Plastiome: Plastisphere-enriched mobile resistome in aquatic environments.

Aquatic microplastics (MPs) act as reservoirs for microbial communities, fostering the formation of a mobile resistome encompassing diverse antibiotic (ARGs) and biocide/metal resistance genes (BMRGs), and mobile genetic elements (MGEs). This collective genetic repertoire, referred to as the "plastiome," can potentially perpetuate environmental antimicrobial resistance (AMR). Our study examining two Japanese rivers near Tokyo revealed that waterborne MPs are primarily composed of polyethylene and polypropylene fibers and sheets of diverse origin. Clinically important genera like Exiguobacterium and Eubacterium were notably enriched on MPs. Metagenomic analysis uncovered a 3.46-fold higher enrichment of ARGs on MPs than those in water, with multidrug resistance genes (MDRGs) and BMRGs prevailing, particularly within MPs. Specific ARG and BMRG subtypes linked to resistance to vancomycin, beta-lactams, biocides, arsenic, and mercury showed selective enrichment on MPs. Network analysis revealed intense associations between host genera with ARGs, BMRGs, and MGEs on MPs, emphasizing their role in coselection. In contrast, river water exhibited weaker associations. This study underscores the complex interactions shaping the mobile plastiome in aquatic environments and emphasizes the global imperative for research to comprehend and effectively control AMR within the One Health framework.

CTX-M-55-type ESBL-producing fluoroquinolone-resistant Escherichia coli sequence type 23 repeatedly caused avian colibacillosis in Kagoshima Prefecture, Japan.

OBJECTIVES: The production of expanded-spectrum beta-lactamases (ESBLs) and fluoroquinolone resistance in Enterobacteriaceae has become a global concern. The aim of this study was to investigate the spread of ESBL-producing and fluoroquinolone-resistant avian pathogenic Escherichia coli (APEC) in Kagoshima, a prefecture with the largest amount of poultry in Japan. METHODS: The antimicrobial susceptibility and genetic characteristics of 228 APEC strains isolated from 57 farms in Kagoshima Prefecture, Japan, between 2005 and 2017 were analysed. Information about the companies with hatcheries connected to the farms was also collected, and the epidemiologic relatedness of APEC strains and the processes of adopting chicks were compared. RESULTS: Seven CTX-M-type ESBL genes, blaCTX-M-1, blaCTX-M-2, blaCTX-M-14, blaCTX-M-15, blaCTX-M-25, blaCTX-M-55, and blaCTX-M-65, were found in 60 (26.3%) of the 228 APEC strains. The ciprofloxacin-resistant strains belonged to 10 different sequence types (ST10, ST23, ST93, ST155, ST156, ST350, ST359, ST602, ST648, and ST9479), and the two ST602 strains showed remarkably high ciprofloxacin resistance (MIC: 128 µg/mL) and had amino acid mutations in GyrA (S83L and D87N), ParC (S80I), and ParE (E460A). A CTX-M-55-type ESBL-producing fluoroquinolone-resistant Og78-ST23 strain was isolated multiple times over two years on a farm. Furthermore, epidemiologically closely related strains were isolated from different farms that used the same common hatcheries. CONCLUSIONS: APEC is often transferred from hatcheries to farms via healthy chicks, and the prudent use of antimicrobials and careful monitoring of resistant strains on poultry farms and hatcheries are important in preventing the selection and spread of high-risk APEC strains such as CTX-M-55-type ESBL-producing Og78-ST23.

The association between farm-level antimicrobial usage and resistance of Staphylococcus spp., as the major genus isolated from aerosol samples, in Japanese piggeries.

Bacteria are the dominant particulate matter in livestock houses and can threaten animal and public health. Antimicrobial resistance (AMR) is a crucial concern worldwide, and nationwide measures established based on the One Health approach are being implemented in many countries. This requires multidisciplinary perspectives and collaboration among the human, animal, and environmental sectors. However, information on the AMR risk in livestock house aerosol is limited, especially its association with antimicrobial usage (AMU). Therefore, this study was conducted to reveal the AMR profile of Staphylococcus, the major bacterial genus in the aerosol of the piggeries of Japanese farms, and the association between farm-level AMU and AMR. The investigation at 10 farrow-to-finish pig farms revealed that regardless of the sampling season and the piggery group, the resistance rate of isolated staphylococci for oxacillin, erythromycin, and lincomycin was more than 40% of the median and tended to be higher than that for other antimicrobials. The AMU adjusted by the defined daily dose (DDD-adjusted AMU) in the fattening piggery group was significantly higher than that in the sow piggery group (p < 0.05). Finally, for the fattening piggery group, the generalized linear mixed model revealed that the AMR rate for oxacillin, erythromycin, tetracycline, and chloramphenicol was positively associated with the corresponding class-based DDD-adjusted AMU of penicillins (odds ratio (OR) = 2.63, p = 0.03), macrolides (OR = 6.89, p = 0.0001), tetracyclines (OR = 2.48, p = 0.04), and amphenicols (OR = 3.22, p = 0.03), respectively. These significant positive associations observed in this study imply that the resistance rate for these antimicrobials may decrease by reducing the corresponding antimicrobials' use. In addition, the resistance rates for erythromycin and chloramphenicol also displayed a positive association with the AMU of antimicrobial classes other than macrolides and amphenicols, respectively. The mechanism underlying these phenomena is unclear; therefore, further evaluation will be needed. As limited studies have reported staphylococci in piggery aerosol and its AMR with quantitative AMU, these results based on on-farm investigations are expected to aid in establishing countermeasures for AMR of aerosol bacteria in pig farms.

mcr-1 remains detectable in various Escherichia coli lineages isolated from healthy swine after withdrawal of colistin use on the farm.

We monitored swine-derived Escherichia coli on a Japanese farm where colistin had been used for the treatment of diseases caused by bacteria and investigated colistin resistance and the presence of mcr-1 in 36 E. coli strains isolated before and after the withdrawal of colistin use. Through the withdrawal of colistin use on the farm, the prevalence of colistin-resistant and mcr-1-positive E. coli was markedly reduced but not eradicated because mcr-1 had been maintained in multiple plasmids and various sequence types of nonpathogenic E. coli carried in healthy swine. The monitoring of sequence types of mcr-1-positive E. coli is expected to be important for controlling colistin resistance in swine or other animals.

Nationwide analysis of antimicrobial resistance in pathogenic Escherichia coli strains isolated from diseased swine over 29 years in Japan.

Pathogenic Escherichia coli strains are important causes of several swine diseases that result in significant economic losses worldwide. In Japan, the use of antimicrobials in swine is much higher than that in other farm animals every year. Antimicrobial resistance in pathogenic E. coli strains also heavily impacts the swine industry due to the limited treatment options and an increase in the potential risk of the One Health crisis. In 2016, we investigated 684 Japanese isolates of swine pathogenic E. coli belonging to four major serogroups and reported the emergence and increase in the highly multidrug-resistant serogroups O116 and OSB9 and the appearance of colistin-resistant strains. In the present study, by expanding our previous analysis, we determined the serotypes and antimicrobial resistance of 1,708 E. coli strains isolated from diseased swine between 1991 and 2019 in Japan and found recent increases in the prevalences of multidrug-resistant strains and minor serogroup strains. Among the antimicrobials examined in this study that have been approved for animal use, a third-generation cephalosporin was found to be effective against the most isolates (resistance rate: 1.2%) but not against highly multidrug-resistant strains. We also analyzed the susceptibilities of the 1,708 isolates to apramycin and bicozamycin, both which are available for treating swine in Japan, and found that the rates of resistance to apramycin and bicozamycin were low (6.7% and 5.8%, respectively), and both antimicrobials are more effective (resistance rates: 2.7% and 5.4%, respectively) than third-generation cephalosporins (resistance rate: 16.2%) against highly multidrug-resistant strains.

Tryptanthrin Reduces Campylobacter jejuni Colonization in the Chicken Gut by a Bactericidal Mechanism.

Campylobacter jejuni is a leading cause of foodborne bacterial gastroenteritis worldwide, and raw or undercooked chicken meat is considered the major source of human campylobacteriosis. In this study, we identified 36 compounds that showed inhibitory effects on C. jejuni growth at low concentrations by screening a chemical compound library. Three of the 36 compounds were herbal compounds, including tryptanthrin (TRP), an indoloquinazoline alkaloid. TRP has been reported to have a variety of biological properties, such as antimicrobial, anti-inflammatory, and antitumor activities, but there was previously no information about its anti-C. jejuni activity. We further conducted in vitro and in vivo experiments to evaluate the potential of TRP for the control of C. jejuni in chicken farms. The MIC of TRP for C. jejuni was much lower than that of 13 other herbal compounds that were previously reported to have anti-C. jejuni activities. Time-kill assays under growing and nongrowing conditions demonstrated that TRP has bactericidal activity against C. jejuni. In addition, TRP showed a narrow-spectrum antimicrobial effect against C. jejuni, and there was little potential for the development of TRP-resistant C. jejuni during serially passaged culture. In chick infection experiments, the administration of TRP in drinking water significantly reduced the cecal colonization of C. jejuni when TRP was used either before or after C. jejuni infection. These data suggest that TRP is effective for the control of C. jejuni in chicken farms. IMPORTANCE Campylobacter is a widespread pathogen in the food chain of chickens. Once chickens become infected, large numbers of Campylobacter cells are excreted in their feces. The development of an effective material for reducing the amount of Campylobacter in the chicken intestinal tract will make it possible to reduce the contamination of the food chain with Campylobacter and to produce safe and secure chicken meat. In the present study, in vivo experiments revealed that the use of an herbal compound, tryptanthrin, significantly reduced the number of Campylobacter cells in the chicken gut by a bactericidal mechanism. Furthermore, our in vitro experiments demonstrated that, compared with the other herbal compounds, tryptanthrin achieved antimicrobial activity against C. jejuni at the lowest concentration. The use of tryptanthrin may lead to the development of a novel control measure for reducing the colonization of C. jejuni in the food chain.

Disinfectant resistance of Salmonella in in vitro contaminated poultry house models and investigation of efficient disinfection methods using these models.

Salmonellaenterica subsp. enterica (Salmonella) shows disinfectant resistance by forming biofilms on solid surfaces. However, efficient disinfection methods to eliminate Salmonella biofilms from farms have not yet been examined in detail. In this study, more than 80% of Salmonella strains from farms in Yamagata prefecture, Japan, were biofilm producers. Regardless of the extent of their biofilm formation ability, their biofilms were highly resistant to hypochlorous acid on plastic surfaces. To establish efficient disinfection methods in farms, we developed in vitro Salmonella-contaminated poultry house models by depositing dust on ceramic and stainless-steel carriers in poultry houses for one month and culturing a representative Salmonella strain on the carriers. Biofilm-like structures, including Salmonella-like cells, were observed on the models by scanning electron microscopy. Salmonella was not efficiently removed from the models even by cleaning with a surfactant at 25/65°C and disinfection with quaternary ammonium compound or hypochlorous acid at 25°C; on the contrary, viable Salmonella cells increased in some tests under these conditions, suggesting that these models successfully simulate the highly persistent characteristics of Salmonella in farms. However, the persistent bacterial cells were markedly decreased by soaking in 65°C surfactant followed by rinsing with 80°C water, additional cleaning using chlorine dioxide or disinfection with dolomitic lime, suggesting the effectiveness of these methods against Salmonella in farms. Since many different disinfection conditions may be easily tested in laboratories, our models will be useful tools for establishing effective and practical disinfection methods in farms.

Intra-macrophage expression of ArtAB toxin gene in Salmonella.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. Worthington, and S. bongori produce ArtAB toxin, which catalyses ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on a prophage in Salmonella, and prophage induction by SOS-inducing agents is associated with increases in ArtAB production in vitro. However, little is known about the expression of artAB in vivo. Here, we showed a significant increase in artAB transcription of DT104 within macrophage-like RAW264.7 cells. Intracellular expression of ArtAB was also observed by immunofluorescence staining. The induced expression of artAB in DT104 and S. bongori was enhanced by treatment of RAW264.7 cells with phorbol 12-myristate 13-acetate (PMA), which stimulates the production of reactive oxygen species (ROS); however, such induction was not observed in S. Worthington. Upregulation of oxyR, a major regulator of oxidative stress, and cI, a repressor of prophage induction, was observed in S. Worthington within RAW264.7 cells treated with PMA but not in the DT104 strain. Although the expression of oxyR was increased, artAB was upregulated in S. bongori, which lacks the cI gene in the incomplete artAB-encoded prophage. Taken together, oxidative stress plays a role in the production of artAB toxins in macrophages, and high expression levels of oxyR and cI are responsible for the low expression of artAB. Therefore, strain variation in the level of artAB expression within macrophages could be explained by differences in the oxidative stress response of bacteria and might be reflected in its virulence.

Duplication of blaCTX-M-1 and a class 1 integron on the chromosome enhances antimicrobial resistance in Escherichia coli isolated from racehorses in Japan.

OBJECTIVES: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae have become a cause for great concern. Although some studies have reported the prevalence of ESBL-producing bacteria and ESBL-encoding genes in horses worldwide, the genetic structure surrounding the ESBL gene has not been analysed in detail. In the present study, we isolated two ESBL-producing Escherichia coli strains from diseased racehorses in Japan and demonstrated the mechanisms underlying the acquisition of their antimicrobial resistance (AMR) genes. METHODS: Two ESBL-producing E. coli strains (E148 and E189) were isolated from the heart and liver of horses with endocarditis and sepsis in 2014 and 2016, respectively, in Japan. Complete genomic sequences of the two strains were analysed using a PacBio RSII sequencer. Antimicrobial susceptibility testing was performed by the agar dilution method. RESULTS: The two isolates possessed a chromosomal AMR gene cluster containing blaCTX-M-1 that was similar to the pEQ1 plasmid found in E. coli isolated from a racehorse in the Czech Republic. In one of the two strains, tandem duplication of the 16-kb region containing blaCTX-M-1 and a class 1 integron, which occurred via IS26-mediated recombination, increased minimum inhibitory concentrations (MICs) associated with the duplicated AMR genes. CONCLUSION: Chromosomal blaCTX-M-1 possibly derived from the pEQ1 or pEQ1-like plasmid was found in Japanese equine E. coli isolates. In Japanese strains, many AMR genes containing blaCTX-M-1 and the class 1 integron are highly accumulated in one region on the chromosome, and the AMR of E. coli was enhanced via the IS26-mediated duplication of the AMR gene cluster.

Identification of a Recently Dominant Sublineage in Salmonella 4,[5],12:i:- Sequence Type 34 Isolated From Food Animals in Japan.

Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) and its monophasic variant (Salmonella 4,[5],12:i:-) are among the most frequently isolated clones from both humans and animals worldwide. Our previous study demonstrated that Salmonella Typhimurium/4,[5],12:i:- strains isolated in Japan could be classified into nine clades and that clade 9 consisted of ST34 strains. In Japan, ST34/clade 9 was first found in the 1990s and has become predominant among food animals in recent years. In the present study, we analyzed the whole genome-based phylogenetic relationships and temporal information of 214 Salmonella Typhimurium/4,[5],12:i:- ST34/clade 9 strains isolated from 1998 to 2017 in Japan. The 214 strains were classified into two sublineages: the newly identified clade 9-2 diverged from clade 9 in the early 2000s and has predominated in recent years. Clonally expanding subclades in clades 9-1 or 9-2 lacked Gifsy-1 or HP1 prophages, respectively, and some strains in these subclades acquired plasmids encoding antimicrobial resistance genes. Additional genome reduction around the fljB gene encoding the phase 2-H antigen was generated by an IS26-mediated deletion adjacent to the transposon in clade 9-2. Although most of the clade 9 strains were isolated from cattle in Japan, the clonally expanding subclades in clade 9-2 (i.e., all and 24% strains of subclades 9-2a and 9-2b, respectively) were isolated from swine. The spread of clade 9 in recent years among food animals in Japan was responsible for the emergence of multiple host-adapted sublineages involving the clonally expanding subclades generated by mobile genetic element-mediated microevolution.

A survey of antimicrobial resistance in Escherichia coli isolated from wild sika deer (Cervus nippon) in Japan.

We examined the antimicrobial susceptibility of 848 Escherichia coli isolates from 237 feces samples of wild sika deer (Cervus nippon) captured between 2016 and 2019 in 39 of the 47 prefectures of Japan. Five of the 237 wild sika deer (2.1%) carried E. coli with resistance to at least one antimicrobial, and all the resistant isolates showed resistance to tetracycline. The resistant isolates contained antimicrobial resistance genes that were similar to those in E. coli derived from humans and farm animals. Although wild sika deer are not currently likely to be a source for the transmission of antimicrobial resistance in Japan, they can potentially mediate antimicrobial resistance spread by coming into contact with humans, animals, and their surroundings.