as standard extraction in identification rate and has the advantage of a shorter processing time.

Nationwide Monitoring of Antimicrobial-Resistant Escherichia coli and Enterococcus spp. Isolated From Diseased and Healthy Dogs and Cats in Japan.

The Japanese Veterinary Antimicrobial Resistance Monitoring System (JVARM) was established for nationwide monitoring of antimicrobial-resistant bacteria isolated from animals. Here, antimicrobial resistance of Escherichia coli and Enterococcus spp. isolates from diseased and healthy dogs and cats was investigated. Isolates were collected from diseased dogs and cats and from healthy dogs and cats in 2018 to 2020. Minimum inhibitory concentrations were determined for 1873 E. coli and 1383 Enterococcus spp. isolates. E. coli isolates were most commonly resistant to nalidixic acid [diseased dog (DD), 62.1%; diseased cat (DC), 59.9%; healthy dog (HD), 23.5%; healthy cat (HC, 24.0%] and ampicillin (DD, 54.4%; DC, 64.1%; HD, 28.4%; HC, 25.2%), followed by ciprofloxacin (DD, 45.0%; DC, 44.0%; HD, 12.9%; HC, 10.4%). Enterococcus spp. isolates were most resistant to tetracycline (DD, 66.9%; DC, 67.8%; HD, 47.0%; HC, 52.0%), followed by erythromycin (DD, 43.2%; DC, 46.6%; HD, 27.8%; HC, 34.0%) and ciprofloxacin (DD, 27.9%; DC, 43.7%; HD, 9.7%; HC 12.9%). Only a few E. coli isolates were resistant to colistin and none were resistant to meropenem. Also, none of the Enterococcus spp. isolates we have tested were resistant to vancomycin. The significantly higher resistance rates of E. coli and Enterococcus spp. isolates from diseased, as opposed to healthy, dogs and cats against most of the tested antimicrobials indicates that the use of antimicrobials could select resistant E. coli and Enterococcus spp.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from pigs in Japan.

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of infection in hospitalized patients and can be prevalent in humans and various animal species. In European countries, MRSA isolates belonging to clonal complex 398 have been detected at high rates in pigs. However, the prevalence of MRSA in pigs and farm environments in Japan remains unclear. MRSA isolates were obtained from pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust. We conducted whole-genome sequencing (WGS) and analyzed the molecular epidemiological relationship between these MRSA isolates using core genome multilocus sequence typing (cgMLST). The prevalence rates of MRSA among pigs in slaughterhouses, diseased pigs on farms, imported breeding pigs, and farm dust were 5.2 %, 3.4 %, 28.8 %, and 0.06 %, respectively. ST 398 isolates that classified as ST398/t034 were isolated from pigs from all sources. The results of cgMLST showed that ST398/t034 isolates originating from domestic pigs clustered into the same cluster as the isolates from imported breeding pigs. However, some clusters only included isolates of domestic pig origin. Most MRSA isolates in this study carried resistance genes for aminoglycosides, ß-lactams, macrolides, tetracyclines, and zinc. None of the MRSA isolates in this study harbored Panton-Valentine leukocidin toxin genes. Molecular epidemiological analysis suggested a relationship between isolates from slaughter pigs and imported breeding pigs and the presence of MRSA isolates of domestic origin. However, more data are needed for elucidation of the origin of these MRSA variants in the pig industry in Japan.

An epidemiological analysis of the level of biosecurity and animal welfare on pig farms in Japan and their effect on the use of veterinary antimicrobials.

In Japan the highest use of veterinary antimicrobials is in pig production. To obtain useful information to achieve the best approach to reducing this use, we analyzed the association between the level of on-farm biosecurity and animal welfare with the level of antimicrobial use as recorded on prescriptions on 38 pig farms under contract to veterinarians of the Japanese Association of Swine Veterinarians. To determine the level of welfare we recorded the risk of pre- and post-weaning deaths and the floor space available per fattening pig (m2/head). Multivariable linear regression analysis was performed, using biosecurity scores and animal welfare indicators as independent variables and the amount of antimicrobial usage as dependent variables. The results showed that the higher scores for the site condition (location) and external biosecurity scores of the sub-categories 'farm contractors' were strongly associated with the lower use of oral antimicrobials (P<0.05). This suggests that in order to reduce the usage of antimicrobials for herd treatments, farmers should consider the location when building a new farm or pig house and strengthen the entrance requirements for high risk visitors. Regression analysis for the respective antimicrobials showed that the site condition, the biosecurity scores of the sub-categories 'farm contractors', 'pen layouts' (e.g. independence of pens and sites), 'pig flows' (e.g. the completeness of all-in/ all-out system) and an animal welfare indicator (i.e. post-weaning mortality risk) were significantly associated with the use of one or more antimicrobials (P<0.05).

Detection of porcine reproductive and respiratory syndrome virus in oral fluid from naturally infected pigs in a breeding herd.

The objectives of the present study were to evaluate the anatomic localization of porcine reproductive and respiratory syndrome virus (PRRSV) in naturally infected pigs and to determine whether oral fluid could be used to detect the virus in infected animals. Two sows, seven 2-month-old grower pigs, and 70 6-month-old gilts were included in this study. PRRSV in sera and oral fluid were identified by nested reverse transcription PCR (nRT-PCR) while lung, tonsil, and tissue associated with oral cavity were subjected to nRT-PCR, immunohistochemistry, and in situ hybridization. In sows, PRRSV was identified in oral fluid and tonsils. PRRSV was also detected in oral fluid, tonsils, salivary glands, oral mucosa, and lungs of all seven grower pigs. However, viremia was observed in only two grower pigs. Double staining revealed that PRRSV was distributed in macrophages within and adjacent to the tonsillar crypt epithelium. In gilts, the North American type PRRSV field strain was detected 3 to 8 weeks after introducing these animals onto the farm. These results confirm previous findings that PRRSV primarily replicates in tonsils and is then shed into oral fluid. Therefore, oral fluid sampling may be effective for the surveillance of PRRSV in breeding herds.

Host cell-specific effects of lentiviral accessory proteins on the eukaryotic cell cycle progression.

Lentiviral accessory proteins are thought to play important roles in regulating the viral replication through modulation of host cell functions. For example, Vpr of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G2 arrest in a host cell-specific manner. Similarly, HIV-2 Vpr, but not Vpx, has been shown to induce G2 arrest in primate cells. It has also been reported that Orf-A of feline immunodeficiency virus (FIV) induces G2 arrest in a simian cell line. However, activities of these non-HIV-1 accessory proteins in different cellular context are unclear. In this study, effects of HIV-2 Vpr, Vpx and FIV Orf-A on cell cycle progression were compared with those of HIV-1 Vpr in various mammalian cell lines and the fission yeast. These non-HIV-1 accessory proteins induced the cell cycle arrest in a host cell-specific manner, and their specificities were different from each other. Interestingly, HIV-2 Vpx-induced G2 arrest in bovine MDBK cells. It was also notable that HIV-2 Vpx and FIV Orf-A appeared to block the cell separation in the fission yeast. The host cell-specific activities of different lentiviral accessory proteins revealed in this study may provide a useful basis for elucidating the mechanism of their functions.

Electrochemical immunoassay at a 17beta-estradiol self-assembled monolayer electrode using a redox marker.

A simple electrochemical immunoassay was demonstrated using a 17beta-estradiol modified electrode. 17beta-estradiol was immobilized on the gold electrode surface with a self-assembly technique. The specific binding between estradiol antibody and 17beta-estradiol on the electrode surface was evaluated by monitoring the change in the electrode response with three hydrophilic redox markers. The decrease in the electrode response for the redox marker was observed, when the antibody was bound to the estradiol self-assembled monolayer (SAM) electrode surface. The change in the electrode response of the redox marker is attributed to the steric hindrance between the antibody on the electrode surface and the redox marker. The relative standard deviation at 30 microg ml(-1) estradiol antibody was 4.1% (n = 3). The competitive reaction between the antigen in the solution and 17beta-estradiol immobilized on the electrode surface for the limited binding sites on the antibody produced an increase in the electrode response with hydroquinone as the marker. The binding affinity of three antigens including 17beta-estradiol to the estradiol antibody was evaluated. Furthermore, the result obtained from this method was compared with the previously reported enzyme binding assay using the biotinylated estradiol and the biotin-immobilized microtiter plate.

Cell cycle arrest induction by an adenoviral vector expressing HIV-1 Vpr in bovine and feline cells.

An accessory protein, Vpr, of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G(2)/M arrest in primate cells, but not in rodent cells, suggesting that a species-specific factor might be involved in the phenomenon. To study whether Vpr can cause G(2)/M arrest in non-primate cells, a novel adenoviral vector, Ad-VIG, co-expressing HIV-1 Vpr and green fluorescent protein (GFP) was constructed and infected on cell lines derived from various mammalian species. With its ability to express GFP, Ad-VIG enabled flow cytometric evaluation of transduction efficiency in the infected cells, and Western blot analysis showed successful expression of Vpr in the vector-transduced cells. Upon Ad-VIG infection, human HeLa, African green monkey Vero, feline CRFK, and bovine MDBK cells manifested cell cycle G(2)/M arrest. This is the first study showing that non-primate feline and bovine cells are susceptible to Vpr-induced cell cycle arrest.