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In order to study the relation of safety level between automated driving vehicle and human driver, analysis of driving behavior of human drivers is necessary. In this research, in order to study driving behavior of human drivers in some conceivable traffic scenes in which it is difficult for automated driving vehicle to avoid a collision, the experimental test by using the driving simulator was conducted. [ABSTRACT FROM AUTHOR]

Abstract The combination of plant extracts with mild heat treatment is a promising strategy for obtaining microbiologically and chemically safe food products. In this study, we evaluated the antimicrobial activity of ethanolic clove extract against Escherichia coli O157: H7 in nutrient broth, buffer, and tomato juice. Clove extract at 0.4% concentration showed significant E. coli O157: H7 population reduction in all media tested. E. coli O157: H7 was most sensitive to the ethanolic clove extract in nutrient-deficient media but demonstrated relative resistance in nutrient-rich media and tomato juice. Moreover, we evaluated the potential of clove extract as a preservative in tomato juice stored at cold storage temperatures of 4 °C and 15 °C. The clove extract exhibited stronger inhibitory effects against the pathogen in tomato juice stored at 15 °C than at 4 °C. Additionally, we assessed the antimicrobial efficacy of clove extract in combination with mild heat treatment against E. coli O157: H7 in tomato juice. The combined treatment of 0.05% clove extract with mild heat at 60 °C for 30 min achieved a 5-log reduction in the E. coli population in tomato juice. Our findings present a potential hurdle technology against E. coli O157: H7 in tomato juice processing by employing a combination of naturally sourced antimicrobial agents from clove and mild heat treatment. This approach can be effectively adopted by large-scale industries and small-scale local juice vendors to ensure the safety of tomato juice products.

Bacterial diseases caused by Vibrio spp. are a significant impediment to the aquaculture industry owing to difficult-to-treat infections and mortality in farmed animals, affecting socioeconomic development. This study aimed to evaluate the role of Japanese retail seafood in the production of antimicrobial-resistant Vibrio spp. and pathogenic factors. One hundred seafood samples purchased from various retail supermarkets in Hiroshima, Japan, were examined using microbiological, phenotypic, and molecular techniques. One hundred and twenty-eight Vibrio isolates belonging to more than 11 species were identified and characterized. Among the 100 samples, 26 (26 %) tested positive for Vibrio spp. with antimicrobial resistance, while 44 (44 %) were positive for those carrying virulence determinants. Out of 128 isolates, V. alginolyticus was predominant (38 %), followed by V. parahaemolyticus (29 %), V. neocaledonicus (7.0 %), V. cholerae (6.3 %), among others. For virulence gene assessment, only six of the 10 virulence attributes, tlh (34.4 %), VPI (29.7 %), ompW (7.0 %), toxR (5.5 %), hlyA (3.9 %), and ompU (3.1 %), were detected in the isolates. tcpA, ctxA, ctxB, and tdh are absent. Eight V. cholerae isolates of international origin were VPI-positive, seven of which harbored the toxR regulon and other virulence genes. The blaTEM resistance gene was identified in V. alginolyticus (22 isolates). All 46 antimicrobial resistance gene-harboring isolates were phenotypically susceptible to cephalosporins, aztreonam, meropenem, tetracycline, chloramphenicol, and ciprofloxacin. Ampicillin resistance was the highest (91.3 %), followed by colistin (30.4 %) and fosfomycin (15.2 %). The multiple-antibiotic resistance index ranged from 0.2 to 0.47, suggesting that some isolates originate from high-risk contamination sources. Our results shed light on the incidence of potentially pathogenic Vibrio spp. and highlighted the importance of continuous monitoring to improve food safety in the seafood supply chain. These findings will be useful for developing microbiological risk assessments for retail seafood management.

This study developed a simple allele specific (AS) PCR assay for the detection of pathogenic Vibrio cholerae strains in seafood. A pair of primers, (1) an AS primer targeting the mutation at site G450 in the hapR2 allele and a (2) common forward primer, were designed to detect the pathogenic O1 and O139 strains. An artificial mismatch was also added to the AS primer to increase its specificity to the target allele. The PCR assays using different DNA polymerases of varying efficiencies were then optimized. The application of the AS primers in both conventional and real-time PCR assays was facilitated with enriched seafood samples spiked with known concentration of pathogenic V. cholerae. The designed AS primers showed high specificity and sensitivity to the hapR2 allele for both conventional and real-time PCR assays. Enrichment of seafood samples for 8 h is recommended to detect viable pathogenic V. cholerae presence in seafood. In this study, we developed an AS-PCR that is simple, rapid, and a low-cost alternative for routine detection of pathogenic V. cholerae in different seafood samples thus aiding in the surveillance and implementation of food safety measures at the seafood post-harvest level.

ABSTRACT Colistin, a last-line antimicrobial agent, is recommended for the treatment of severe infections caused by multidrug-resistant (MDR) microorganisms. However, reports on plasmid-mediated mobilized colistin resistance (mcr) genes have prompted the importance of routine colistin resistance surveillance. Herein, we report the emergence of Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1 carrying an mcr-9.1 gene in vegetables from Japan. A total of 308 colistin-resistant isolates were retrieved from 200 fresh vegetables in Hiroshima Prefecture, Japan. PCR detection of mcr-1 to mcr-9 was conducted. While none of the isolates detected positive for mcr-1 to mcr-8 genes, we found two (0.65%) positive strains, E. cloacae CST17-2 and R. ornithinolytica CST129-1, that harbored mcr-9.1 allele. These isolates were subjected to phenotypic susceptibility testing, whole-genome sequencing (WGS), PCR-based replicon typing, and conjugation experiment. We found that both isolates had high colistin resistance [minimum inhibitory concentration (MIC) 16 or >64 µg/mL] and showed MDR phenotypes. WGS of both isolates revealed mcr-9 on a plasmid of the IncHI2/HI2A backbone. The mcr-9-bearing plasmid, pCST17-2_1, was self-transferable, although the pCST129-1_1 plasmid was not. Despite being colistin-resistant, the so-called two-component regulatory operon, qseBC, which induces polymyxin resistance, was absent from the genetic arrangements downstream of mcr-9 in R. ornithinolytica CST129-1. Nonetheless, a conjugation experiment demonstrated that mcr-9 in a Raoultella-type background is capable of mediating colistin resistance. In silico genomic analysis and comparison revealed distinct genetic structures surrounding mcr-9, especially in the downstream vicinities. The E. cloacae CST17-2 strain is of sequence-type ST738, a sequence type that has emerged in mcr-9.1-containing E. cloacae. Remarkably, we report the first mcr-9-carrying colistin-resistant Enterobacteriaceae isolated from Japanese vegetables, which is a grave public health concern. Our findings highlight the importance of strict epidemiological monitoring to track and/or prevent further dissemination of mcr homologs across the vegetable industry. IMPORTANCE Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

Reports have documented antimicrobial usage in aquaculture, and the aquatic ecosystem can be considered a genetic storage site for antibiotic-resistant bacteria. This study assessed the prevalence of antimicrobial resistance (AMR) among Gram-negative bacteria recovered from retail seafood in Hiroshima, Japan. A total of 412 bacteria were isolated and screened for the presence of β-lactamases, acquired carbapenemases, and mobile colistin-resistance (mcr) genes. Forty-five (10.9%) isolates were dominated by Morganella (28%), Proteus (22%), Aeromonas (14%), Citrobacter (8%), and Escherichia (8%) and carried AMR genes. The identified AMR genes included those encoded in integrons (19), aac(6՛)-Ib (11), blaTEM-1 (7), blaCTX-M-like (12), blaCTX-M-65 (2), blaSHV-12 (1), blaSHV-27 (1), blaOXA-10 (1), blaOXA-2 (1), and mcr (2). The most common clinical resistances were against ampicillin, colistin, sulfamethoxazole/trimethoprim, tetracycline, and ciprofloxacin. Multidrug resistance (MDR) occurred in 27 (60%) AMR isolates, and multiple antibiotic resistance indices ranged from 0.2 to 0.8. A conjugation experiment showed that 10 of the 11 selected MDR strains harbored conjugable plasmids, although PCR-based replicon typing described seven strains as untypable. IncF replicon was identified in MDR extended-spectrum β-lactamase-producing Escherichia coli of the pathogenic B2 phylogroup. Our findings suggest that retail seafood harbors MDR bacteria of human interest that require strict resistance surveillance in the seafood production continuum.

Colistin is a last-resort antibiotic used in the treatment of multidrug resistant Gram-negative bacteria. However, the activity and efficacy of colistin has been compromised by the worldwide spread of the mobile colistin resistance genes (mcr-1 to mcr-10). In this study, two clinical Escherichia coli strains, named EcCAI51, and EcCAI73, harbored mcr-1, showed multidrug-resistant phenotypes (with colistin MIC = 4 μg/ml), and belonged to phylogroup D: multilocus sequence type 1011 (ST1011) and phylogroup A: ST744, respectively. Findings revealed the existence of mcr-1 gene on two conjugable plasmids, pAMS-51-MCR1 (∼122 kb IncP) and pAMS-73-MCR1 (∼33 kb IncX4), in EcCAI51, and EcCAI73, respectively. The mcr-1-pap2 element was detected in the two plasmids. Additionally, the composite transposon (ISApl1-IS5D-pap2-mcr-1-ISApl1) was identified only in pAMS-51-MCR1 suggesting the potential for horizontal gene transfer. The two strains carried from 16 to 18 different multiple acquired antimicrobial resistance genes (ARGs). Additionally, two different multireplicon virulence plasmids (∼117 kb pAMS-51-Vr and ∼226 kb pAMS-73-Vr) carrying the sit operon, the Salmochelin siderophore iroBCDE operon and other several virulence genes were identified from the two strains. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of EcCAI73, and EcCAI51 with global E. coli lineages at HC levels of 50 (HC50) to 100 (HC100) core genome allelic differences. To the best of our knowledge, this study presented the first complete genomic sequences of mcr-1-carrying IncP and IncX4 plasmids from human clinical E. coli isolates in Egypt. In addition, the study illustrated the mcr-1 broad dissemination in diverse plasmids and dissimilar E. coli clones.

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6′)-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.

Objectives: This study describes the first draft genome sequence of a multidrug-resistant (MDR) Escherichia coli D-ST69 clinical isolate from Egypt carrying blaNDM-1 and blaOXA-244. Methods: The strain was isolated in December 2014 from a wound pus swab of a male patient in the city of Kafr El-Sheikh using MacConkey agar containing 2 μg/mL meropenem. The strain was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole-genome sequencing using an Illumina MiSeq platform. Results: The draft genome of the strain (HR14_AS) was 5.08 Mbp in size containing a total of 90 contigs encoding 4677 predicted genes with an average G+C content of 50.7%. Strain HR14_AS belongs to sequence type 69 (ST69), phylogroup D and exhibits an MDR phenotype, with minimum inhibitory concentrations (MICs) of 64 μg/mL and 32 μg/mL for meropenem and doripenem, respectively. Multiple acquired antimicrobial resistance genes conferring resistance to macrolides [mdf(A)], fluoroquinolones [aac(6')-Ib-cr], quinolones (qnrS1), trimethoprim (dfrA14), β-lactams (blaNDM-1, blaOXA-244, blaCTX-M-15, blaOXA-9 and blaTEM-1B) and aminoglycosides [aac(3)-IId, aac(6')-Ib, aadA1 and aph(3')-VI] were detected. The blaOXA-244 and blaNDM-1 genes were located on the chromosome (Tn6237) and on an IncI1-type self-conjugative plasmid of >93 kb in size, respectively. Conclusions: Here we report the first draft genome sequence of a MDR E. coli D-ST69 isolate carrying blaNDM-1 and blaOXA-244. Besides clonal expansion of the E. coli ST38 pandemic clone, this study further identified that the spread of OXA-244-producing E. coli could be related to mobilisation of the IS1R-made composite transposon (Tn6237) carrying blaOXA-244.

The aim of this study was to assess the hygienic status of raw milk cheese and determine the trends of virulence and antimicrobial resistance in thermotolerant Escherichia coli. Two hundred samples of karish, a popular Egyptian fresh raw milk cheese, were analyzed for coliforms and fecal coliforms using a standard most probable number (MPN) technique. Overall, 85% of samples were unsuitable for consumption, as they exceeded Egyptian standards for coliforms (10 MPN/g), and 65% of samples exhibited coliforms at 44.5 °C. Of 150 recovered thermotolerant strains, 140 (93.3%) were identified as E. coli. Importantly, one Shiga toxin-producing E. coli (STEC) strain carrying a striking virulence pattern, stx1−, stx2+, eae−, was detected. Eleven strains (7.8%, 11/140) showed resistance to third-generation cephalosporins. Antibiotic resistance genes included blaSHV, blaCTX-M, qnrS, tet(A), and tet(B), which were present in 4.3%, 2.8%, 0.71%, 2.1%, and 0.71% of isolates, respectively. In conclusion, this study indicated that hygienic-sanitary failures occurred throughout the production process of most retail karish cheese. Furthermore, our findings emphasize the need for adopting third-generation cephalosporin-resistant E. coli as an indicator for monitoring antimicrobial resistance in raw milk cheese to identify the potential public health burden associated with its consumption.

Objectives: The aim of this study was to investigate the occurrence of plasmid-encoded colistin resistance among Gram-negative bacteria isolated from meat and meat products in Egypt and to report the draft genome sequence of anmcr-1/IncI2-carrying multidrug-resistant (MDR) Escherichia coli B1:ST101 isolate. Methods: A total of 128 colistin-resistant strains were isolated from various meat and meat product samples in different cities in Egypt. Multiplex PCR screening for plasmid-mediated colistin resistance genes was performed. Whole-genome sequencing was performed using an Illumina NextSeq platform and the genome was assembled using CLC Genomics Workbench 7.5.1. Results: A singlemcr-1-positive MDR E. coli strain was isolated from beef sausages. The genome size of the E. coli strain was calculated at 5 044 715 bp, with a total of 226 contigs and a G + C content of 50.5%. The strain belonged to ST101 (phylogroup B1). The mcr-1 gene was located on an IncI2-type self-conjugative plasmid of 64.6 kb in size. The strain showed a MDR phenotype, with a colistin MIC of 4 mg/L. A large number of acquired antimicrobial resistance genes was identified, including genes encoding resistance to colistin (mcr-1), β-lactams (blaTEM-1), phenicols (floR), trimethoprim (dfrA12), aminoglycosides [aac(3)-IIa, aph(3”)-Ib and aadA2], macrolides (mphA and mdfA), tetracyclines (tetA), sulfonamides (sul1 and sul2) and quinolones (qnrS1). Conclusion: Here we report the first draft genome sequence of anmcr-1/IncI2-carrying MDR E. coli B1:ST101 isolated from beef sausage in Egypt. This study highlights the potential role played by food products in the spread of colistin resistance to humans.

The emergence and spread of multidrug-resistant Salmonella enterica (S. enterica) to humans through food of animal origin are considered a major global public health concern. Currently, little is known about the prevalence of important antimicrobial resistance genes in S. enterica from retail food in Africa. Therefore, the screening and characterization of the extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes in S. enterica isolated from retail meats and slaughterhouses in Egypt were done by using PCR and DNA sequencing techniques. Twenty-eight out of thirty-four (82.4%) non-duplicate S. enterica isolates showed multidrug-resistance phenotypes to at least three classes of antimicrobials, and fourteen (41.2%) exhibited an ESBL-resistance phenotype and harbored at least one ESBL-encoding gene. The identified β-lactamase-encoding genes included blaCTX-M-1, blaCTX-M-3, blaCTX-M-13, blaCTX-M-14, blaCTX-M-15, and blaSHV-12 (ESBL types); blaCMY-2 (AmpC type); and blaTEM-1 and blaOXA-1 (narrow-spectrum types). PMQR genes (included qnrA, qnrB, qnrS, and aac(6′)-Ib-cr) were identified in 23 (67.6%) isolates. The presence of ESBL- and PMQR-producing S. enterica with a high prevalence rate in retail meats and slaughterhouses is considered a major threat to public health as these strains with resistance genes could be transmitted to humans through the food chain.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

We describe here the complete genome sequence of an Enterobacter hormaechei ST279 coharbouring blaVIM-1 and mcr-9 recovered from uncooked beef patty in June 2017, Egypt. The tested isolate was resistant to carbapenem but susceptible to colistin (minimum inhibitory concentration (MIC), 0.5 μg/mL). The antimicrobial susceptibility profile and conjugation experiments were performed. The entire genome was sequenced by the Illumina MiniSeq and Oxford Nanopore methods. The blaVIM-1 and mcr-9 genes are carried on the same IncHI2/pMLST1 plasmid, pMS37a (Size of 270.9 kb). The mcr-9 gene was located within the physical boundaries demarcated by two insertion elements IS903 (upstream) and IS1 (downstream) but did not possess the downstream regulatory genes (qseC/qseB) which regulate the expression of mcr-9. Therefore, the mcr-9 might be silently disseminated among carbapenem-resistant Enterobacterales. In addition to blaVIM-1 and mcr-9, plasmid pMS37a harbored various antibiotic resistance genes including aac(6’)-Il, ΔaadA22, aac(6’)-Ib-cr, sul1, dfrA1 and tetA. To the best of our knowledge, this is the first report of a blaVIM-1 and mcr-9-coharbouring E. hormaechei isolate of food origin worldwide. The identification of a multidrug-resistant VIM-1 and mcr-9 positive Enterobacter hormaechei isolate from food is worrisome as retail meat and meat products could serve as a vehicle for these MDR bacteria, which could be transferred between animals and humans through the food chain. It further highlights that Enterobacterales co-producing MCR and carbapenemases being found in the food chain indeed correspond to a One-Health issue, highlighting the need for serious steps to prevent their further dissemination.

This study describes the first full genomic sequence of an mcr-9 and blaVIM-4-carrying multidrug-resistant Enterobacter hormaechei clinical isolate from Egypt. The strain was isolated in April 2015 from the sputum of a patient in Cairo, Egypt. The mcr-9 and blaVIM-4 genes were identified by PCR screening and DNA sequencing; the isolate was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole genomic sequencing. mcr-9 and blaVIM-4 were carried by an IncHI2 plasmid, pAMS-38a (281,121 bp in size); the plasmid also carried genes conferring resistance against sulfonamides (sul1), quinolones (qnrA1), trimethoprim (dfrA1), β-lactams (blaTEM-1B), aminoglycosides (aac (6’)-II, aadA23, aadA2b, and ant(2’’)-Ia). The strain was susceptible to colistin (MIC, qseC/qseB regulatory system located downstream of mcr-9 in Enterobacterales, which is involved in the induction of colistin-resistance. The genetic context of mcr-9 and blaVIM-4 was identified as IS1-mcr-9-IS903-pcoS-∆pcoE-rcnA and intI1-blaVIM-4—aac (6’)-II-dfrA1-∆aadA23-smr-ISPa21-qacE∆1, respectively. This is the first report of an mcr-9 and blaVIM-4 /IncHI2-carrying multidrug-resistant E. hormaechei clinical isolate from Africa and the Middle East. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Further screening for mcr-9 is essential for identifying its incidence and to prevent its dissemination.

The first occurrence of New Delhi metallo-β-lactamase 5 (NDM-5), carried on an IncI1-Iγ-type plasmid of >93 kb in a multidrug-resistant Escherichia coli strain in Kafr El-Sheikh, Egypt, is reported. The strain was isolated from a wound pus swab from a patient diagnosed with a fracture of the right femur. This E. coli strain was found to belong to sequence type (ST) 5018 and also to carry other resistance genes, including blaCTX-M-15, blaCMY-42, blaOXA-1, and aac(6′)-Ib-cr.

If highly precise elucidation of the blood flow characteristics in a carotid bifurcation was possible, it would be widely applicable to diagnosis of circulatory diseases such as arteriosclerosis and cerebrovascular disease. This study was conducted to establish a new flow-dividing ratio estimation method applicable to an unsteady flow on a two-dimensional ultrasonic-measurement-integrated simulation of a carotid artery bifurcation for which it has been previously difficult to obtain a stable solution. In this new method, the flow-dividing ratio was directly adjusted by specifying the flow rate in a branch so that the difference of the Doppler velocities in the external carotid artery was decreased. The effectiveness of the proposed method was confirmed by a numerical experiment using the actual shape of a carotid artery bifurcation, and the superiority of the two-dimensional ultrasonic-measurement-integrated simulation over the ordinary simulation in terms of the reproducibility of the blood flow structure was clarified by analysis using clinical ultrasound data.