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.

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.

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.

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.

Occurrence and molecular characteristics of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius in an academic veterinary hospital.

Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.

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.

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.

National monitoring for antimicrobial resistance among indicator bacteria isolated from food-producing animals in Japan.

The antimicrobial susceptibilities of 2,205 isolates of Escherichia coli and 1,181 isolates of Enterococcus faecalis (n=610) and E. faecium (n=571) from apparently healthy cattle, pigs and broiler and layer chickens collected from 2000 to 2003 were examined using an agar dilution method. Overall, the isolates from cattle and layer chickens showed a lower incidence of resistance to almost all antimicrobials studied compared with those from pigs and broiler chickens. Fluoroquinolone resistance was found at a low level in isolates of E. coli from four animal species and in E. faecalis from pigs and broiler and layer chickens. Resistance to cephalosporin was identified in isolates of E. coli from broiler chickens in 2000-2002 and from four animal species in 2003. Incidence of antimicrobial resistance in the bacteria did not vary from year to year during the investigation period.

Association of antimicrobial resistance in Campylobacter isolated from food-producing animals with antimicrobial use on farms.

On 1,374 food-producing animal farms in 2001-2004, we investigated the influence of antimicrobial usage within 6 months before a survey conducted on antimicrobial resistance in Campylobacter. Tetracyclines and fluoroquinolones were used for therapeutic purposes on 107 and 21 farms, respectively. Oxytetracycline-resistant C. jejuni and C. coli were found in 57.1% (8/14) and 92.5% (37/40) of the farms, respectively, when the animals were treated with tetracyclines. However, they were found in 43.2% (112/259) and 74.3% (122/164) of the farms, respectively, when the animals were not treated with tetracyclines. Enrofloxacin-resistant C. jejuni and C. coli were found in 66.7% (2/3) and 16.7% (1/6) of the farms, respectively, when the animals were treated with fluoroquinolones. However, they were found in 15.5% (42/270) and 28.8% (57/198) of the farms, respectively, when the animals were not treated with fluoroquinolones. The percentage of oxytetracycline resistance in C. coli was significantly elevated when the animals were treated with tetracyclines (P < 0.05). The farms on which antimicrobials included by their resistance pattern were used accounted for 9.1% (4/44) and 24.1% (14/58) of the farms where enrofloxacin-resistant C. jejuni and C. coli were isolated, respectively. It is likely that several factors account for the presence of fluoroquinolone-resistant Campylobacter.

Changes of multi-drug resistance pattern in Salmonella enterica subspecies enterica serovar typhimurium isolates from food-producing animals in Japan.

A total of 153 isolates of Salmonella Typhimurium derived from food-producing animals in Japan between 2002 and 2005 were investigated for antimicrobial resistance and phage types related to definitive phage type 104 (DT104). The predominant resistance type was resistance to ampicillin, dihydrostreptomycin, kanamycin, and oxytetracycline in bovine (45.2%, 48/104) and resistance to dihydrostreptomycin and oxytetracycline in porcine isolates (58.7%, 27/48). DT104-related phage type was found in 32 of 104 bovine isolates, two of 48 porcine isolates, and one of eight isolates from poultry, showing that the proportion of the phage type in S. Typhimurium isolates from cattle and pigs significantly (P<0.01) decreased from 71.9% and 31.4% in 1999-2001 to 30.8% and 4.1% in 2002-2005, respectively.

Antimicrobial resistance types and genes in Salmonella enterica infantis isolates from retail raw chicken meat and broiler chickens on farms.

Salmonella enterica subsp. enterica serotype Infantis isolates from retail raw chicken meat (n = 98) and broiler chickens on farms (n = 70) were examined for antimicrobial susceptibility and antimicrobial resistance genes. A total of 15 antimicrobial resistance types, 14 in meat and 10 in broiler isolates, were identified, and 9 of the 15 types were indistinguishable between meat and broiler isolates. Resistance to both oxytetracycline and dihydrostreptomycin accounted for 94.0% of the resistance types in meat and broiler isolates, and each type harbored aadA1 within 1.0 kb of class 1 integron and tetA. Of nalidixic acid resistance types, point mutations at 87Asp (GAC) to Tyr (TAC) in the quinolone resistance-determining region of gyrA was detected in 10 of 13 meat isolates and at 87Asp to Asn (AAC) in four of seven broiler isolates. These findings suggest that the antimicrobial resistance of Salmonella Infantis in retail chicken meat predominantly originates from broiler chickens.

Antimicrobial resistance in Salmonella isolates from apparently healthy food-producing animal from 2000 to 2003: the first stage of Japanese veterinary antimicrobial resistance monitoring (JVARM).

Antimicrobial susceptibility of 183 Salmonella isolates from apparently healthy food-producing animals obtained during the period from 2000 to 2003 throughout Japan was examined. Of 29 serovars identified, Salmonella Infantis (37.7%) was the most prevalent, followed by S. Typhimurium (19.7%). Salmonella bacteria resistant to dihydrostreptomycin (77.6%) were about 10% more prevalent than those resistant to oxytetracycline (67.8%), though the nation-level veterinary use of tetracycline antibiotics is much greater than that of streptomycin in Japan. In seventeen isolates (9.3%) resistant to nalidixic acid, single point mutations were detected at 84 or 87 in the quinolone resistance-determining region of the gyrA gene.

The dynamics of antimicrobial-resistant Campylobacter jejuni on Japanese broiler farms.

We investigated for dynamics of Campylobacter clones on 2 different managerial broiler farms. Campylobacter isolates were differentiated by resistance typing and molecular typing methods. On farm I, the same C. jejuni clones resistant to fluoroquinolone and oxytetracycline were isolated after one and half years again and another susceptible clone was invaded. The susceptible clone was isolated again after half year. Broiler flocks on the farm may be repeatedly infected with a few C. jejuni clones. On farm II, new clones including antimicrobial resistant one, were often invaded. The change of predominant C. jejuni clone in each flock on both the farms was observed, in the absence of antimicrobial selective pressure.

Role of coresistance in the development of resistance to chloramphenicol in Escherichia coli isolated from sick cattle and pigs.

OBJECTIVE: To determine the cause of persistent resistance to chloramphenicol (CP) after the ban on its use in food-producing animals in several countries. SAMPLE POPULATION: 71 CP-resistant and 104 CP-susceptible Escherichia coli strains isolated from sick cattle and pigs in Japan. PROCEDURE: Susceptibility of all bacterial strains to thiamphenicol (TP) and florfenicol (FFC) was tested by use of an agar dilution method. The CP-resistance genes and variable region within class 1 integrons in CP-resistant strains were identified by use of a PCR assay. RESULTS: The CP acetyltransferase gene (ie, cat1) was identified as the predominant CP-resistance gene in strains isolated from cattle, and the cat1and nonenzymatic CP-resistance gene (ie, cmlA) were the predominant CP-resistance genes in strains isolated from pigs. Additionally, strains with cat1 isolated from cattle often were resistant to ampicillin, dihydrostreptomycin (DSM), oxytetracycline, and trimethoprim (TMP), whereas strains with cat1 or cmlA isolated from pigs often were resistant to DSM and TMP. Class 1 integrons were significantly more prevalent in strains with CP-resistance genes, compared with prevalence in strains without CP-resistance genes. All gene cassettes within the integrons were involved in resistance to DSM, TMP, or both. CONCLUSIONS AND CLINICAL RELEVANCE: Coresistance that develops because of the use of DSM and TMP in cattle and pigs apparently contributes to the selection of CP-resistant strains of E coli. Thus, it is possible that bacterial resistance to CP in animals would persist despite a ban on the use of CP in cattle and pigs.