Influence of an improvement in frontal lobe hemodynamics on neurocognitive function in adult patients with moyamoya disease.

BACKGROUND: In adults, moyamoya disease (MMD) often presents with slight neurocognitive impairment, which may result from frontal lobe hemodynamic insufficiency. METHODS: In this study, we performed revascularization surgery by superficial temporal artery-anterior cerebral artery (ACA) direct bypass in 20 adults with MMD with poor anterograde ACA flow (Group M). The pre- and postoperative neurocognitive test results of these patients were retrospectively analyzed. The comparative group (Group C) included 23 patients with unruptured aneurysms or brain tumors who underwent craniotomy, as well as the same neurocognitive tests as Group M. We calculated the compositive frontal lobe function index (CFFI) based on the results of seven neurocognitive tests for each patient, and the difference between the pre- and postoperative CFFI values (CFFI Post - Pre) was compared between the two groups. RESULTS: Frontal perfusion improved postoperatively in all patients in Group M. The CFFI Post - Pre was significantly higher in Group M than in Group C (0.23 ± 0.44 vs. - 0.20 ± 0.32; p < 0.001). After adjusting for postoperative age, sex, preoperative non-verbal intelligence quotient, and preoperative period of stress, Group M had a significantly higher CFFI Post - Pre than Group C in the multiple regression analysis (t value = 4.01; p < 0.001). CONCLUSION: Improving frontal lobe hemodynamics might be the key for improving neurocognitive dysfunction in adults with MMD. The surgical indication and method should be considered from the perspective of both stroke prevention and neurocognitive improvement or protection.

Changes in alanine aminotransferase and body composition and metabolic factors among individuals receiving medical health checkups.

AIM: To examine the relationship between changes in alanine aminotransferase (ALT) and those in body composition and metabolic factors in participants receiving medical health checkups (4350 men [mean age 52.5 years] and 5398 women [mean age 50.5 years]) METHODS: We divided the participants into four types based on their ALT value at baseline and 1 year: A, ALT ≤30 (baseline) and ≤30 (1 year); B, ALT ≥31 (baseline) and ≤30 (1 year); C, ALT ≤30 (baseline) and ≥31 (1 year); and D, ALT ≥31 (baseline) and ≥31 (1 year). The change in each body composition-related parameter (waist circumference, fat mass, fat-free mass, fat mass to fat-free mass ratio, etc.) after 1-year was defined as Δ. RESULTS: The mean changes in waist circumference (cm) in the four types (A, B, C, and D) were -0.33, -1.54, 0.66, and -0.29 (overall p < 0.0001) in men, and -0.19, -0.90, 0.30, and 0.090 (overall p < 0.0001) in women. The mean changes in fat mass (kg) in the four types were -0.027, -0.86, 0.62, and 0.092 (overall p < 0.0001) in men, and 0.0067, -0.48, 0.39, and 0.063 (overall p < 0.0001) in women. The mean changes in fat-free mass (kg) in the four types were -0.028, -0.55, 0.42, and -0.034 (overall p < 0.0001) in men, and -0.0091, -0.34, 0.12, and -0.045 (overall p = 0.0012) in women. The mean changes in fat mass to fat-free mass ratio in the four types were -0.00042, -0.0120, 0.00837, and 0.00171 (overall p < 0.0001) in men, and -0.00013, -0.00817, 0.00730, and 0.00628 (overall p < 0.0001) in women. CONCLUSION: A decrease in ALT to ≤30 IU/L may be associated with improved body composition balance, but caution should be exercised for the decrease in muscle mass.

Body Composition in Patients With Metabolic Dysfunction-associated Steatotic Liver Disease: Impact of Body Surface Area.

BACKGROUND/AIM: To examine the relationship between the body surface area (BSA) and body composition in patients with metabolic dysfunction-associated steatotic liver disease (MASLD, 2,141 men and 986 women). MATERIALS AND METHODS: BSA and body composition parameters were examined. RESULTS: The median body mass index (BMI) was 25.0 kg/m2 for both men and women (p=0.7754). The median body surface area (BSA) was 1.854 m2 for men and 1.618 m2 for women (p<0.0001). In men, the median fat mass was 17.7 kg, whereas in women, it was 22.1 kg (p<0.0001). Additionally, the median fat-free mass was 55.4 kg in men and 39.3 kg in women (p<0.0001).). In male cases, BSA significantly correlated with fat mass (r=0.82, p<0.0001) and fat-free mass (r=0.95, p<0.0001). In female cases, BSA significantly correlated with fat mass (r=0.87, p<0.0001) and fat-free mass (r=0.94, p<0.0001). CONCLUSION: BSA could be a useful marker for the estimation of body composition in patients with MASLD.

Low-frequency transmission and persistence of antimicrobial-resistant bacteria and genes from livestock to agricultural soil and crops through compost application.

Livestock excrement is composted and applied to agricultural soils. If composts contain antimicrobial-resistant bacteria (ARB), they may spread to the soil and contaminate cultivated crops. Therefore, we investigated the degree of transmission of ARB and related antimicrobial resistance genes (ARGs) and, as well as clonal transmission of ARB from livestock to soil and crops through composting. This study was conducted at Rakuno Gakuen University farm in Hokkaido, Japan. Samples of cattle feces, solid and liquid composts, agricultural soil, and crops were collected. The abundance of Escherichia coli, coliforms, ß-lactam-resistant E. coli, and ß-lactam-resistant coliforms, as well as the copy numbers of ARG (specifically the bla gene related to ß-lactam-resistant bacteria), were assessed using qPCR through colony counts on CHROMagar ECC with or without ampicillin, respectively, 160 days after compost application. After the application of the compost to the soil, there was an initial increase in E. coli and coliform numbers, followed by a subsequent decrease over time. This trend was also observed in the copy numbers of the bla gene. In the soil, 5.0 CFU g-1 E. coli was detected on day 0 (the day post-compost application), and then, E. coli was not quantified on 60 days post-application. Through phylogenetic analysis involving single nucleotide polymorphisms (SNPs) and using whole-genome sequencing, it was discovered that clonal blaCTX-M-positive E. coli and blaTEM-positive Escherichia fergusonii were present in cattle feces, liquid compost, and soil on day 0 as well as 7 days post-application. This showed that livestock-derived ARB were transmitted from compost to soil and persisted for at least 7 days in soil. These findings indicate a potential low-level transmission of livestock-associated bacteria to agricultural soil through composts was observed at low frequency, dissemination was detected. Therefore, decreasing ARB abundance during composting is important for public health.

Association of toxin-producing Clostridioides difficile with piglet diarrhea and potential transmission to humans.

The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST) 11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.

Carbapenem and colistin-resistant hypervirulent Klebsiella pneumoniae: An emerging threat transcending the egyptian food chain.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great public health problem and is associated with many disease outbreaks and high mortality rates. Alarmingly, K. pneumoniae has been isolated from food in several recent studies. This study aimed to investigate the prevalence and characteristics of CRKP in food samples from Egypt. METHODS: A total of 311 food samples (including 116 minced meat, 92 chicken meat, 75 diced meat, and 28 mutton) were collected from local markets in Egypt and were screened for CRKP with the determination of their antimicrobial resistance profiles. The whole genome sequence was done for 23 CRKP isolates to clarify the relationship between CRKP from food and human cases in Egypt using the SNP core genome. The conjugation probability of the blaNDM-5 harboring plasmid was identified using oriTfinder RESULTS: CRKP was isolated from 11% (35/311) of the samples, with 45.71% (16/35) of them showing resistance to colistin, one of the last-resort options for treating CRKP-mediated infections. In addition to the carbapenem and colistin resistance, the CRKP isolates frequently exhibited resistance to multiple antimicrobials including ß-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol. In addition, most of the CRKP were potentially hypervirulent K. pneumoniae (HvKP) identified as phylogroup Kp1 and of high-risk groups as detected in STs reported in many human outbreaks globally, such as ST383 and ST147. The core-genome phylogeny showed similarities between the isolates from this study and those previously isolated from clinical human samples in Egypt. In addition, analysis of the plasmid on which blaNDM is encoded revealed that several antimicrobial resistance genes such as blaOXA-9, blaCTX-M-15, aac(6')-Ib, qnrS1, and several virulence genes are encoded on the same plasmid. CONCLUSIONS: This study is significant for food safety and public health and is important to further identify the change in the epidemiology of CRKP infections, especially the consumption of contaminated food products.

Case report of belt electrode-skeletal muscle electrical stimulation for acute heart failure with severe obesity: a novel therapeutic option for acute phase rehabilitation.

Background: Belt electrode skeletal muscle electrical stimulation (B-SES) is an emerging therapy anticipated to yield more favorable outcomes than conventional neuromuscular electrical stimulation (NMES), owing to its larger stimulation area. However, information on its efficacy and safety in patients with heart failure remains limited. Case presentation: A 43-year-old man with a body mass index of 41 kg/m2 was admitted to our hospital for acute heart failure due to dilated cardiomyopathy. The patient required prolonged catecholamine support owing to poor cardiac function, and heart transplantation was considered. We initiated a mobilization program, but the patient's mobility was highly limited due to severe obesity and symptomatic orthostatic hypotension. B-SES was introduced to accomplish weight loss and early ambulation. We applied an intensive monitoring program for safe use and modulated the intensity of B-SES according to physical function. During the B-SES program, the patient's body weight decreased from 89.6 kg to 78.6 kg. Sequential evaluations of body composition and skeletal muscle ultrasonography revealed improved muscle mass, quality, and physical function. Furthermore, we explored the workload of B-SES using expiratory gas analysis. No adverse events were observed during B-SES. Discussion: We successfully used B-SES to improve muscle function and morbidity in the treatment of acute heart failure. B-SES could be an option for patients with heart failure who have limited mobility and obesity.

Transferable linezolid resistance genes (optrA and poxtA) in enterococci derived from livestock compost at Japanese farms.

OBJECTIVES: Linezolid is a last-resort antimicrobial in human clinical settings to treat multidrug-resistant Gram-positive bacterial infections. Mobile linezolid resistance genes (optrA, poxtA, and cfr) have been detected in various sources worldwide. However, the presence of linezolid-not-susceptible bacteria and mobile linezolid resistance genes in Japan remains uncertain. Therefore, we clarified the existence of linezolid-not-susceptible bacteria and mobile linezolid resistance genes in farm environments in Japan. METHODS: Enterococci isolates from faeces compost collected from 10 pig and 11 cattle farms in Japan in 2021 were tested for antimicrobial susceptibility and possession of mobile linezolid resistance genes. Whole-genome sequencing of optrA and/or poxtA genes positive-enterococci was performed. RESULTS: Of 103 enterococci isolates, 12 from pig farm compost were not-susceptible (2 resistant and 10 intermediate) to linezolid. These 12 isolates carried mobile linezolid resistance genes on plasmids or chromosomes (5 optrA-positive Enterococcus faecalis, 6 poxtA-positive E. hirae or E. thailandicus, and 1 optrA- and poxtA-positive E. faecium). The genetic structures of optrA- and poxA-carrying plasmids were almost identical to those reported in other countries. These plasmids were capable of transferring among E. faecium and E. faecalis strains. The optrA- and poxtA-positive E. faecium belonged to ST324 (clade A2), a high-risk multidrug-resistant clone. The E. faecalis carrying optrA gene on its chromosome was identified as ST593. CONCLUSIONS: Although linezolid is not used in livestock, linezolid-not-susceptible enterococci could be indirectly selected by frequently used antimicrobials, such as phenicols. Moreover, various enterococci species derived from livestock compost may serve as reservoirs of linezolid resistance genes carried on globally disseminated plasmids and multidrug-resistant high-risk clones.

Antimicrobial Resistant Bacteria Monitoring in Raw Seafood Retailed: a Pilot Study Focused on Vibrio and Aeromonas.

In aquaculture, bacterial infections in sea animals are treated using antimicrobials. As seafood is frequently consumed in its raw form, seafood contaminated with water-borne antimicrobial-resistant bacteria presents a potential transmission route to humans and can influence food safety. In this study, we aimed to determine the abundance of water-borne bacteria in retail raw seafood and to characterize their antimicrobial resistance profiles. In total, 85 retail raw seafood samples (32 fish, 26 shellfish, 25 mollusks, and two crustaceans) were purchased from supermarkets in Japan, and water-borne bacteria were isolated. The isolated bacterial species predominantly included Vibrio spp. (54.1%) and Aeromonas spp. (34.1%). Vibrio or Aeromonas spp. were isolated from more than 70% of the seafood samples. Tetracycline-, sulfamethoxazole-, and/or trimethoprim/sulfamethoxazole-resistant Vibrio or Aeromonas spp. isolates were detected in seven (21.9%) fish samples (two wild-caught and five farm-raised) harboring tet, sul, and/or dfr genes. Sulfamethoxazole- and trimethoprim/sulfamethoxazole-resistant isolates were only detected in farm-raised fish. Tetracycline and sulfamethoxazole are commonly used in aquaculture. These results suggest that water-borne bacteria like Vibrio and Aeromonas spp. should be the primary focus of antimicrobial-resistant bacteria monitoring to effectively elucidate their spread of bacteria via seafood.

Insulin Resistance: A Marker for Fat-to-Lean Body Composition in Japanese Adults.

We sought to investigate the relationship between insulin resistance (IR) and body composition as assessed by bioelectrical impedance analysis in Japanese health check-up recipients (1186 men and 1441 women). IR was defined as a Homeostasis Model Assessment of IR (HOMA-IR) ≥ 2.5. In body-composition-related parameters, the fat mass index (F index) was defined as fat mass divided by the height squared (kg/m2). The fat-free mass index (FF index) was defined as fat-free mass divided by the height squared (kg/m2). The F index to FF index ratio (F-FF ratio) was defined as the F index divided by the FF index. Factors related to HOMA-IR were examined. The median HOMA-IR was 1.54 in men and 1.30 in women (p < 0.0001). The median F index was 4.9 kg/m2 in men and 6.1 kg/m2 in women (p < 0.0001). The median FF index was 18.2 kg/m2 in men and 15.1 kg/m2 in women (p < 0.0001). The median F-FF ratio was 0.272 in men and 0.405 in women (p < 0.0001). The F-FF ratio was an independent factor associated with HOMA-IR in the multivariate analysis in both genders, while the F index and FF index were not in both genders. In conclusion, fat and skeletal muscle balance can be controlled by IR in Japanese adults.

Association between the Suita Score and Body Composition in Japanese Adults: A Large Cross-Sectional Study.

The purpose of this study was to clarify the relationship between the Suita score (a prediction model for the development of cardiovascular disease) and body composition in Japanese health check-up subjects (6873 men and 8685 women). The Suita score includes 8 items (age, gender, smoking, diabetes, blood pressure, low-density lipoprotein, high-density lipoprotein, and chronic kidney disease). Factors associated with the Suita score within body composition-related parameters (body mass index (BMI), waist circumference (WC), fat mass index, fat-free mass index, fat mass to fat-free mass ratio (F-FF ratio), and water mass index) as assessed by bioelectrical impedance analysis were examined. The mean age of subjects was 54.8 years in men and 52.8 years in women (p < 0.0001). The mean BMI was 23.9 kg/m2 in men and 21.8 kg/m2 in women (p < 0.0001). Diabetes mellitus was found in 1282 subjects (18.7%) among men and 816 subjects (9.4%) among women (p < 0.0001). The mean Suita score was 42.0 in men and 29.6 in women (p < 0.0001). In multivariate analysis, WC (p < 0.0001), F-FF ratio (p < 0.0001), and water mass index (p < 0.0001) were independent factors linked to the Suita score for both genders. In conclusion, body composition can be associated with the Suita score in Japanese adults receiving health check-ups.

Body Composition Analysis in Patients with Metabolic Dysfunction-Associated Fatty Liver Disease.

We sought to examine body composition using bioimpedance analysis in patients with metabolic dysfunction-associated fatty liver disease (MAFLD, 2014 males and 949 females). Factors linked to the fat-free mass index (FF index) were examined using univariate and multivariate analysis. An FF index < 18 kg/m2 in males and an FF index < 15 kg/m2 in females were defined as having decreased skeletal muscle mass. The median age and body mass index (BMI) were 55 years and 25.4 kg/m2 in males, and 57 years and 25.4 kg/m2 in females, respectively. The FF index strongly correlated with muscle mass index both in males (r = 0.999) and females (r = 0.999). The prevalence of patients with an FF index < 18 kg/m2 in males and an FF index < 15 kg/m2 in females was well stratified according to age, BMI, severity of FL, and FIB4 index. In the males, in the multivariate analysis, BMI (p < 0.0001), fat mass index (p < 0.0001), and waist circumference (p = 0.0050) were found to be significant factors linked to FF index. In the females, in the multivariate analysis, BMI (p < 0.0001) and fat mass index (p < 0.0001) were found to be significant. In conclusion, fat accumulation as reflected by BMI, which is an easily available marker, could be a useful indicator for the skeletal muscle mass in MAFLD.

Advantages of enhanced recovery after surgery program in robot-assisted radical cystectomy.

Radical cystectomy is a gold-standard treatment for muscle-invasive bladder cancer. We recently introduced robot-assisted radical cystectomy (RARC) with perioperative enhanced recovery after surgery (ERAS). The medical records of patients with bladder cancer who underwent open radical cystectomy (ORC) or RARC/ERAS at NTT Medical Center Tokyo were retrospectively reviewed to compare the surgical outcomes, hospital stay, and medical costs between groups. Multidisciplinary full ERAS items were provided for the RARC/ERAS group. The median estimated blood losses in the ORC and RARC/ERAS groups were 650 and 100 mL, and the median operative times were 312 and 445 min, respectively. In addition, the median times to liquid food intake in these groups were 6 and 0 days, the median times to first flatus and first defecation were 2 and 1 day, and 3 and 1.5 days, respectively. The rates of postoperative ileus in the ORC and RARC/ERAS groups were 27.5% and 4.5%, and the median postoperative hospital stays was 26.5 and 12 days, respectively. Medical costs excluding surgery were significantly lower in the RARC/ERAS group. In conclusion, RARC/ERAS represents a safe treatment option for muscle-invasive bladder cancer with decreased perioperative complications and lower medical costs.

Hand Grip Strength Differences in Geriatric Subjects with and without Hand Diseases.

Objective: The aim of this study was to investigate the impact of untreated hand diseases on hand grip strength, a value that is commonly used as a diagnostic parameter for sarcopenia and frailty in geriatric populations. We hypothesized that individuals with untreated hand diseases would have lower grip strength than those without hand diseases. Methods: A total of 240 individuals aged at least 65 years were recruited and divided into two groups based on the presence or absence of typical hand diseases. Grip strength was compared between the two groups separately for men and women using a t-test, with each group consisting of 60 women or 60 men. Results: Both women and men in the hand disease group exhibited significantly lower grip strength than those in the control group. Conclusions: These findings suggest that untreated hand diseases have a negative impact on grip strength, and this may introduce bias in the screening or diagnosis of sarcopenia and frailty. It is essential to consider the presence of hand diseases when measuring hand grip strength in older adults.

16S rRNA nanopore sequencing for rapid diagnosis of causative bacteria in bovine mastitis.

The rapid identification of specific bacterial pathogens in bovine mastitis is crucial for appropriate antimicrobial treatment. Sequencing of 16S rRNA gene amplicons is a proven, useful strategy for diagnosing bacterial infections. In this study, the use of 16S rRNA analysis with nanopore sequencer for the rapid identification of causative bacteria in bovine mastitis, was evaluated. DNA was extracted from 122 milk samples from cattle with suspected mastitis based on clinical symptoms. 16S rRNA gene amplicon sequencing was conducted using a nanopore sequencer. The efficacy of bacterial identification was verified by comparison with conventional culture methods. Nanopore sequencing identified the causative bacteria with high accuracy within approximately 6 h from the time of sample collection. When the major causative bacteria of bovine mastitis (Escherichia coli, Streptcoccus uberis, Klebsiella pneumoniae, and Staphylococcus aureus) were detected by nanopore sequencing, 98.3% of the results were consistent with identification through conventional culturing methods. 16S rRNA gene analysis using a nanopore sequencer enabled the rapid and accurate identification of bacterial species in bovine mastitis.

Association Between Frontal Lobe Hemodynamics and Neurocognitive Dysfunction in Adults With Moyamoya Disease: Retrospective Cohort Analysis.

BACKGROUND: Adult patients with moyamoya disease (MMD) may present with mild cognitive dysfunction, even those without evidence of conspicuous brain parenchymal damage. This cognitive dysfunction might be caused by local frontal lobe ischemia. OBJECTIVE: To explore the relationship between frontal lobe hemodynamic insufficiency and cognitive dysfunction in patients with MMD. METHODS: Thirty adult patients with MMD without conspicuous brain parenchymal damage were retrospectively examined. Patients with MMD with frontal lobe intracerebral steal phenomenon on single photon emission computed tomography were defined as group S (n = 13) and those without it were defined as group P (n = 17). A comparative group comprising patients with unruptured intracranial aneurysm was defined as group C (n = 30). The results of various cognitive and intelligence tests and a composite cognitive score were compared between groups. RESULTS: The digit span test forward version ( P = .041), frontal assessment battery ( P = .022), and composite cognitive score ( P = .015) z-scores were significantly lower in group S than group C. Adjusting for sex and age, patients in group S had a significantly lower composite cognitive score compared with those in group C in multiple regression analysis ( P = .037). Executive dysfunction and working memory dysfunction may be involved in the cognitive decline observed in group S. CONCLUSION: Mild cognitive dysfunction in MMD was associated with frontal lobe hemodynamic insufficiency. Future studies should examine whether revascularization can improve cerebral hypoperfusion and neurocognitive function in these patients.

Antimicrobial resistance and molecular epidemiological analysis of Escherichia fergusonii harboring the mcr gene in pigs and broiler chickens in Okinawa, Japan.

The dissemination of mcr-harboring Enterobacteriaceae, e.g., Escherichia fergusonii, with resistance to colistin via animal products is a public health concern. In our previous study, E. fergusonii harboring the mcr gene were isolated from 11 pigs and 43 chickens. To understand the spread of mcr-harboring E. fergusonii in Okinawa, Japan, and to gain further insights into how they can be controlled, an antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), a conjugation test for the transferability of mcr-harboring plasmids, and PCR-based replicon typing (PBRT) were performed using the 54 strains. According to the disk-diffusion and broth microdilution methods, 9 of the 11 strains from pigs and 9 of the 43 strains from chickens had multidrug resistance (MDR). The broth microdilution method showed that all strains were resistant to colistin, and the minimum inhibitory concentration of colistin was 4-16 µg/mL. PFGE suggested identical PFGE types were being transmitted within one pig farm, within one chicken farm, and among several chicken farms. These findings showed that some mcr-harboring E. fergusonii in Okinawa exhibited MDR, and these had spread within farms and between farms. In the mcr gene conjugation test and PBRT, a type IncI2 plasmid replicon was detected in all mcr-1-harboring transconjugants. Therefore, evidence suggests that the IncI2 plasmid is probably involved in the transmission of the mcr-1 gene. It is important to monitor the antimicrobial resistance profile and dissemination of the IncI2 plasmid in mcr-harboring E. fergusonii.

Infiltration of hidden antimicrobial resistance among healthy people in a Japanese community.

Background: Under non-antimicrobial selective pressure, antimicrobial-resistant bacteria do not easily become dominant in the microbiota. Furthermore, their low levels prevent detection by isolation, resulting in an underestimation of the prevalence of antimicrobial-resistant bacteria. Objectives: We evaluated the infiltration of antimicrobial-resistant bacteria and their related ß-lactamase genes among healthy people in non-clinical settings. Methods: Cephalosporin- and fluoroquinolone-resistant Escherichia coli and bla genes were quantified in 217 faecal samples from healthy people in non-clinical settings in Japan. E. coli colonies grown on deoxycholate hydrogen sulphide-lactose (DHL) agar, with and without antimicrobials (cefotaxime and ciprofloxacin), were quantified, and E. coli isolates were analysed for their susceptibility to antimicrobials and the presence of bla genes. DNA extracted from faecal samples was used to quantify bla genes using quantitative PCR (qPCR). Results: The isolation rates of cefotaxime- and ciprofloxacin-resistant E. coli were 6.9% and 12.4%, respectively, using agars without antimicrobials, and 12.0% and 24.4%, respectively, using agars with antimicrobials. For samples from which cefotaxime- and ciprofloxacin-resistant E. coli were isolated only using agars with antimicrobials, the ratios of cfu on DHL agars with and without antimicrobials were below -2 log. E. coli harbouring bla genes were isolated from 35.0% of the faecal samples using agars, and bla genes were detected in 65.0% of faecal DNA samples using qPCR. Conclusions: Among people carrying cefotaxime- and ciprofloxacin-resistant E. coli in non-clinical settings, cefotaxime- and ciprofloxacin-resistant E. coli were not dominant in half of the subjects. These individuals may play a role as reservoirs of antimicrobial-resistant bacteria.

Decreasing the abundance of tetracycline-resistant Escherichia coli in pig feces during nursery using flavophospholipol as a pig feed additive.

Tetracyclines (TCs) are widely used for livestock, and the high prevalence of TC-resistant Escherichia coli in livestock has become a serious concern worldwide. In Japan, the National Action Plan on Antimicrobial Resistance in 2016 aimed to reduce the TC resistance rate in E. coli derived from livestock. Flavophospholipol (FPL), used as a feed additive, has an inhibitory effect on the spread of plasmid-mediated antimicrobial resistance. The number of TC-resistant E. coli was determined in pigs administered TCs and/or FPL to clarify the effect of FPL on reducing the number of TC-resistant E. coli in pigs. TC-resistant E. coli and their plasmids were then analyzed. The pigs were divided into four groups: control, doxycycline (DOXY; a TC), FPL, and a DOXY-FPL combination. Their feces were collected from the nursing period to the day before being transported to the slaughterhouse, followed by estimation of TC-resistant E. coli (colony-forming units [CFU]/g). The number of TC-resistant E. coli increased with the use of DOXY, suggesting that DOXY administration provides a selective pressure for TC-resistant E. coli. Supplementation with FPL as a feed additive significantly suppressed the increase in the number of TC-resistant E. coli, especially during the DOXY administration period. Transfer and growth inhibition analyses were performed for TC-resistant isolates. FPL inhibited the conjugational transfer and growth of a few TC-resistant E. coli isolates. These results suggest that FPL is effective against the spread of TC-resistant E. coli.

Conjugative IncHI2/HI2A plasmids harbouring mcr-9 in colistin-susceptible Escherichia coli isolated from diseased pigs in Japan.

Colistin is a last resort antimicrobial used for the treatment of gram-negative bacterial infections. Plasmid-mediated colistin resistance (mcr) genes are a cause of global concern, and, thus far, mcr-1-10 have been identified. In a previous study, we screened mcr-1-5 in Escherichia coli derived from diseased pigs in Japan and reported a high prevalence of mcr-1, -3 and -5. However, the previous report on the prevalence of mcr genes was inaccurate. In the present study, we aimed to clarify the prevalence of all reported variants of mcr in E. coli derived from the diseased pigs, which were previously screened for mcr-1-5. Additionally, we also characterized the mcr-9-positive E. coli , which was detected in this study. We screened mcr in 120 E. coli strains from diseased pigs and mcr-positive E. coli and an mcr-carrying plasmid were also characterized. One mcr-9-positive colistin-susceptible E. coli strain was detected (0.8 %). Plasmid-mediated mcr-9 was transferred to E. coli ML4909 as the recipient strain, and it was located on IncHI2/HI2A plasmid p387_L with other antimicrobial resistance genes (ARGs). The region harbouring ARGs including mcr-9, was similar to that on the Klebsiella pneumoniae chromosome harbouring mcr-9 isolated in Japan. mcr-3, -5 and -9 were detected (4.2 %) in colistin-susceptible strains. mcr-9 was found to be disseminated via the plasmid IncHI2/HI2A p387_L and transferred and inserted into chromosomes via a transposon. Our results suggest that mcr genes should be monitored regularly, regardless of their susceptibility to colistin.