Contribution of different class 2 integron elements to fitness costs in multi-drug resistant Escherichia coli and evaluation of their adaptability in "farm-to-table" environments.

Integrons play a pivotal role in the dissemination of antimicrobial resistance, because they can capture and express exogenous antimicrobial resistance genes. This study aimed to elucidate the structure and contribution of different elements of class 2 integrons to fitness costs in their host bacteria and evaluate their adaptability to the "farm-to-table" process. We mapped 27 typical class 2 integrons of Escherichia coli isolated from aquatic foods and pork products, each harboring an inactive truncated class 2 integrase gene and the gene cassette (GC) array dfrA1-sat2-aadA1 with strong Pc2A/Pc2B promoters. Notably, the fitness costs associated with class 2 integrons depended on the Pc promoter strength and quantity and content of GCs in the array. Additionally, the costs of integrases were activity-dependent, and a balance was identified between GC capture ability and integron stability, which could explain the inactive truncated integrase identified. Although typical class 2 integrons exhibited low-cost structures in E. coli, the bacteria incurred biological costs, including decreasing growth rates and biofilm formation, in farm-to-table environments, especially under low-nutrient conditions. Nevertheless, sub-inhibitory antibiotic concentrations led to the selection of class 2 integron-carrying bacteria. This study provides important insights into how integrons may travel from preharvest to consumer goods.

Fitness cost and compensation mechanism of sulfonamide resistance genes (sul1, sul2, and sul3) in Escherichia coli.

The fitness cost of antibiotic resistance is a crucial factor to determine the evolutionary and transmission success of resistant bacteria. Exploring the fitness cost and compensation mechanism of antibiotic resistance genes (ARGs) in bacteria may effectively reduce the transmission of drug-resistant genes in the environment. Engineered bacteria with the same genetic background that carry sulfonamide resistance gene were generated to explore the fitness cost of sulfonamide resistance gene in Escherichia coli. There were significant differences in the protein expression of the two-component system pathway (fliZ, fliA, fliC and lrhA), folate biosynthesis pathway (sul1, sul2 and sul3), ABC transporter system (ugpC, rbsA and gsiA), and outer membrane pore protein OmpD through the comparative analysis of differential proteins compared to sensitive bacteria. Thus, we could speculate the possible fitness compensation mechanism. Finally, quantitative Real-time PCR (qRT-PCR) was used to verify the functions of some differential proteins at the transcriptional level. The fitness cost and compensatory evolution of antibiotic resistance are an essential part of bacterial evolution.

A simple and efficient method for potential point-of-care diagnosis of human papillomavirus genotypes: combination of isothermal recombinase polymerase amplification with lateral flow dipstick and reverse dot blot.

Cervical cancer is the second most common cancer in the world's woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1 h with a sensitivity of low to 100 fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.

Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

BACKGROUND: Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. METHODS: A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. RESULTS: The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. CONCLUSIONS: It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA.

BACKGROUND: Cervical cancer is a common gynecologic malignant tumor and has a great impact on women's health. Human papillomavirus (HPV) is implicated in cervical cancer and precancerous lesions and the two are possibly two stages of disease progression. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important means to prevent cervical cancer. METHODS: Here we present a novel, rapid, sensitive and specific isothermal method of recombinase polymerase amplification (RPA), which is established to detect the two most common high-risk human papillomavirus type 16 and type 18 DNA. In this study, we evaluate the efficacy of the RPA assay, incubating clinical specimens of HPV16 and HPV18 using plasmids standard. It operates at constant low temperature without the thermal instrumentation for incubation. The products can be detected via agarose gel electrophoresis assay, reverse dot blot assay, and quantitative real-time assay with SYBR Green I. We assess the diagnostic performance of the RPA assay for detecting of HPV16 and HPV18 in 335 clinical samples from patients suspected of cervical cancer. RESULTS: The results revealed no cross-reaction with other HPV genotypes and the RPA assay achieve a sensitivity of 100 copies. Compared with TaqMan qPCR, the RPA technique achieves exponential amplification with no need for pretreatment of sample DNA at 37°C for 20 minutes, which reveals more satisfactory performance. The agreement between the RPA and qPCR assays was 97.6% (κ = 0.89) for HPV16 positivity and 98.5% (κ = 0.81) for HPV18 positivity, indicating very good correlation between both tests. CONCLUSIONS: Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.

Rapid on-site nucleic acid testing: On-chip sample preparation, amplification, and detection, and their integration into all-in-one systems.

As nucleic acid testing is playing a vital role in increasingly many research fields, the need for rapid on-site testing methods is also increasing. The test procedure often consists of three steps: Sample preparation, amplification, and detection. This review covers recent advances in on-chip methods for each of these three steps and explains the principles underlying related methods. The sample preparation process is further divided into cell lysis and nucleic acid purification, and methods for the integration of these two steps on a single chip are discussed. Under amplification, on-chip studies based on PCR and isothermal amplification are covered. Three isothermal amplification methods reported to have good resistance to PCR inhibitors are selected for discussion due to their potential for use in direct amplification. Chip designs and novel strategies employed to achieve rapid extraction/amplification with satisfactory efficiency are discussed. Four detection methods providing rapid responses (fluorescent, optical, and electrochemical detection methods, plus lateral flow assay) are evaluated for their potential in rapid on-site detection. In the final section, we discuss strategies to improve the speed of the entire procedure and to integrate all three steps onto a single chip; we also comment on recent advances, and on obstacles to reducing the cost of chip manufacture and achieving mass production. We conclude that future trends will focus on effective nucleic acid extraction via combined methods and direct amplification via isothermal methods.

Molecular insights into how a deficiency of amylose affects carbon allocation--carbohydrate and oil analyses and gene expression profiling in the seeds of a rice waxy mutant.

BACKGROUND: Understanding carbon partitioning in cereal seeds is of critical importance to develop cereal crops with enhanced starch yields for food security and for producing specified end-products high in amylose, ß-glucan, or fructan, such as functional foods or oils for biofuel applications. Waxy mutants of cereals have a high content of amylopectin and have been well characterized. However, the allocation of carbon to other components, such as ß-glucan and oils, and the regulation of the altered carbon distribution to amylopectin in a waxy mutant are poorly understood. In this study, we used a rice mutant, GM077, with a low content of amylose to gain molecular insight into how a deficiency of amylose affects carbon allocation to other end products and to amylopectin. We used carbohydrate analysis, subtractive cDNA libraries, and qPCR to identify candidate genes potentially responsible for the changes in carbon allocation in GM077 seeds. RESULTS: Carbohydrate analysis indicated that the content of amylose in GM077 seeds was significantly reduced, while that of amylopectin significantly rose as compared to the wild type BP034. The content of glucose, sucrose, total starch, cell-wall polysaccharides and oil were only slightly affected in the mutant as compared to the wild type. Suppression subtractive hybridization (SSH) experiments generated 116 unigenes in the mutant on the wild-type background. Among the 116 unigenes, three, AGP, ISA1 and SUSIBA2-like, were found to be directly involved in amylopectin synthesis, indicating their possible roles in redirecting carbon flux from amylose to amylopectin. A bioinformatics analysis of the putative SUSIBA2-like binding elements in the promoter regions of the upregulated genes indicated that the SUSIBA2-like transcription factor may be instrumental in promoting the carbon reallocation from amylose to amylopectin. CONCLUSION: Analyses of carbohydrate and oil fractions and gene expression profiling on a global scale in the rice waxy mutant GM077 revealed several candidate genes implicated in the carbon reallocation response to an amylose deficiency, including genes encoding AGPase and SUSIBA2-like. We believe that AGP and SUSIBA2 are two promising targets for classical breeding and/or transgenic plant improvement to control the carbon flux between starch and other components in cereal seeds.

Development of a loop-mediated isothermal amplification assay for detection of Cronobacter spp. (Enterobacter sakazakii).

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/µL (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/µL, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula.

Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick.

The worrying emergence of multiple resistance genes to last-resort antibiotics in food animals and human populations throughout the food chain and relevant environments has been increasingly reported worldwide. Enterobacteriaceae pathogens are considered the most common reservoirs of such antibiotic resistance genes (ARGs). Thus, a rapid, efficient and accurate detection method to simultaneously screen and monitor such ARGs in Enterobacteriaceae pathogens has become an urgent need. Our study developed a recombinase polymerase amplification (RPA) assay combined with a lateral flow dipstick (LFD) for simultaneously detecting predominant resistance genes to last-resort antibiotics of Enterobacteriaceae pathogens, including mcr-1, blaNDM-1 and tet(X4). It is allowed to complete the entire process, including crude DNA extraction, amplification as well as reading, within 40 min at 37°C, and the detection limit is 101 copies/µl for mcr-1, blaNDM-1 and tet(X4). Sensitivity analysis showed obvious association of color signals with the template concentrations of mcr-1, blaNDM-1 and tet(X4) genes in Enterobacteriaceae pathogens using a test strip reader (R 2 = 0.9881, R 2 = 0.9745, and R 2 = 0.9807, respectively), allowing for quantitative detection using multiplex RPA-LFD assays. Therefore, the RPA-LFD assay can suitably help to detect multiple resistance genes to last-resort antibiotics in foodborne pathogens and has potential applications in the field.

Draft genome of a multidrug and multi-heavy metal resistant Vibrio parahaemolyticus ST165 strain of Penaeus vannamei from seawater farms in Zhejiang, China.

OBJECTIVES: Vibrio parahaemolyticus ST165 strain (FB-11) was first isolated in 2019 in China from Penaeus vannamei from seawater farms. The strain includes multidrug resistant (MDR) and multi-heavy metal resistant (MHMR) phenotypes. In this study, we aimed to determine the draft genome sequence of strain FB-11 and analyze the genetic features with a special focus on MDR and MHMR genes. METHODS: The genomic DNA was sequenced using Nanopore PromethION and the Illumina Novaseq6000 platform, and the reads were de novo assembled into contigs using Unicycler. Genome function elements were predicted, the coding sequences were annotated, and whole-genome sequencing (WGS) analysis was performed. RESULTS: WGS analysis revealed that the genome comprised two chromosomes of 3,328,286 bp (GC content, 45.37%) and 1,805,825 bp (GC content, 45.36%). It harbored 26 important drug resistant genes and six important heavy metal resistant genes; all of these were located on the chromosomes. Multilocus sequence type of the strain was ST165. CONCLUSIONS: This is the first report of the complete genome sequence of a V. parahaemolyticus ST165 strain isolated from P. vannamei in China. This genome sequence provides useful information on the genomic features associated with antimicrobial and heavy metal resistance in V. parahaemolyticus ST165.

Occurrence of Virulence Factors and Antibiotic and Heavy Metal Resistance in Vibrio parahaemolyticus Isolated from Pacific Mackerel at Markets in Zhejiang, China.

ABSTRACT: Vibrio parahaemolyticus is a widespread bacterium in the marine environment and is one of the leading causes of food-derived bacterial poisoning in humans worldwide. The main objective of this study was to determine the prevalence, virulence factors, and antibiotic and heavy metal resistance profiles of V. parahaemolyticus in Pacific mackerel (Pneumatophorus japonicus) from different markets in Zhejiang Province, People's Republic of China. In total, 112 (31.11%) V. parahaemolyticus isolates were identified from 360 Pacific mackerel samples, with an extremely low occurrence of the virulence genes trh (1.79%, 2 of 112) and tdh (0%, 0 of 112). Antibiotic resistance testing revealed that most isolates showed resistance to ampicillin (96.43%, 108 of 112) and streptomycin (90.18%, 101 of 112), whereas all strains were sensitive to kanamycin, florfenicol, cefamandole, and trimethoprim-sulfamethoxazole. Furthermore, 46.43% (52 of 112) of isolates, which had 12 different phenotypes, were classified as multidrug resistant. In addition, the multiple antibiotic resistance index values of isolates were between 0.05 and 0.63, and the maximum multiple antibiotic resistance index was attributed to two isolates that exhibited resistance to 12 antibiotics. Heavy metal resistance patterns were similar among the six different markets. The majority of isolates showed resistance to Cd2+ (78.57%) and Pb2+ (51.79%), and fewer were resistant to Cu2+ (37.50%), Zn2+ (25.00%), Co2+ (9.82%), Ni2+ (6.25%), and Mn2+ (4.46%). No isolates were resistant to Cr3+. In total, 22.32% (25 of 112) of strains were multiheavy metal resistant. Furthermore, multidrug resistance and multiheavy metal resistance were found to be positively correlated in the V. parahaemolyticus strains by using Pearson's correlation analysis (P = 0.008; R = 0.925). This information will contribute to the monitoring of variations in the antibiotic and heavy metal resistance profiles of V. parahaemolyticus strains from seafood and provide insight into the appropriate use of antibiotics and the safe consumption of seafood.

Corrigendum: Diverse Mobile Genetic Elements and Conjugal Transferability of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Escherichia coli Isolates From Penaeus vannamei and Pork From Large Markets in Zhejiang, China.

[This corrects the article DOI: 10.3389/fmicb.2019.01787.].

Co-occurrence of Antibiotic and Heavy Metal Resistance and Sequence Type Diversity of Vibrio parahaemolyticus Isolated From Penaeus vannamei at Freshwater Farms, Seawater Farms, and Markets in Zhejiang Province, China.

Vibrio parahaemolyticus is the leading cause of seafood-borne bacterial poisoning in China and is a threat to human health worldwide. The aim of this study was to assess the antibiotic resistance profiles and distribution of heavy metal resistance of V. parahaemolyticus isolates from Penaeus vannamei from freshwater farms, seawater farms, and their corresponding markets in Zhejiang, China and to assess the relationship between multidrug resistance (MDR) and multi-heavy metal resistance (MHMR). Of the 360 P. vannamei samples that we tested, 90 (25.00%) were V. parahaemolyticus positive, but the occurrence of pathogenic isolates carrying the toxin genes tdh (4.44%) and trh (3.33%) was low. None of the tested isolates harbored both the tdh and trh genes. However, antibiotic resistance profiles varied among different sampling locations, levels of resistance to the antibiotics ampicillin (76.67%) and streptomycin (74.44%) were high overall, and MDR isolates were common (40.00% of all isolates). Heavy metal resistance patterns were similar among the different sampling locations. Overall, the majority of V. parahaemolyticus isolates displayed tolerance to Cd2+ (60.00%), and fewer were resistant to Cu2+ (40.00%), Zn2+ (38.89%), Ni2+ (24.44%), Cr3+ (14.44%), and Co2+ (8.89%). In addition, 34.44% (31/90) of isolates tested in this study were found to be MHMR. Using Pearson's correlation analysis, MDR and MHMR were found to be positively correlated (P = 0.004; R = 0.759). The 18 V. parahaemolyticus isolates that were both MDR and MHMR represented 18 sequence types, of which 12 were novel to the PubMLST database, and displayed a high level of genetic diversity, suggesting that dissemination may be affected by mobile genetic elements via horizontal gene transfer. However, a low percentage of class 1 integrons without gene cassettes and no class 2 or 3 integrons were detected in the 18 MDR and MHMR isolates or in the 90 V. parahaemolyticus isolates overall. Thus, we suggest that future research focus on elucidating the mechanisms that lead to a high prevalence of resistance determinants in V. parahaemolyticus. The results of this study provide data that will support aquatic animal health management and food safety risk assessments in the aquaculture industry.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine.

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC(50)) of the test strip under an optical density scanner were calculated to be 0.1 +/- 0.01 ng mL(-1) and 0.1 +/- 0.01 ng mL(-1), 0.56 +/- 0.08 ng mL(-1), and 0.71 +/- 0.06 ng mL(-1), respectively, the cut-off levels with the naked eye of 1 ng mL(-1) and 1 ng mL(-1) for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.

Employing DNA binding dye to improve detection of Enterocytozoon hepatopenaei in real-time LAMP.

Enterocytozoon hepatopenaei (EHP) is a pathogen in the pancreatic tissue of prawn, as reported in recent years. Enterosporidiosis caused by EHP in Penaeus genus is spreading widely, which seriously threatens the sustainable development of shrimp aquaculture in the world. Empolying the DNA binding dye of SYTO-16, a real-time quantitative loop-mediated isothermal amplification (LAMP) method has been established herein to detect EHP. The primer sequences used in the LAMP reaction were according to the SSU rRNA gene. The LAMP assay has reached a sensitivity of 101 copies/µL and no cross-reaction with other aquatic pathogens. Compared with normal PCR, the efficiency of the LAMP technique is more sensitive, which has a shorter detection time. The use of fluorescent nucleic acid dye (SYTO-16) reveals a more satisfactory performance relative to calcein. The quantitative LAMP assay can be considered as a valid tool for rapid detection of microsporidian in prawns. Our study provides a scientific basis to follow the effect of the pathogen infection on growth of cultured penaeid shrimp.

Antibiotic Resistance and Characteristics of Integrons in Escherichia coli Isolated from Penaeus vannamei at a Freshwater Shrimp Farm in Zhejiang Province, China.

Our study was conducted to investigate the antibiotic susceptibility profiles, integrons and their associated gene cassettes (GCs), and insertion sequence common regions of Escherichia coli isolates from Penaeus vannamei collected at a large-scale freshwater shrimp farm in Zhejiang Province, People's Republic of China. A total of 182 E. coli isolates were identified from 200 samples. With the exception of imipenem, isolates were most commonly resistant to ß-lactams, followed by tetracylines and sulfonamides. Fifty-two (28.6%) E. coli isolates were classified as multidrug resistant, and the patterns were highly diverse, with 29 types represented. The multiple-antibiotic resistance indices of the isolates were 0.17 to 0.56; 9.3% (17) of the 182 isolates were positive for class 1 integrons, 0.5% (1 isolate) was positive for class 2 integrons, and an insertion sequence common region 1 element was found upstream of the intI1 (integrase) gene in one of the intI1-positive isolates. Four GC arrays were detected in class 1 integrons, and one GC array was detected in class 2 integrons. Although the overall prevalence of antimicrobial-resistant bacteria in P. vannamei was lower than that previously reported for poultry and livestock farms in China, concerns about the inappropriate use of antibiotics and the transmission of antimicrobial-resistant bacteria in aquaculture were raised. Alternative approaches to reducing or replacing the use of antibiotics should be further studied.

Diverse Mobile Genetic Elements and Conjugal Transferability of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Escherichia coli Isolates From Penaeus vannamei and Pork From Large Markets in Zhejiang, China.

High prevalence rates of sulfonamide resistance genes sul1, sul2, and sul3 have been observed in Gram-negative bacteria isolated from humans, domestic animals, and aquaculture species worldwide. We investigated the distribution characteristics, location, conjugative transferability, and genetic environments of sul genes from Escherichia coli isolates collected from Penaeus vannamei and pork samples from three large markets in Zhejiang, China. The prevalence rates of sul genes in sulfonamide-resistant E. coli isolates from P. vannamei and pork samples were 90.0 and 88.6%, respectively, and the prevalence of sul1 and sul2 was significantly higher than that of sul3 (p < 0.05). Twenty-four representative sul-positive E. coli isolates were analyzed in detail. Southern blot hybridization confirmed that sul genes of E. coli isolates were located on plasmids and/or chromosomes. Transfer of resistance through conjugation was observed in all 18 E. coli isolates harboring sul genes on plasmids. Replicon typing identified seven different incompatibility groups and IncF was the dominant replicon type among sul gene-containing plasmids from both sources. PCR walking analysis indicated that 87.5% (35/40) of sul gene-related fragments carried insertion sequences (ISs) belonging to a variety of families in diverse sites, with IS26 occurring most frequently. In addition, the sul1 gene was detected mainly in fragments carrying class 1 integrons. Co-location on the same fragment with resistance genes that may contribute to the persistence and dissemination of sul1 and/or sul2 genes. The diversity of mobile genetic elements and resistance genes adjacent to sul3 was much lower than those adjacent to sul1 and sul2, especially those located in chromosomes, which reduced the transmission potential of the sul3 gene. In conclusion, combined with the results of clonal relatedness analysis by PFGE and MLST of 24 representative E. coli isolates from P. vannamei and pork samples, it showed that a small number of sul genes were vertically transmitted among E. coli from P. vannamei and that horizontal gene transfer was likely the main transmission mechanism of sul genes from both sources. Our results provide important information to better understand the risk of transmission of sul genes from seafood and meat to humans.

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food.

Salmonella can cause serious foodborne diseases. We have developed a lateral flow immunoassay combined with recombinase polymerase amplification (LFD-RPA) for detection of Salmonella in food. The conserved fragment (fimY) was selected as the target gene. Under an optimal condition (37 °C, 10 min), the sensitivity was 12 colony-forming units (CFU)/mL in a pure culture. Testing with 16 non-Salmonella strains as controls revealed that LFD-RPA was specific to the fimY gene of Salmonella. The established assay could detect Salmonella at concentrations as low as 1.29 × 102 CFU/mL in artificially contaminated samples. This detection was at a slightly higher level than that for a pure bacterial culture. Combined with the test strip reader, the LFD-RPA is a feasible method for quantitative detection of Salmonella based on the test line intensity, which was the ratio for the test line and control line of the reflected light. The method could be a potential point-of-care test in limited resource areas and provides a new approach and technical support for the diagnosis of food safety.

Strategies for screening, purification and characterization of bacteriocins.

Bacteriocins are bioactive antimicrobial peptides synthesized in the ribosome of numerous bacteria and released extracellularly. Bacteriocins have the ability to kill or inhibit the growth of prokaryotes and could potentially be useful against pathogens and antibiotic-resistant strains of bacteria. The antimicrobial mechanisms and relatively narrow killing spectrums of bacteriocins distinguish them from traditional broad-spectrum antibiotics, making them possible candidates to replace antibiotics in the future. In recent years, researchers have discovered many new bacteriocins from different sources. However, the discovery of new bacteriocins involves complicated screening, identification, purification, and characterization processes. This review describes the strategies currently employed for screening, identification, purification, and characterization of bacteriocins. Approximately 40 novel bacteriocins are reviewed in this paper. Our conclusions about the research process will guide the development of novel methods and allow for the faster, easier, and more efficient discovery of novel bacteriocins.

Purification, Characterization, and Mode of Action of Pentocin JL-1, a Novel Bacteriocin Isolated from Lactobacillus pentosus, against Drug-Resistant Staphylococcus aureus.

Staphylococcus aureus and its drug-resistant strains, which threaten public health and food safety, are in need of effective control by biopreservatives. A novel bacteriocin, pentocin JL-1, produced by Lactobacillus pentosus that was isolated from the intestinal tract of Chiloscyllium punctatum, was purified by a four-step chromatographic process. Mass spectrometry based on MALDI-TOF indicated that pentocin JL-1 has a molecular mass of 2987.23 Da. Only six of the twenty-five amino acids could be identified by Edman degradation. This bacteriocin is thermostable and tolerates a pH range of 5-7. Also, it is sensitive to proteinase K, trypsin, pepsin, and alkaline protease. This bacteriocin has a broad inhibitory spectrum against both Gram-positive and Gram-negative strains and in particular is effective against multidrug-resistant S. aureus. Additionally, we showed that the cell membrane is the target of pentocin JL-1 against methicillin-resistant S. aureus (MRSA), causing a loss of proton motive force. Furthermore, pentocin JL-1 has a drastic impact on the structure and integrity of MRSA cells. These results suggest that pentocin JL-1 has potential as a biopreservative in the food industry.