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.

Simultaneous determination of glyphosate, glufosinate, and their metabolites in honey using liquid chromatography-tandem mass spectrometry and solid-phase extraction.

Here, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of residual glyphosate, glufosinate, and their metabolites N-acetylglyphosate (Gly-A), 3-methylphosphinicopropionic acid (MPPA) and N-acetylglufosinate (Glu-A) in honey using a mixed mode column of reversed-phase and anion exchange without derivatization. The target analytes were extracted from honey samples using water, cleaned up on a reverse phase C18 cartridge column and an anion exchange NH2 cartridge column, and quantified using LC-MS/MS. Glyphosate, Glu-A, Gly-A, and MPPA were detected in negative ion mode based on deprotonation, whereas glufosinate was detected in positive ion mode. The coefficients of determination (R2) of the calibration curve, calculated in the range of 1-20 µg/kg for glufosinate, Glu-A, and MPPA, and 5-100 µg/kg for glyphosate and Gly-A, were higher than 0.993. The developed method was evaluated using honey samples spiked with glyphosate and Gly-A at 25 µg/kg and glufosinate, and MPPA and Glu-A at 5 µg/kg, based on the maximum residue levels. The validation results show good recoveries (86-106%) and precision (< 10%) for all target compounds. The limit of quantification of the developed method is 5 µg/kg for glyphosate, 2 µg/kg for Gly-A, and 1 µg/kg for glufosinate, MPPA and Glu-A. These results suggest that the developed method is applicable for quantifying residual glyphosate, glufosinate, and their metabolites in honey in compliance with Japanese maximum residue levels. Moreover, the proposed method was applied to the analysis of honey samples and glyphosate, glufosinate, and Glu-A were detected in some samples. The proposed method will be a useful tool for the regulatory monitoring of residual glyphosate, glufosinate, and their metabolites in honey.

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.

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.

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius, and a survey of foulbrood pathogen contamination in Japanese honey.

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.

Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples.

To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1-196.2 ng/ml and 0-12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.

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.

Association between antimicrobial treatment and resistance of pathogenic Escherichia coli isolated from diseased swine in Kagoshima Prefecture, Japan.

Pathogenic Escherichia coli is an important cause of diarrhea, edema disease, and septicemia in swine. In Japan, the volume of antimicrobial drugs used for animals is highest in swine, but information about the prevalence of antimicrobial-resistant bacteria is confined to apparently healthy animals. In the present study, we determined the O serogroups, virulence factors, and antimicrobial resistance of 360 E. coli isolates from swine that died of disease in Kagoshima Prefecture, Japan, between 1999 and 2017. The isolates of the predominant serogroups O139, OSB9, O149, O8, and O116 possessed virulence factor genes typically found in diarrheagenic E. coli. We further found five strains of third-generation cephalosporin-resistant E. coli that each produced an extended-spectrum ß-lactamase encoded by blaCTX-M-14, blaCTX-M-15, blaCTX-M-24, blaCTX-M-61, or blaSHV-12. In 218 swine with a clear history of antimicrobial drug use, we further analyzed associations between the use of antimicrobials for the treatment of diseased swine and the isolation of resistant E. coli. We found significant associations between antimicrobial use and selection of resistance to the same class of antimicrobials, such as the use of ceftiofur and resistance to cefotaxime, cefazolin, or ampicillin, the use of aminoglycosides and resistance to streptomycin, and the use of phenicols and resistance to chloramphenicol. A significant association between antimicrobial use and the resistance of E. coli isolates to structurally unrelated antimicrobials, such as the use of ceftiofur and resistance to chloramphenicol, was also observed.

Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia.

Entamoeba suis and E. polecki subtype (ST) 1 and ST3 recently have been inferred to be virulent in pigs. However, because relevant molecular epidemiological surveys have been limited, the prevalences of these species remain unknown and their pathogenicities are still controversial. We surveyed 196 fecal samples of pigs (118 of adults, 78 of piglets) at Tangerang in West Java, Indonesia, in 2017, employing PCR using porcine Entamoeba-specific primers. E. suis was the more frequently detected species, observed in 81.1% of samples, while E. polecki ST1 and ST3 were detected in 18.4% and 17.3% of samples, respectively; mixed infections (harboring 2-3 species or subtypes of Entamoeba) were confirmed in 29.3% of positive samples. Statistically significant differences in the positive rates were not seen between adult pigs and piglets, except for those of E. polecki ST3. The prevalences of Eimeria spp. and/or Cystoisospora suis (79.1%), strongyles (55.6%), and Strongyloides spp. (6.1%) were also observed morphologically in the samples. Further chronological or seasonal investigations of pigs and humans in these high-prevalence areas are needed to assess the virulence of the Entamoeba parasites, including the effects on pig productivity, and to evaluate the zoonotic impacts of these organisms.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Detections of gastrointestinal parasites, including Giardia intestinalis and Cryptosporidium spp., in cattle of Banten province, Indonesia.

Gastrointestinal parasites can induce low productivity in livestock by causing acute or chronic enteritis. Veterinarians make great efforts to design rational and effective hygienic protocols for both the prevention and treatment of diarrhea. Although prevalences can vary depending on the examined areas or the ages of the hosts, and the methods used for detections, it is helpful to accumulate data across many areas to evaluate parasitic distribution. A coprological survey in cattle was conducted in Tangerang, Banten Province of Indonesia, in order to determine the prevalence of the parasites, including those of diarrhea-associated diseases. Furthermore, the risk of transmission of Giardia intestinalis and Cryptosporidium spp. to human was genetically analyzed. Gastrointestinal parasites were detected in 87 of 109 cattle samples, including 85 carrying Eimeria spp., 36 carrying Fasciola gigantica, 35 carrying Strongyloides spp., 33 carrying Paramphistomum spp., and 15 carrying Capillaria spp. Giardia intestinalis and Cryptosporidium spp., parasites with zoonotic potential, were detected in 9 and 1 cattle samples, respectively. Molecular analyses identified the G. intestinalis isolate as a member of Assemblage E, which has been recently detected in humans in another country. These results may be helpful in understanding the hygienic risk affecting the livestock productivity and zoonotic potential of cattle in Indonesia.

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3' end of the tRNA genes pheV or pheR The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Transcriptomic analysis of Campylobacter jejuni grown in a medium containing serine as the main energy source.

Campylobacter jejuni is one of the most important causes of food-borne diseases in industrialized countries. Amino acids are an important nutrient source for this pathogen because it lacks enzymes related to glycolysis. However, the metabolic characteristics of C. jejuni grown in a nutrient-restricted medium with specific amino acids have not been fully elucidated. This study shows that C. jejuni NCTC 11168 grows well in a nutrient-restricted medium containing serine, aspartate, glutamate, and proline. Subtracting serine significantly reduced growth, but the removal of the three other amino acids did not, suggesting that serine is a priority among the four amino acids. A transcriptomic analysis of C. jejuni NCTC 11168 grown in a medium with serine as the main energy source was then performed. Serine seemed to be sensed by some chemoreceptors, and C. jejuni reached an adaptation stage with active growth in which the expression of flagellar assembly components was downregulated and the biosyntheses of multiple amino acids and nucleotide sugars were upregulated. These data suggest that C. jejuni NCTC 11168 requires serine as a nutrient.

A frameshift mutation in the rRNA large subunit methyltransferase gene rlmA II determines the susceptibility of a honey bee pathogen Melissococcus plutonius to mirosamicin.

American foulbrood (AFB) and European foulbrood (EFB) caused by Paenibacillus larvae and Melissococcus plutonius, respectively, are major bacterial infections of honey bees. Although macrolides (mirosamicin [MRM] and tylosin) have been used to prevent AFB in Japan, macrolide-resistant P. larvae have yet to be found. In this study, we revealed that both MRM-resistant and -susceptible strains exist in Japanese M. plutonius and that a methyltransferase gene (rlmA II ) was disrupted exclusively in MRM-susceptible strains due to a single-nucleotide insertion. The M. plutonius RlmAII modified G748 of 23S rRNA, and the deletion of rlmA II resulted in increased susceptibility to MRM and the loss of modification at G748, suggesting that methylation at G748 by RlmAII confers MRM resistance in M. plutonius. The single-nucleotide mutation in MRM-susceptible strains was easily repaired by spontaneous deletion of the inserted nucleotide; however, intact rlmA II was only found in Japanese M. plutonius and not in a Paraguayan strain tested or any of the whole-genome-sequenced European strains. MRM has been used in apiculture only in Japan. Although M. plutonius is not the target of this drug, the use of MRM as a prophylactic drug for AFB may have influenced the antibiotic susceptibility of the causative agent of EFB.

Phylogenetic Characterization of Salmonella enterica Serovar Typhimurium and Its Monophasic Variant Isolated from Food Animals in Japan Revealed Replacement of Major Epidemic Clones in the Last 4 Decades.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and its monophasic variant (Salmonella 4,[5],12:i:-) are the major causes of gastroenteritis in both humans and animals. Pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis have been used widely as subtyping methods for these pathogens in molecular epidemiological analyses, but the results do not precisely reflect phylogenetic information. In this study, we performed a phylogenetic analysis of these serovars using whole-genome sequencing data and identified nine distinct genotypic clades. Then, we established an allele-specific PCR-based genotyping method detecting a clade-specific single nucleotide polymorphism to rapidly identify the clade of each isolate. Among a total of 815 isolates obtained from cattle in Japan between 1977 and 2017, clades 1, 7, and 9 contained 77% of isolates. Obvious replacement of the dominant clone was observed five times in this period, and clade 9, which mostly contains Salmonella 4,[5],12:i:-, is currently dominant. Among 140 isolates obtained from swine in Japan between 1976 and 2017, clades 3 and 9 contained 64% of isolates. Clade 9 is the latest clone as is the case in cattle isolates. Clade 9 is similar to an epidemic clone from Europe, which is characterized by sequence type 34 (ST34), chromosomal Salmonella genomic island 3, and a composite transposon containing antimicrobial resistance genes. The increased prevalence of clade 9 among food animals in Japan might be a part of the pandemic of the European Salmonella 4,[5],12:i:- clone.