Role of multidrug resistance and co-resistance on a high percentage of streptomycin resistance in Escherichia coli isolated from chicken meats in Japan.

We focused on streptomycin resistance because of the high percentage of streptomycin-resistant Escherichia coli concerning the amount used of streptomycin. Antimicrobial resistance and horizontal transfer were identified in 117 isolates of coliform bacteria from chicken meat to identify the factors that increase streptomycin resistance. Escherichia (45 isolates) was the predominant genus. Most streptomycin-resistant Escherichia isolates were resistant to other antimicrobials (17/18), suggesting that using various antimicrobials could select streptomycin-resistant Escherichia isolates. Resistance was transferred from 7 out of the 18 streptomycin-resistant isolates. The transconjugants acquired strA/strB (7/7), blaTEM (5/7), aphA1 (5/7), tetB (3/7), dfrA14 (1/7) and/or dfrA17 (1/7). The co-resistance of streptomycin resistance with other resistances would also increase streptomycin resistance.

Surveillance, Isolation, and Genetic Characterization of Bat Herpesviruses in Zambia.

Bats are of significant interest as reservoirs for various zoonotic viruses with high diversity. During the past two decades, many herpesviruses have been identified in various bats worldwide by genetic approaches, whereas there have been few reports on the isolation of infectious herpesviruses. Herein, we report the prevalence of herpesvirus infection of bats captured in Zambia and genetic characterization of novel gammaherpesviruses isolated from striped leaf-nosed bats (Macronycteris vittatus). By our PCR screening, herpesvirus DNA polymerase (DPOL) genes were detected in 29.2% (7/24) of Egyptian fruit bats (Rousettus aegyptiacus), 78.1% (82/105) of Macronycteris vittatus, and one Sundevall's roundleaf bat (Hipposideros caffer) in Zambia. Phylogenetic analyses of the detected partial DPOL genes revealed that the Zambian bat herpesviruses were divided into seven betaherpesvirus groups and five gammaherpesvirus groups. Two infectious strains of a novel gammaherpesvirus, tentatively named Macronycteris gammaherpesvirus 1 (MaGHV1), were successfully isolated from Macronycteris vittatus bats, and their complete genomes were sequenced. The genome of MaGHV1 encoded 79 open reading frames, and phylogenic analyses of the DNA polymerase and glycoprotein B demonstrated that MaGHV1 formed an independent lineage sharing a common origin with other bat-derived gammaherpesviruses. Our findings provide new information regarding the genetic diversity of herpesviruses maintained in African bats.

Molecular Characteristics and Antimicrobial Resistance of Salmonella enterica Serovar Schwarzengrund from Chicken Meat in Japan.

Our previous study revealed that Salmonella enterica serovar Schwarzengrund-contaminated areas of broiler chickens have expanded from West Japan to East Japan. The present study investigated the antimicrobial resistance and molecular characteristics of 124 S. Schwarzengrund isolates obtained from chicken meat produced in East and West Japan from 2008 to 2019. Comparing the isolates obtained in 2008 and 2015-2019, an increase in the proportion of those resistant to kanamycin [51.4-89.7% (p < 0.001)] was observed. In contrast, the proportion of isolates resistant to both streptomycin and tetracycline and those that harbored a 1.0-kb class 1 integron, aadA1, and tetA, significantly decreased from 100% in 2008 to 47.1% in 2015-2019 (p < 0.001). A 1.0-kb class 1 integron containing aadA1, harbored by 78 isolates, was different from that reported in globally distributed S. Schwarzengrund strains (1.9 kb, containing the dfrA12-aadA2 gene cassette). Twenty-five isolates from different product districts and years of isolation were typed as sequence type (ST) 241 with multilocus sequence typing. Our results suggest that S. Schwarzengrund, which contaminates chicken meat in Japan, shares a common ancestor regardless of the product district from 2008 to recent years. Moreover, S. Schwarzengrund ST241 may have spread from western to eastern Japan.

Surveillance of Shiga toxin-producing Escherichia coli and Campylobacter spp. in wild Japanese deer (Cervus nippon) and boar (Sus scrofa).

Increasing game meat consumption in Japan requires the dissemination of safety information regarding the presence of human pathogens in game animals. Health information regarding the suitability of these animals as a meat source is not widely available. In this study, we aimed to evaluate the safety of game meat and detect potential human pathogens in wild deer (Cervus nippon) and boar (Sus scrofa) in Japan. Fecal samples from 305 wild deer and 248 boars of Yamaguchi, Kagoshima, and Tochigi prefectures collected monthly for 2 years were examined for the prevalence of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter spp. STEC was isolated from 51 deer consistently throughout the year and from three boars; O-antigen genotype O146, the expression of stx2b, and eaeA absence (n=33) were the major characteristics of our STEC isolates. Other serotypes included the medically important O157, stx2b or stx2c, and eaeA-positive (n=4) and O26, stx1a, and eaeA-positive strains (n=1). Campylobacter spp. were isolated from 17 deer and 31 boars. Campylobacter hyointestinalis was the most common species isolated from 17 deer and 25 boars, whereas Campylobacter lanienae and Campylobacter coli were isolated from three and two boars, respectively. Seasonal trends for the isolation of these bacteria were not significant. This study demonstrates that wild game animals carry human pathogens; therefore, detailed knowledge of the safe handling of game meat is needed to prevent foodborne infections.

Comparison of antimicrobial susceptibilities of bacterial isolates between cured and uncured cases of bovine mastitis.

To evaluate the effect of antimicrobial susceptibility on outcomes, we compared the minimum inhibitory concentrations (MICs) for Staphylococcus, Streptococcus, and the family Enterobacteriaceae from cured and uncured mastitis cases; milk shipment for uncured cases could not be resumed within 3 weeks after initial clinical examination. A higher MIC50 of ampicillin and a higher MIC90 of cefazolin for Enterobacteriaceae isolates were observed for cured rather than uncured cases with differences in ≥2 tubes. Endotoxins are generally released from Enterobacteriaceae upon antimicrobial treatment; their amounts are presumed to be greater in mastitis cases resulting from ß-lactam antibiotic-susceptible rather than -resistant microbes. For staphylococcal and streptococcal isolates, the MIC50 and MIC90 of ß-lactam antibiotics were similar for cured and uncured cases.

Effects of climatic elements on Salmonella contamination in broiler chicken meat in Japan.

The effects of climatic elements on Salmonella contamination of chicken meat were investigated. Logistic regression analysis was performed to evaluate the association between Salmonella isolation, for 240 chicken samples purchased from March 2015 to February 2017, and climatic elements, over 65 days of chicken rearing. Salmonella was isolated from 143 samples (59.6%), and the most dominant serovars identified were Infantis (77/240, 32.1%) and Schwarzengrund (56/240, 23.3%). Previous studies have reported S. Schwarzengrund contamination of broiler chickens only in western Japan; however, in the present study, S. Schwarzengrund was also isolated from meat produced in eastern Japan-20% (12/60) in the C prefecture to 36.4% (8/22) in the Y prefecture-suggesting that S. Schwarzengrund-contaminated areas have expanded towards eastern Japan. Air temperature showed a significant negative association with S. Schwarzengrund isolation for chicken meat produced during periods with rising temperature (spring and summer) [odds ratio (OR), 0.894 to 0.935; P<0.01]. Moreover, the risk of S. Schwarzengrund contamination of chicken meat was higher during spring (OR, 3.951; P=0.008) and winter (OR, 4.071; P=0.006) than during summer. Effects of climatic elements and differences in contamination risk across seasons were not observed for any Salmonella serovars and only S. Infantis, which could be attributed to differences in transmission patterns and vehicles among Salmonella serovars. These findings are valuable for understanding the dynamics of S. Schwarzengrund dissemination in broiler farms.

Rapid and Selective Discrimination of Gram-Positive and Gram-Negative Bacteria by Boronic Acid-Modified Poly(amidoamine) Dendrimer.

There is an urgent need to develop a rapid and selective method for the detection of bacteria because delayed diagnosis and the overuse of antibiotics have triggered drug resistance in bacteria. To this end, we prepared boronic acid-modified poly(amidoamine) generation 4 (B-PAMAM(G4)) dendrimer as cross-linking molecules that form aggregates with bacteria. Within 5 min of adding B-PAMAM(G4) dendrimer solution to a bacterial suspension, large aggregates were observed. Interestingly, the aggregate formation with various bacteria was pH-dependent. In basic pH, both Gram-positive and Gram-negative bacteria formed aggregates, but in neutral pH, only Gram-positive bacteria formed aggregates. We revealed that this bacteria-selective aggregation involved the bacterial surface recognition of the phenylboronic acid moiety of B-PAMAM(G4) dendrimer. In addition, we demonstrated that the spherical structure of B-PAMAM(G4) was one of the important factors for the formation of large aggregates. The aggregation was also observed in the presence of ≤10 mM fructose. B-PAMAM(G4) dendrimer is expected to be a powerful tool for the rapid and selective discrimination between Gram-positive and Gram-negative bacteria.

Adult-onset Krabbe disease presenting with an isolated form of peripheral neuropathy.

INTRODUCTION: Adult-onset Krabbe disease is clinically rare and usually affects the pyramidal tracts in the central nervous system. Patients develop a spastic gait, and peripheral neuropathy sometimes occurs simultaneously. METHODS: A 55-year-old woman with consanguineous parents developed slowly progressive, asymmetric muscle weakness and atrophy in her forearms, while her ability to walk remained unaffected without pyramidal tract signs after onset at age 51 years. RESULTS: Nerve conduction studies demonstrated an asymmetric demyelinating-type peripheral neuropathy, and sural nerve biopsy documented reduced myelinated nerve fiber density with uniformly thin myelin sheaths, suggesting hypomyelination. Brain MRI demonstrated minor white-matter injury along the optic radiations, which was associated with asymptomatic, mild, prolonged latency on visual evoked potentials. Laboratory analysis documented low enzyme activity of galactocerebrosidase (GALC) and a known mutation of the GALC gene. CONCLUSION: Isolated peripheral neuropathy occurs very rarely in adult-onset Krabbe disease. Muscle Nerve 54: 152-157, 2016.

Genetic diversity in the prion protein gene (PRNP) of domestic cattle and water buffaloes in Vietnam, Indonesia and Thailand.

There has been an accumulation of information on frequencies of insertion/deletion (indel) polymorphisms within the bovine prion protein gene (PRNP) and on the number of octapeptide repeats and single nucleotide polymorphisms (SNPs) in the coding region of bovine PRNP related to bovine spongiform encephalopathy (BSE) susceptibility. We investigated the frequencies of 23-bp indel polymorphism in the promoter region (23indel) and 12-bp indel polymorphism in intron 1 region (12indel), octapeptide repeat polymorphisms and SNPs in the bovine PRNP of cattle and water buffaloes in Vietnam, Indonesia and Thailand. The frequency of the deletion allele in the 23indel site was significantly low in cattle of Indonesia and Thailand and water buffaloes. The deletion allele frequency in the 12indel site was significantly low in all of the cattle and buffaloes categorized in each subgroup. In both indel sites, the deletion allele has been reported to be associated with susceptibility to classical BSE. In some Indonesian local cattle breeds, the frequency of the allele with 5 octapeptide repeats was significantly high despite the fact that the allele with 6 octapeptide repeats has been reported to be most frequent in many breeds of cattle. Four SNPs observed in Indonesian local cattle have not been reported for domestic cattle. This study provided information on PRNP of livestock in these Southeast Asian countries.

Methicillin-resistant Staphylococcus aureus carriage among veterinary staff and dogs in private veterinary clinics in Hokkaido, Japan.

To explore the prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medical practices, MRSA carriage was tested among 96 veterinarians (Vets), 70 veterinary technicians (VTs) and 292 dogs with which they had contact at 71 private veterinary clinics (VCs) in Hokkaido, Japan. MRSA isolates were obtained from 22 Vets [22.9%] and 7 VTs [10%]. The prevalence of MRSA among Vets was as high as that found in an academic veterinary hospital in our previous study. In contrast, only two blood donor dogs and one dog with liver disease (1.0%, 3/292) yielded MRSA. All MRSA-positive dogs were reared or treated in different VCs, in each of which at least one veterinary staff member carrying MRSA worked. Sequence types (ST) identified by multilocus sequence typing, spa types, and SCCmec types for canine MRSA isolates (ST5-spa t002-SCCmec II [from two dogs] or ST30-spa t021-SCCmec IV [from a dog]) were concordant with those from veterinary staff members in the same clinics as the MRSA-positive dogs, with which they had potentially had contact. Most MRSA isolates from veterinary staff were the same genotype (SCCmec type II and spa type t002) as a major hospital-acquired MRSA clone in Japan. The remaining MRSA was the same genotypes as domestic and foreign community-associated MRSA. Measures against MRSA infection should be provided in private VCs.

Epidemiological analysis of methicillin-resistant Staphylococcus aureus carriage among veterinary staff of companion animals in Japan.

Veterinary staff carrying methicillin-resistant Staphylococcus aureus(MRSA) can be a source of MRSA infection in animals. To identify risk factors of MRSA carriage among veterinary staff, MRSA carriage and epidemiological information (sex, career, contact with MRSA-identified animal patients and others) were analyzed from 96 veterinarians and 70 veterinary technicians working at 71 private veterinary clinics in Japan. Univariate analysis determined sex (percentage of MRSA carriage, male (29.2%) vs. female (10%); P=0.002) and career (veterinarians (22.9%) vs. veterinary technicians (10%); P=0.030) as risk factors. Multivariable analysis revealed that sex was independently associated with MRSA carriage (adjusted odds ratio, 3.717; 95% confidence interval, 1.555-8.889; P=0.003). Therefore, male veterinary staff had a higher risk of MRSA carriage than female staff.

Distribution of antimicrobial-resistant lactic acid bacteria in natural cheese in Japan.

To determine and compare the extent of contamination caused by antimicrobial-resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P=0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm-made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm-made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to >512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial-resistant LAB in imported and Japanese farm-made cheeses on the Japanese market, but not in Japanese commercial cheeses.

Contribution of the AcrAB-TolC efflux pump to high-level fluoroquinolone resistance in Escherichia coli isolated from dogs and humans.

Fluoroquinolone resistance is mainly caused by mutations in quinolone resistance-determining regions of DNA gyrase and topoisomerase IV in Escherichia coli. The AcrAB-TolC efflux pump contributes to resistance against fluoroquinolone and other antimicrobials. In this study, we investigated a high-level mechanism of fluoroquinolone resistance in E. coli that was isolated from human clinical samples and canine fecal samples. E. coli strains with high levels of fluoroquinolone resistance have been found to be frequently resistant to cephalosporins. Strains with high-level fluoroquinolone resistance exhibited lower intracellular enrofloxacin (ENR) concentrations, higher expression of AcrA, and a greater reduction in the fluoroquinolone minimum inhibitory concentration for treatment with an efflux pump inhibitor. The frequency of strains with enhanced ENR resistance selection and the survival rate of E. coli in the presence of ENR in vitro were correlated well with AcrA protein expression levels in the parental strains. These results suggest that AcrAB-TolC efflux pump over-expression is related to high-level fluoroquinolone resistance and the selection of strains with enhanced fluoroquinolone resistance.

Presence of Staphylococcus aureus ST398 and ST9 in Swine in Japan.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is mainly associated with swine and is capable of causing zoonotic infections. The methicillin-resistant S. aureus (MRSA) multilocus sequence type (ST) 398 of swine origin is predominant in Europe and North America, whereas ST9 is predominant in Asia. To evaluate the possible emergence of MRSA in swine, we examined the ST and spa type of 15 methicillin-susceptible S. aureus (MSSA) isolates obtained from swine in 8 different prefectures from north to south Japan between 2003 and 2009. Sequence analyses revealed that 6 porcine MSSA isolates belonged to ST398; 6 to ST9; and 1 each to ST5, ST97, and ST705. Of the 6 MSSA ST398 strains, 4 were classified as spa type t034. This study illustrated that there is a reservoir in Japanese swine of livestock-associated MSSA types.

Factors associated with antimicrobial-resistant Escherichia coli in zoo animals.

Factors associated with the carriage of antimicrobial-resistant Escherichia coli isolates were analysed among zoo animals. An association was observed between selection of amoxicillin as the first-line therapy and a significantly higher percentage of resistance to ampicillin (54.5%) from 11 animals treated with antimicrobials, compared with isolates from 32 untreated animals (9.4%). In addition, the percentage resistance to kanamycin (36.4%), gentamicin (27.3%), trimethoprim (27.3%) and tetracycline (63.6%) from 11 treated animals was significantly higher than those from 32 untreated animals (3.1%, 3.1%, 3.1% and 25%, respectively), although these antimicrobials were rarely used. All kanamycin-, gentamicin- and trimethoprim-resistant isolates and more than half of the tetracycline-resistant isolates from treated animals were also resistant to ampicillin. Co-resistance to other antimicrobials with ampicillin was suggested to contribute to an increasing of resistance towards antimicrobials that were rarely administered. The present investigation revealed an association of antimicrobial treatment with the spread of antimicrobial-resistant bacteria among zoo animals.

Seasonal variation in Campylobacter-contaminated retail chicken products: a year-round investigation in Japan.

Campylobacter was isolated from retail meat samples collected during the fiscal year 2009 in Japan. The higher percentages of contamination of chicken products were observed from June (39.3%) to November (83.3%). However, the highest number of human campylobacteriosis cases was reported in June in the Infectious Agents Surveillance Report. The chicken isolates with distinct clusters IVb and I, based on the restriction fragment length polymorphism of the flaA gene, were predominantly obtained during the periods between April and November 2009 and between February and March 2010, respectively. Extensive monitoring of Campylobacter contamination in chickens produced in various places is needed to analyse the seasonal variations between contamination of the meat products and the number of human cases with campylobacteriosis.

Prevalence and mechanism of antimicrobial resistance in Staphylococcus aureus isolates from diseased cattle, swine and chickens in Japan.

Antimicrobial administration is essential for the control and treatment of diseases in animals, but the emergence and prevalence of antimicrobial-resistant Staphylococcus aureus is a significant concern during animal production. Here we investigated the antimicrobial susceptibility of S. aureus from diseased food-producing animals and molecularly characterized the methicillin-resistant and fluoroquinolone-resistant isolates. A total of 290 S. aureus isolates obtained from cattle (n=246), swine (n=16), and chickens (n=28) between 2003 and 2009 were examined for antimicrobial susceptibility against 9 antimicrobials using an agar dilution method. Resistance to penicillin (PC) was most frequently found (24.8%), followed by oxytetracycline (OTC, 10.0%), dihydrostreptomycin (4.1%), erythromycin (EM, 3.1%), enrofloxacin (ERFX, 2.1%), and kanamycin (1.7%). The PC resistance rate was significantly higher in swine than in cattle (P<0.01) and chickens (P<0.01). The resistance rates to OTC, EM and ERFX were significantly higher in swine and chickens than in cattle (P<0.05). Methicillin-resistant S. aureus (MRSA) was recovered from milk derived from a cow with mastitis in 2003; sequence type 8, SCCmec type IV and spa type t024. In the six ERFX-resistant strains isolated after 2003, amino acid substitutions in ParC with/without GyrA were detected. As the prevalence of MRSA and FQ-resistant S. aureus in the animals should be noticed, continuous monitoring is necessary to control resistance to clinically important antimicrobials in S. aureus from food-producing animals.

Antimicrobial susceptibility of Escherichia coli isolates from wild mice in a forest of a natural park in Hokkaido, Japan.

To reveal the antimicrobial susceptibilities of Escherichia coli isolates from wild mice, 81 E. coli isolates were obtained from 109 voles (Clethrionomys spp.), 52 large Japanese field mice (Apodemus speciosus) and 19 small Japanese field mice (A. argenteus) captured in a forest of a natural park in Hokkaido, Japan. Seventy-eight of the 81 E. coli isolates were susceptible to all 10 antimicrobial agents tested. One E. coli isolate was resistant to ampicillin, dihydrostreptomycin, kanamycin, chloramphenicol and oxytetracycline. Two isolates were resistant to oxytetracycline. A low prevalence of antimicrobial resistance was maintained among wild mice that inhabited the forest.