RESUMO
The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/µL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and -20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.
RESUMO
The emergence of extended-spectrum ß-lactamase-producing Salmonella enterica serovar Enteritidis (ESBL-SE) in humans and foods has gained global attention. In particular, CTX-M-type ESBL-SE are increasingly being detected from various sample types. The aim of this study was to comprehensively analyze the epidemiology and characteristics of blaCTX-M-55-carrying ESBL-SE isolates of clinical origin in Shanghai, China. A total of 292 S. Enteritidis isolates were recovered from the feces and blood of outpatients and inpatients between 2006 and 2014. Overall, there was a high frequency of cefotaxime-resistant isolates (97.3%), which was significantly higher (p < 0.01) than that of isolates resistant to the other tested antibiotics. All S. Enteritidis isolates exhibited resistance to ≥1 antibiotic, and 98.0% were multidrug resistant. A total of 233 isolates were identified as ESBL-SE, 166 of which were CTX-M type. Six subtypes of CTX-M-encoding genes were detected, among which blaCTX-M-55 (91.6%, 152/166) was the most prevalent genotype. There was high genetic similarity among blaCTX-M-55-positive ESBL-SE. The blaCTX-M-55 gene in the ESBL-SE donor strains could be easily transferred into Enterobacteriaceae recipient strains. This study highlights that CTX-M-55 should be considered an important surveillance target in Shanghai, China. Cephalosporins, especially cefotaxime, must be used with caution in empirical treatment for Salmonella infections.
RESUMO
Poultry products such as eggs provide essential nutrients to the human body and thus play vital roles in the human food network. Salmonella is one of the most notorious foodborne pathogens and has been found to be prevalent in eggs. To better understand the characteristics of Salmonella in eggs, we investigated the prevalence of Salmonella spp. in 814 fresh eggs collected from poultry farms and retail marketplaces in Yangling, Shaanxi Province, China. The serotype, genotype, and antibiotic susceptibilities of 61 Salmonella isolates recovered from the eggs were analyzed. The average detection rate of Salmonella-positive eggs was 5.6%, with 6.6% of the eggs collected from poultry farms and 5.1% from marketplaces. Thirteen serotypes were identified from the 61 isolates, among which Salmonella Typhimurium (24.5%) and Salmonella Indiana (22.9%) were the most prevalent serotypes. Other dominant serotypes included Salmonella Thompson (13.1%) and Salmonella Enteritidis (11.4%), with the remaining nine serotypes detected at low rates (1.6-4.9%). All the Salmonella isolates tested were resistant to sulfisoxazole (100.0%). The majority (77.1%) of the isolates were resistant to nalidixic acid, amoxicillin-clavulanate, and ampicillin, while nearly two-thirds (63.9-68.9%) were resistant to trimethoprim-sulfamethoxazole, kanamycin, tetracyclines, and chloramphenicol. The rate of resistance to ciprofloxacin was 40.1%; the resistance rates to streptomycin, ceftiofur, and ceftriaxone ranged from 21.3 to 26.2%; and those to gentamicin, amikacin, and cefoxitin were relatively low (3.3-16.4%). Forty-nine (80.3%) Salmonella isolates exhibited resistance to multiple antibiotics, 20 (32.8%) of which were resistant to at least 10 antibiotics. Subtyping by pulse-field gel electrophoresis revealed a close genetic relatedness of Salmonella isolates from poultry farms, in striking contrast to the high diversity of the isolates obtained from marketplaces. Isolates of the same serotype always shared identical genotype and antibiotic resistance profiles, even the ones that were recovered from eggs sampled at different locations and times. These findings indicate that diverse Salmonella spp. with high rates of multidrug resistance are prevalent in fresh eggs in the study area. More attention should be paid to egg production, transportation, and storage to prevent foodborne outbreaks caused by Salmonella.
