Sample collection and bacterial isolation
During April to July 2018, a total number of 134 samples, containing 37 cooked meat, 53 raw meat, 26 environmental samples and 18 human samples, were collected from 24 markets in four districts of Beijing (Table 1). Among 674 isolates, 172 (composed by 26 different genera) were isolated from cooked meat, 330 (26 different genera) were recovered from raw meat, 91 (22 different genera) from environment samples and 81 (15 different genera) from human samples.
Difference in bacterial composition of different samples
Samples from different sources show differences in bacterial composition. For example, the major bacterial species in cooked meat, raw meat, environmental samples and human samples are Proteus (41/172, 23.84% of the total isolated strains in cooked meat), Proteus (74/330, 22.42%), Macrococcus (18/91, 19.78%) and Staphylococcus (26/81, 32.10%), respectively. The ten most diverse bacteria in different samples are shown in supplementary Figure S1. On the other hand, there are several genus, such as Aeromonas, Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Macrococcus, Proteus and Staphylococcus are common in different samples (Figure 2A).
According to the 2013 CDC report on antibiotic resistance threats and the 2017 WHO list of multidrug-resistant bacteria [3, 27], we select 220 isolates, which belonging to the following genus Bacillus, Enterococcus, Staphylococcus, Acinetobacter, Aeromonas, Escherichia and Klebsiella, as the potential foodborne pathogens for further studies. The major pathogenic bacteria are Klebsiella and Escherichia in the meat samples. However, Staphylococcus shows the main threat in environmental and human-derived samples (Figure 2B).
Antimicrobial susceptibility tests of isolated potential pathogenetic strains
To understand the profiles of antimicrobial resistance in the 220 selected strains, all isolates were tested with the antimicrobial which were frequently used in clinical treatment. Generally, isolates mainly show resistance to amoxicillin + clavulanate, tetracycline and erythromycin with the rate of 46.78% (103/220), 44.66% (98/220), 32.73% (72/220), respectively (Table 2). It is noteworthy that Acinetobacter, Escherichia and Klebsiella isolates show resistance to ceftazidime with the rate of 37.50% (3/8), 5.26% (2/38), 9.09% (5/55), respectively. 12.50% (1/8) of the Acinetobacter isolates, 5.56% (2/36) of Aeromona isolates, 2.63% (1/38) of Escherichia isolates and 9.09% (5/55) of Klebsiella isolates are resistant to polymyxin B. None of the isolates show resistance to meropenem and vancomycin. Additionally, according to our results, the Gram-positive strains are more resistant to broad-spectrum antimicrobials (P value = 0.02), and the isolates from human samples shows higher resistance rate (P value < 0.01), as indicated in Table 2.
Antimicrobial resistance genes and their mobility
According to the results of antimicrobial susceptibility tests, Acinetobacter, Aeromonas, Escherichia and Klebsiella isolates showing resistance to ceftazidime and polymyxin B, were subjected to whole genomic sequencing, and their whole genomic sequences were searched against ResFinder to screen ARGs. 40.00% (2/5) of the analyzed Klebsiella isolates carry ESBL genes, such as blaOKP-B-10, blaSHV28, blaCTX-M-15 and blaTEM-1B. In the genomes of Acinetobacter isolates, the specific ESBL genes blaOXA-130 and blaOXA-51 were detected with rate of 66.67% (2/3). Interestingly, the presence of mcr-7.1 like gene, leading the resistance to polymyxin B, was found in two Aeromonas hydrophila strains (MIC value = 128 μg/mL), both isolated from raw meat. And their sequence identity with the mcr-7.1 gene (GenBank accession number: MG267386.1) is 81.34% and 77.24%, respectively (Table S1).
We screened MGEs and annotated the flanking genetic contexts of the selected ARGs to predict their mobility. According to the results, two K. pneumoniae isolates harbor the ESBL genes which are located on mobile elements, as shown in Figure 3. The K. pneumoniae CNN2-10 is isolated from raw meat. It carries the transposon Tn2 harboring blaCTX-M-15 and blaTEM-1B, and transposon Tn2003 harboring blaSHV-28. And the K. pneumoniae CYXC3-2 from cooked meat carries the transposon Tn2003, which harboring the blaOXA-B-10 gene. The genetic constitutions of the two transposons are siminar with former reports [28, 29].
Prevalence of virulence factors
In our study, bacterial genus, such as Bacillus, Enterococcus, Staphylococcus, Escherichia and Klebsiella which reported showing various virulence factors, were also selected for whole genomic sequencing. The presence of toxin gene ces, encoding the synthesis of cereulide synthetase, were found in two B. cereus strains (2/6, 33.33%). Additionally, the virulence factors related to adhesion and invasion were identified in various bacterial species (Table 3). For instance, all the Acinetobacter isolates carry ompA, which is involved in bacterial adherence and invasion. In Escherichia isolates, the detection rates of adherence and invasion factors like fdeC, fimH, ibeC and ompA were 33.33% (1/3), 33.33% (1/3), 33.33% (1/3), 66.67% (2/3), respectively. The major adherence and invasion factors in Klebsiella isolates are wabG, fimH, and mrkD with the rate of 91.67% (11/12), 66.67% (8/12) and 58.33% (7/12) respectively.