High qnrS retention of ESBL-producing and mcr-harbouring colistin-resistant Escherichia coli in Vietnamese food products.

Plasmid-mediated antibiotic-resistant bacteria's transmission is fatal and a major threat to public health. This study aimed to clarify the presence of plasmid-mediated quinolone resistance（PMQR）genes in extended-spectrum ß-lactamase（ESBL）-producing or/and mcr-harbouring colistin（COL）-resistant Escherichia coli（ESBL-COL-EC）isolates from Vietnamese and Japanese chicken meat. Resistance towards ciprofloxacin（CIP）was examined in 308 ESBL-COL-EC isolates; CIP-resistant ESBL-COL-EC isolates were examined for the PMQR gene. Approximately, 71.1% and 38.1% of ESBL-COL-EC and ESBLproducing E. coli isolates from Vietnamese and Japanese chicken meat were CIP-resistant, respectively. Multiplex PCR led PMQR detection showed that 35.2% of CIP-resistant ESBL-COL-EC isolates from Vietnamese food contained PMQR gene, whereas CIP-resistant ESBL-COL-EC isolates from Japanese chicken meat did not. Conjugation assays showed that the transmission of qnrS gene carried by E. coli to Salmonella. In conclusion, ESBL-COL-EC isolates from Vietnamese food are associated with a high frequency of fluoroquinolone resistance and a high distribution of the qnrS gene.

Prevalence of streptomycin and tetracycline resistance and increased transmissible third-generation cephalosporin resistance in Salmonella enterica isolates derived from food handlers in Japan from 2006 to 2021.

AIMS: The increasing prevalence of AmpC ß-lactamase (AmpC)- and extended-spectrum ß-lactamase (ESBL)- producing food pathogens is a serious public health concern. AmpC- and ESBL-producing Salmonella species pose a high risk of food contamination. This study aimed to investigate changes in the prevalence of Salmonella among food handlers in Japan from 2006 to 2021 using 100 randomly selected isolates from 2006, 2012, 2018, and 2021 with different serotypes and antimicrobial resistance patterns. METHODS AND RESULTS: The average Salmonella isolation rate was 0.070% (19 602/27 848 713). Serotyping revealed that the most common serotypes were Enteritidis in 2006, Infantis in 2012, Agoueve/Cubana in 2018, and Schwarzengrund in 2021. Antimicrobial susceptibility testing showed that Salmonella isolates exhibited the highest resistance to streptomycin (<40%), followed by tetracycline (<20%-40%). Moreover, 6% of the Salmonella isolates produced cephalosporinases with the blaCMY-2, blaCTX-M-14, and blaTEM genes. The annual incidence of cephalosporin resistance has increased. Plasmid conjugation assays revealed that cephalosporin-resistant Salmonella spp. transmitted their resistance to Escherichia coli. Additionally, plasmid genome analysis showed that the insertion sequence IS26 was encoded in the upstream and downstream regions of blaCTX-M-14 and qnrS1 in the IncHI1 plasmid, which could be transmitted to other bacteria. CONCLUSIONS: The tested Salmonella isolates showed high resistance to specific antibiotics, with differences in resistance depending on the serotype. Further increase and spread of transmissible cephalosporin-resistant strains should be noted.

Edible river fish-derived extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales harboring transferable plasmids encoding bla CTX-M-15, bla CTX-M-27, and bla CTX-M-55.

Transmission of extended-spectrum ß-lactamase (ESBL) genes has increased the global prevalence of ESBL-producing bacteria, especially in developing countries. Human infection with these bacteria may be food-mediated but has not been fully elucidated. Therefore, we aimed to examine ESBL-producing bacteria in edible river fish and elucidate their potential for horizontal gene transfer. A total of 173 ESBL-producing Enterobacterales were isolated (Escherichia coli [n = 87], Klebsiella pneumoniae [n = 52], Enterobacter cloacae complex [n = 18], Citrobacter freundii complex [n = 14], Atlantibacter hermannii [n = 1] and Serratia fonticola [n = 1]) from 56 of 80 fish intestinal contents sampled. Among the bacterial bla CTX-M genotypes, bla CTX-M-55 was the most predominant, followed by bla CTX-M-15, bla CTX-M-27, and bla CTX-M-65. Furthermore, we found that ESBL-producing Enterobacterales were able to transfer their bla CTX-M genes to E. coli. In summary, our results suggest that ESBL-producing Enterobacterales transfer bla CTX-M to indigenous gut E. coli in humans, following the consumption of contaminated fish.

ESBL-producing Atlantibacter hermannii harboring a plasmid encoding IS26 up and down stream of blaCTX-M-27 ,blaLAP-2, and qnrS1 isolated from an edible river Anabas testudineus fish.

Extended-spectrum ß-lactamase-producing Atlantibacter hermannii was isolated from an edible river fish, Anabas testudineus, which was sold in a market located in Vietnam. The genome sequence was obtained by using next-generation sequencing, which involved Oxford Nanopore and Illumina technologies. The 92 kb plasmid encodes the gene blaCTX-M-27.

IncA/C plasmid encoding blaCTX-M-55 in non-O1 Vibrio cholerae isolates from the edible river fish Mastacembelus sp.

Extended-spectrum ß-lactamase-producing non-O1 Vibrio cholerae was isolated from edible Mastacembelus sp. in Vietnam. The genome sequence was sequenced using DNBSEQ-G400 and MinION Mk1b. A plasmid of approximately 183-kb encoding blaCTX-M-55 and blaTEM-1 was detected.

Detection of chromosome-mediated blaNDM-1-carrying Aeromonas spp. in the intestinal contents of fresh water river fish in Ho Chi Minh City, Vietnam.

The spread of antibiotic-resistant bacteria is a global problem that should be addressed through the perspective of the "one health" concept. The purpose of this study was to determine the contamination rate of antibiotic-resistant Aeromonas spp. in fresh water river fish purchased from a fish market in Vietnam. We then defined the pattern of antibiotic resistance to assess antibiotic-resistant contamination. Antibiotic-resistant Aeromonas spp. were detected in the intestinal contents of 32 of 80 fish. blaNDM-1 was detected in seven strains. Extended-spectrum ß-lactamase and AmpC ß-lactamase-related genes were detected in 28 strains, including blaCTX-M-55, blaCTX-M-15, blaCTX-M-1, and blaDHA,blaFOX, and blaMOX. The blaNDM-1 detected in the seven Aeromonas spp. strains were found chromosomally. This finding suggests that the blaNDM gene is stable in the natural environment and may spread widely into animals and humans via Aeromonas spp. with a transposon. Our results suggest the importance of continuing to monitor carbapenemase genes in Aeromonas spp. to evaluate the possibility that they may spread in other Enterobacterales, and to elucidate the mechanism of spread.

Common presence of plasmid encoding blaCTX-M-55 in extended-spectrum ß-lactamase-producing Salmonella enterica and Escherichia coli isolates from the same edible river fish.

The transmission of potentially life-threatening plasmid-mediated antibiotic-resistant bacteria poses a major threat to public health. This study aimed to determine the presence of commonly observed plasmids encoding plasmid-mediated antibiotic-resistance genes in Salmonella and Escherichia coli isolates from fishery products. Eighty river fishes were purchased from retail stores and supermarkets in Vietnam. Only Salmonella-positive fishes were used for antibiotic-resistant E. coli isolation. Salmonella serotyping was performed using Salmonella antisera. Isolated bacterial DNA was extracted, and antibiotic susceptibility, resistance genes, and replicon typing were determined. Our results showed that Salmonella was isolated from 12.5% (10/80) of the river fishes. Cefotaxime-resistant Salmonella was isolated from 3.8% (3/80) of the fishes and colistin-resistant Salmonella from 1.3% (1/80) . Salmonella serotyping revealed Potsdam, Schwarzengrund, Bardo/Newport, Give, Infantis, Kentucky, and Typhimurium. Multiplex polymerase chain reaction revealed the presence of extended-spectrum ß-lactamase-related genes blaCTX-M-55 and blaCTX-M-65 and the colistin resistance gene mcr-1. To date, no study has reported an antibiotic-resistance plasmid present in multiple bacteria collected from the same food. Thus, horizontal transmission of antibiotic-resistance plasmids may occur at the food level.

IncHI2 Plasmid Encoding blaCTX-M-55 and mcr-1.1 in Salmonella enterica SE20-C72-2 and Escherichia coli EC20-C72-1 Isolates from the Edible River Fish Anabas testudineus.

Salmonella enterica SE20-C72-2 and Escherichia coli EC20-C72-1 were isolated from the edible fish Anabas testudineus in Vietnam. The chromosomes and plasmids from both strains were sequenced using Oxford Nanopore and Illumina sequencing. Plasmids approximately 250 kbp long, encoding blaCTX-M-55 and mcr-1.1, were detected in both strains.

ESBL-producing Vibrio vulnificus and V. alginolyticus harbour a plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

In recent years, trade liberalisation has led to the spread of antibiotic-resistant bacteria (ARB) in food products. Because ARB has reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern. Here, we report the complete genome sequences of ESBL-producing Vibrio vulnificus and V. alginolyticus strains harbouring a plasmid isolated from imported seafood. First, V. vulnificus and V. alginolyticus were isolated from purchased frozen and thawed Litopenaeus vannamei shrimp, and genome extraction and sequencing were performed. Hybrid genome assemblies were performed using Unicycler and annotated using DFAST. Then genome analysis was performed using BRIG. Plasmid comparisons showed that the plasmids carried by both Vibrios are remarkably similar and encode the same antibiotic-resistance genes. The 270-310 kb region specific to both Vibrios were isolated in this study and encodes the antibiotic-resistance genes blaCTX-M and qnr. Furthermore, the mobile genetic factors ISEc9, ISVch4, and ISVpa4 are located upstream and downstream of these genes. This is the first report of ESBL-producing V. vulnificus and V. alginolyticus harbouring a common plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

Carbapenem-Resistant Citrobacter freundii and Escherichia coli Harboring a Common IncC-Type Plasmid Encoding IS26 Upstream of blaNDM-1, sul1, aph(3')-VI, and qacE Isolated from Edible Mastacembelidae Fish.

Carbapenem-resistant Citrobacter freundii CF20-4P-1 and Escherichia coli EC20-4B-2 were isolated from edible Mastacembelidae in Vietnam. We present the draft genome sequences, and the complete plasmid genome sequencing was also performed by hybrid assembly sequencing of Oxford Nanopore and Illumina. The 137-kbp plasmid encoding the assembled blaNDM-1 was detected in both strains.

Simple and quick detection of extended-spectrum ß-lactamase and carbapenemase-encoding genes using isothermal nucleic acid amplification techniques.

The spread of plasmid-mediated antibiotic-resistant bacteria must be controlled; to this end, developing kits for simple and rapid detection in food and clinical settings is desirable. This review describes the detection of antibiotic resistance genes in extended-spectrum ß-lactamase （ESBL）- and carbapenemase-producing bacteria. Loop-mediated isothermal amplification （LAMP）, a technique developed in Japan, is a useful diffusion amplification method that does not require equipment like thermal cyclers, and amplifies the target gene in 30 min at about 65℃. Although most reports targeting ESBL and carbapenemase genes are intended for clinical use, environmental and food samples have also been targeted. Recombinase polymerase amplification （RPA） has recently been developed; in RPA, the reaction proceeds under the human skin with reaction conditions of 30 min at 37℃. Detection of ESBL and carbapenemase-encoding genes in food and clinical samples using RPA has been reported in limited studies. However, research on RPA has just begun, and further development is expected.

Frequent contamination of edible freshwater fish with colistin-resistant Escherichia coli harbouring the plasmid-mediated mcr-1 gene.

The threat of antimicrobial resistance is increasing. Microbial food contamination poses a serious public health risk; however, there are only a few studies on the prevalence of colistin-resistant Escherichia coli (COL-E) contamination in freshwater fish. This study aimed to characterise the antibiotic resistance genes and antibiotic susceptibility profiles of COL-E in freshwater fish in Vietnam. In total, 103 fish were collected and 63 COL-E were isolated. COL-E was investigated by genotyping mcr and AmpC/extended-spectrum ß-lactamase (ESBL)-related genes. The results show that COL-E and AmpC/ESBL-producing COL-E were confirmed in 24.3 % and 14.6 % of the fish, respectively. Multiplex PCR for mcr-1-9 showed that all 63 COL-E harboured mcr-1, while mcr-3 was detected in 7.9 % of COL-E. The minimum inhibitory concentration of colistin ranged from 2 to 256 µg/mL. Meanwhile, antibiotic susceptibility results show that all COL-E were resistant to ampicillin, streptomycin, and chloramphenicol.

Prevalence and Characterization of Gentamicin Resistance Genes in Escherichia coli Isolates from Beef Cattle Feces in Japan.

Gentamicin is an important antibiotic for the treatment of opportunistic infections in the clinical field. Gentamicin-resistant bacteria have been detected in livestock animals and can be transmitted to humans through the food supply or direct contact. We have previously revealed that gentamicin-resistant Escherichia coli are distributed at a comparatively high rate from beef cattle in Japan, but few studies have focused on the molecular epidemiology of gentamicin-resistant bacteria. To understand these bacteria, this study examined the prevalence of various gentamicin resistance genes in gentamicin-resistant E. coli isolates from beef cattle feces. Of the 239 gentamicin-resistant E. coli isolates, the presence of the aacC2, aadB, or aac(3)-VIa genes was confirmed in 147, 84, and 8 isolates, respectively. All aac(3)-VIa-harboring isolates had an MIC value of 64 µg/mL for gentamicin and exhibited resistance to 11 antibiotic agents. An analysis of the representative aac(3)-VIa-harboring E. coli strain GC1-3-GR-4 revealed that the aac(3)-VIa gene was present on the IncA/C plasmid together with the aadA and blaCMY genes. Furthermore, the upstream region of the aac(3)-VIa gene contained the aadA gene and the class 1 integron-integrase gene (intI1). The aac(3)-VIa gene was detected for the first time in Japan and is expected to be able to transfer between bacteria via the IncA/C plasmid and integron. These results reveal the expansion of the distribution or diversity of gentamicin resistance genes in Japan.

Genome Sequence of Carbapenemase-Producing Enterobacter cloacae 0102-4P-1 Harboring the IncC-Type Plasmid with a Multidrug Resistance Site Encoding blaNDM-1, Isolated from Commercially Imported Shrimp.

A carbapenem-resistant Enterobacter cloacae 0102-4P-1 strain was isolated from commercially imported shrimp in Japan. Here, we present a draft genome sequence. The complete plasmid sequence was also determined by hybrid assembly sequencing using Oxford Nanopore and Illumina methods. The assembled whole genome and plasmid were 5,164,033 bp and 162,852 bp long, respectively.

Comprehensive lipidomics of lupus-prone mice using LC-MS/MS identifies the reduction of palmitoylethanolamide that suppresses TLR9-mediated inflammation.

Lipid mediators are known to play crucial roles not only in the onset of the inflammatory response but also in the induction of resolution of inflammation. Here, we report that palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, can suppress the inflammation induced by Toll-like receptor (TLR) signaling both in vitro and in vivo. PEA was found to be significantly reduced in the serum and spleen of lupus-prone MRL/lpr mice analyzed by lipidomics. PEA suppressed pro-inflammatory cytokine production in a mouse macrophage cell line stimulated with TLR ligands such as lipopolysaccharide, peptidoglycan, poly (I:C), imiquimod, and CpG-ODN. PEA also inhibited both mRNA and protein levels of IL-6 in bone marrow-derived dendritic cells (BMDCs) and B cells stimulated with CpG-ODN. Augmentation of cell surface CD86 and CD40 on BMDCs and B cells, IgM production, and cell proliferation of B cells in response to CpG-ODN were attenuated by PEA. Moreover, PEA treatment significantly reduced mortality and serum IL-6 levels in mice injected with CpG-ODN plus D-galactosamine. Taken together, PEA ameliorates inflammation induced by TLR signaling, which could be a novel therapeutic target for inflammatory disorders.

Abundance of colistin-resistant Escherichia coli harbouring mcr-1 and extended-spectrum ß-lactamase-producing E. coli co-harbouring blaCTX-M-55 or -65 with blaTEM isolates from chicken meat in Vietnam.

Although the spread of plasmid-mediated antibiotic-resistant bacteria is a public health concern, food contamination with plasmid-mediated antibiotic-resistant Escherichia coli in Vietnam has not been well investigated. This study aimed to describe the prevalence of colistin-resistant, carbapenem-resistant, and endemic blaCTX-M in extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Colistin and carbapenem-resistant ESBL-producing E. coli were isolated from chickens in Vietnam and Japan. Colistin-resistant and AmpC/ESBL-producing E. coli (52% and 93%, respectively) were detected in chickens from Vietnam, in comparison to 52.7%, AmpC/ESBL-producing E. coli found in chicken from Japan. Carbapenem-resistant E. coli has not been isolated in Vietnam and Japan. Genotyping revealed that colistin-resistant E. coli harboured mcr-1, and most of the AmpC/ESBL-related genes were blaCTX-M-55 and blaCTX-M-65 together with blaTEM in Vietnamese chickens and blaCMY-2 in Japanese chickens. Multi-drug resistance analysis showed that ESBL-producing E. coli isolates had greater resistance to quinolones, streptomycin, and chloramphenicol than colistin-resistant E. coli isolates from Vietnam, suggesting the selection of multiple antibiotic resistance genes in ESBL-producing E. coli. In conclusion, colistin-resistant E. coli was detected in approximately half of the chicken samples, the majority of which harboured mcr-1. The high prevalence of ESBL-producing E. coli has remained constant in the last 5 years. The predominant blaCTX-M in ESBL-producing E. coli was blaCTX-M-55 or blaCTX-M-65, with the coexistence of blaTEM in Vietnam. These results can be implemented in monitoring systems to overcome the development of antimicrobial resistance.

Streptococcal meningitis reveals the presence of residual streptococci and down-regulated aquaporin 4 in the brain.

The pathology of streptococcal meningitis is poorly understood, even though streptococcal infection induces meningitis. The aim of this study was to clarify the relationship between streptococcal meningitis and aquaporin 4 (AQP4) in the mouse brain. After Streptococcus suis infection, the streptococcal number was calculated, and AQP4 mRNA expression in the brain was quantified at 2 and 7 days after infection. At 7-day post-infection, mice with neurological symptoms showed significantly higher S. suis levels in the brain than mice without neurological symptoms. AQP4 expression was significantly decreased in mice with neurological symptoms than in mice without neurological symptoms. Image analysis demonstrated that S. suis progressed to invade the white matter. Pathological analysis revealed that infected mouse brains had higher inflammation and neurological damage scores than uninfected mouse brains. Therefore, mice with neurological symptoms caused by streptococcal meningitis had high S. suis levels in the brain and reduced AQP4 expression.

Long-Term Grow-Out Affects Campylobacter jejuni Colonization Fitness in Coincidence With Altered Microbiota and Lipid Composition in the Cecum of Laying Hens.

Campylobacter jejuni is one of the leading causes of gastrointestinal illness worldwide and is mainly transmitted from chicken through the food chain. Previous studies have provided increasing evidence that this pathogen can colonize and replicate in broiler chicken during its breeding; however, its temporal kinetics in laying hen are poorly understood. Considering the possible interaction between C. jejuni and gut microbiota, the current study was conducted to address the temporal dynamics of C. jejuni in the cecum of laying hen over 40 weeks, with possible alteration of the gut microbiota and fatty acid (FA) components. Following oral infection with C. jejuni 81-176, inocula were stably recovered from ceca for up to 8 weeks post-infection (p.i.). From 16 weeks p.i., most birds became negative for C. jejuni and remained negative up to 40 weeks p.i. 16S rRNA gene sequencing analyses revealed that most of the altered relative rRNA gene abundances occurred in the order Clostridiales, in which increased relative rRNA gene abundances were observed at >16 weeks p.i. in the families Clostridiaceae, Ruminococcaceae, Lachnospiraceae, and Peptococcaceae. Lipidome analyses revealed increased levels of sterols associated with bile acid metabolisms in the cecum at 16 and/or 24 weeks p.i. compared with those detected at 8 weeks p.i., suggesting that altered microbiota and bile acid metabolism might underlie the decreased colonization fitness of C. jejuni in the gut of laying hens.

Untargeted Phylogenetic Group III of Multi-drug-Resistant Bacillus cereus Isolated Using Fraser Medium from Retail Chickens in Ho Chi Minh City.

The prevalence of food-borne bacteria in developing countries is less well understood than in developed countries. The ISO11290-1 isolation method is commonly used to study Listeria contamination in chicken; however, all isolates are identified as untargeted Bacillus cereus. This study aimed to determine the classification, antibiotic susceptibility, and virulence genes of B. cereus isolated from retail chickens in Vietnam. Bacterial isolation using the ISO11290-1 method yielded 12 strains of B. cereus from seven out of 60 chickens. For determining bacterial diversity, panC and multilocus sequence typing (MLST) analyses were performed. PanC analysis showed that all seven strains belong to the phylogenetic group III, to which the highest risk of foodborne illnesses was associated. MLST analysis showed that most strains contained a ST205 complex; further, all strains were found to be resistant to ampicillin, ciprofloxacin, and tetracycline. Virulence genes were also investigated. ces, a cereulide-related gene, was detected in 50% of the isolated strains, followed by cytK, nheA, and hblA enterotoxins in 41.7%, 16.7%, and 25% of the strains, respectively. In conclusion, B. cereus may be erroneously detected when attempting to detect Listeria in food using the ISO11290-1 method. Further study of the prevalence of B. cereus in Vietnamese food is needed to improve food safety.

Draft Genome Sequence of Stenotrophomonas maltophilia CRB139-1, Isolated from Poultry Meat in Japan.

Stenotrophomonas maltophilia is a nosocomial pathogen that primarily causes respiratory infection in humans. This pathogen is widely distributed in the environment, including in foods. Here, we report the draft genome sequence of S. maltophilia strain CRB139-1, isolated from poultry meat in Japan. The genome size was 4,619,918 bp at 90× coverage.