A Preliminary Study of Antibiotic Resistance Genes in Domestic Honey Produced in China

Abstract

Antibiotic resistance genes (ARGs) are emerging contaminants that pose a health risk to humans worldwide. Little information on ARGs in bee honey is available. This study profiles ARGs in bee honey samples produced in China, the biggest producer in the world. Of 317 known ARGs encoding resistance to 8 classes of antibiotics, 212 were found in collected honey samples by a real-time quantitative polymerase chain reaction approach. Occurrence frequencies of genes providing resistance to FCA (fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol) and aminoglycosides were 21.0% and 18.5%, respectively. Frequencies of genes encoding efflux pumps were 42.5% and those of destructase genes 36.6%, indicating that these two mechanisms were predominant for resistance. Nine plasmid-mediated quinolone resistance genes were detected. Of the nine transposase genes known to be involved in antibiotic resistance, eight were found in the samples examined, with tnpA-4, tnpA-5, and tnpA-6 being more abundant. The abundance of the transposase genes was associated with genes conferring resistance to tetracyclines (r = 0.648, p < 0.01), macrolide–lincosamide–streptogramin B (r = 0.642, p < 0.01), FCA (r = 0.517, p < 0.01), and aminoglycosides (r = 0.401, 0.01 < p < 0.05). This is the first study on the abundance and diversity of ARGs in Chinese bee honey products. These findings suggest that bee honey may be a significant source of ARGs that might pose threat to public health. Further research is required to collect more samples in diverse geographic regions in China to make a more comprehensive judgment of ARG in bee honey.

Introduction

Antibiotic resistance is one of the most relevant global health challenges (www.who.int/glass/) (Mattar et al., 2020). To mitigate this problem, it is important to identify sources and dissemination routes of antibiotic resistance genes (ARGs) (Ashbolt et al., 2013). Because food is a reservoir of ARGs (Rolain, 2013), and food consumption is an important route of ARGs' transmission to humans (Holmes et al., 2016; Pan et al., 2018).

Bee honey is a natural substance produced by honey bees and has been used in many cosmetics, pharmaceuticals, and food products due to its nutritional and medicinal value (Kumar et al., 2020). The honey bees are important agricultural pollinators, the forager bees can visit an average of 1000 flowers within 1.5 km flight radius from the hive in 1 day to find pollen, nectar, propolis, honeydew, and water. At the same time, these bees can encounter a large number of pollutants in the air, soil, vegetation, and water especially within 7 km² surrounding their apiary (Fakhimzadeh and Lodenius, 2000).

Therefore, the honey bees can easily come into contact with bacterial carriers of antimicrobial resistance genes present in the environments during their foraging travels (Goretti et al., 2020). Cenci-Goga et al. (2020) reported that 24 of 35 (68.6%) sample bee bodies were positive for at least 1 antimicrobial resistance gene, with 2 samples positive for aph (5.7%), 8 for blaZ (22.9%), 3 for tetM (8.6%), 10 for sul1 (28.6%), and 18 for sul2 (51.4%). Long-term exposure to antibiotics has caused the accumulation of resistance determinants in the gut microbiota of honeybees and honey (Tian et al., 2012). In the USA, eight tetracycline resistance genes, including tetB, tetC, tetD, tetH, tetL, tetY, tetM, and tetW, were found at high frequencies in the gut bacteria of farmed honeybees (Tian et al., 2012), and streptomycin-resistant genes (strA–strB) were found to be prevalent in the honeybee gut symbiont, Snodgrassella alvi (Ludvigsen et al., 2018). Recently, 11 different ARGs were identified in the genomic sequences of 10 Gilliamella isolates from Chinese bumblebee gut (Zhang et al., 2021). In addition, Murray et al. (2007) discovered a natural tetracycline resistance plasmid called pMA67 containing a tetL gene from Paenibacillus larvae, a Gram-positive bacterial pathogen of honey bees. Lopez et al. (2008) reported that Bacillus cereus isolates from Argentinean honey samples contain a variety of tetracycline and oxytetracycline resistance genes, including tetK, tetL, tetM, and otrA.

According to FAOSTAT (www.fao.org/faostat/en/#home), a total of 1,850,868 t of honey was produced in 2018, with China (446,900 t) being the biggest producer. However, the abundance and diversity of ARGs in honey products produced in China has not been reported. This study aimed to find out the extent and characteristics of the prevalence of ARGs in Chinese honey. To detect ARGs in this study, we tried to purchase as many brands of honey as possible from Chinese supermarkets. Real-time quantitative polymerase chain reaction (qPCR) was employed with 327 validated primer sets previously used (Wang et al., 2014; Yan et al., 2017).

Materials and Methods

Sample source and pretreatment

Thirty-eight domestic honey samples (designated as H01–H38) produced in different provinces of China were purchased from supermarkets (Supplementary Table S1). Honey (25 mL) was thoroughly mixed with an equal volume of phosphate-buffer solution (PBS), centrifuged (5000 g, 40 min), and washed twice. The final pellet was resuspended in 1 mL PBS and used instantly or stored at −80°C.

DNA extraction

DNA was extracted from the honey samples using the MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0 (Cat #9763, Takara, Japan) according to the manufacturer's guidelines. DNA quality and concentration were determined using 1% agarose gel electrophoresis and a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific, Waltham, MA), respectively. The quality (OD₂₆₀/OD₂₈₀ ratio) and quantity (OD₂₆₀) of the DNA were measured using a NanoDrop 2000 Spectrophotometer. An OD₂₆₀/OD₂₈₀ ratio of ∼1.8 was considered good DNA quality for qPCR.

Real-time qPCR

A total of 327 validated primer sets (16S rDNA, 9 transposase genes, and 317 ARGs) were synthesized by Sangon Biotech, Inc. (Shanghai, China). The ARGs included those encoding resistance to aminoglycosides (38 genes), β-lactams (54 genes), FCA (fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol; 35 genes), MLSB (macrolide–lincosamide–streptogramin B; 53 genes), sulfonamides (6 genes), tetracyclines (45 genes), vancomycin (31 genes), and others (55 genes) (Supplementary Table S2) (Wang et al., 2014; Yan et al., 2017). Real-time qPCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). All qPCR assays were run in 96-well plates with a total of 20 μL mixtures per well containing 6 μL ddH₂O, 10 μL TB Green Premix Ex Taq II (Tli RNaseH Plus; 2 × ), 1.6 μL forward and reverse primers (10 μM each), 0.4 μL ROX Reference Dye II (50 × ), and 2 μL template DNA (negative control contained 2 μL DNA-free water, instead). Each DNA sample was assayed with three technical replicates. qPCR conditions were as follows: initial enzyme activation at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 30 s, and annealing at 60°C for 30 s. Amplification of different plasmid-mediated quinolone resistance (PMQR) genes was based on Yan et al. (2017). A melt curve was automatically generated, and C _T = 31 was determined as the detection limit of the threshold cycle (Wang et al., 2014). Wells with multiple melt peaks as well as those with a threshold cycle beyond 31 were counted as negative, meanwhile, genes detected in only 1 of the 3 technical replicates were considered false positives and removed.

Statistical analysis

We used the 2^−ΔCT (Schmittgen et al., 2008) values to compare the relative abundances of ARGs between different samples: $Δ C_{T} = C_{T, (A R G)} - C_{T, (16 S r D N A)}$

where C _T,(ARG) and C _{T,(16S rDNA)} are the threshold cycles, for ARGs and 16S rDNA from qPCR experiments, respectively. Data visualization was conducted using the Circos software (http://circos.ca/). Based on the 2^−ΔCT values, heatmap graphs and Pearson correlation were generated using R3.6.0 with the pheatmap package and Hmisc package, respectively.

Results

The abundance and diversity of ARGs detected in honey samples

Among the 38 honey samples, a total of 212 different ARGs were detected, with a mean of 64.9 different ARGs per sample (Supplementary Table S3). While 108 different ARGs were detected in both H07 and H08, only 21 different ARGs were detected in H12 (Supplementary Fig. S1 and Supplementary Table S3). Furthermore, 98, 63, and 104 different ARGs were found in H36, H37, and H38 of the same brand, respectively (Supplementary Fig. S1 and Supplementary Table S3). Moreover, the relative abundance of specific resistance gene also varied among samples (Fig. 1). These results suggest that we should focus on the occurrence of ARGs, as well as the abundance of resistance genes in honey samples.

FIG. 1.

Resistance gene profile from 38 honey samples. Each column is labeled with the sample name, and each row displays the results from a single primer set. FCA, fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol; MLSB, macrolide–lincosamide–streptogramin B.

The diversity of ARG types in each honey sample was analyzed by Circos software according to the corresponding antibiotics (Fig. 2). The 212 ARGs cover the genes conferring resistance to eight major classes of antibiotics commonly administered to humans and animals. Frequency of each type of ARGs varied among different honey samples, FCA (10.5–40.0%), aminoglycosides (8.5–34.4%), and tetracyclines (3.1–28.2%), being the most dominant in the honey samples, followed by MLSB (not detected [ND]—17.7%), β-lactams (1.6–32.8%), vancomycin (ND—15.4%), and sulfonamides (ND—6.3%). The specific genes with highest detection rate in each type of ARGs were strB (35, 92.1%) conferring resistance to aminoglycosides; bla _CTX-M-5 (30, 78.9%), β-lactams; qnrD (32, 84.2%) and qnrS (30, 78.9%), FCA; lnuA-1 (27, 71.1%), MLSB; sul2 (30, 78.9%), sulfonamides; tetR-2 (31, 81.6%), tetracyclines; and vanSC-2 (29, 76.3%), vancomycin (Supplementary Table S3). The above results indicated that the spectrum of ARGs varied in samples.

FIG. 2.

Distribution of each ARG type in 38 honey samples. The circle diagram is divided into two parts, the sample information on the right and the antibiotic to which they confer resistance on the left. The length of the bars on the outer-ring represents the percentage of ARGs in each sample, and the number on the bars in the inner-ring indicates the relative abundance of ARGs in each sample. ARG, antibiotic resistance gene.

In the 212 ARGs detected, efflux pumps (42.5%) and antibiotic deactivation (36.6%) were the major resistance mechanisms, followed by protection (19.7%) (Fig. 3B). ARGs in resistance mechanisms also varied among honey samples (Fig. 3C). For example, there was a 75.4% detection rate of ARGs in the antibiotic deactivation mechanism in H18, but only 20.5% in H31.

FIG. 3.

ARGs detected in all samples were classified based on the mechanism of resistance. (A) The proportion of antibiotic resistance mechanism in 317 ARGs to be detected; (B) the proportion of resistance mechanism of detected ARGs; (C) the proportion of resistance mechanisms detected in each honey sample. ARG, antibiotic resistance gene.

High occurrence of PMQR genes in honey samples

All nine PMQR genes were detected (Fig. 4A) and the abundance of each gene varied among samples (Fig. 4B). Similarly, the occurrence of the PMQR genes in each sample also varied. For example, eight PMQR genes were detected in H11, but none in H35 (Supplementary Table S3).

FIG. 4.

PMQR genes detected in honey samples. (A) The number of different PMQR genes detected in all the honey samples. (B) PMQR gene profile for all honey samples. Each row is labeled with the sample name, and each column displays results from a single primer set. PMQR, plasmid-mediated quinolone resistance.

Correlation between ARGs and transposase genes

As shown in Figure 5 and Supplementary Table S3, eight of the nine transposase genes investigated were found in at least one sample. Only Tp614 was not found. The dominant transposase genes were tnpA-4 and tnpA-5 (both at 92.1%), followed by tnpA-6 (84.2%). Correlations between various ARG types and transposase genes were explored (Table 1 and Supplementary Table S4). The results showed that transposase genes had positive correlation with the genes conferring resistance to tetracyclines (r = 0.648, p < 0.01), MLSB (r = 0.642, p < 0.01), FCA (r = 0.517, p < 0.01), and aminoglycosides (r = 0.401, 0.01 < p < 0.05).

FIG. 5.

Transposase genes in the honey samples. (A) The number of each transposase gene detected in all the honey samples. (B) Transposase gene profile in each honey sample. Each row is labeled with the sample name, and each column displays the results from a single primer set.

Table 1.

Correlation Analysis Between Antibiotic Resistance Genes and Transposase

Resistance genes	Transposase
Aminoglycoside	0.401^a
β-Lactam	0.311
FCA	0.517^b
MLSB	0.642^b
Sulfonamide	0.298
Tetracycline	0.648^b
Vancomycin	0.042
PMQR	0.095
Other	0.777^b

Correlation is significant at the 0.05 level (0.01 < p < 0.05).

Correlation is significant at the 0.01 level (0.001 < p < 0.01).

FCA, fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol; MLSB, macrolide–lincosamide–streptogramin B; PMQR, plasmid-mediated quinolone resistance.

Discussion

In this study, to preliminarily investigate the ARGs in Chinese bee honey, 327 primer sets were used for ARG detection in 38 honey samples produced in 31 provincial-level administrative regions of Chinese mainland. The results indicated that 212 different ARGs were detected from the collected samples, demonstrating that ARGs exist widely in honey. Previous studies generally focused on resistance genes of specific microorganisms in bees and bee honey, for instance several resistance genes of Gilliamella isolates from Chinese bumblebee gut (Zhang et al., 2021), P. larvae containing a tetracycline resistance plasmid from honey bee (Murray et al., 2007), and tetracycline resistance genes in B. cereus isolates from Argentinean honey samples (Lopez et al., 2008). Without isolating visible colonies, we used the total DNA from honey samples as templates to detect the ARGs using qPCR, to give a relatively full picture of ARG contaminations in our samples. The diverse and abundant ARGs found in honey needs special attention, as these ARGs may spread to human by direct honey consumption. However, no limits have been set on ARGs as part of the standards in any honey products worldwide, although residual limits of lots of antibiotics have been laid down.

Previous study showed a direct link between prevalence of acquired streptomycin resistance genes, strA-strB, in the gut microbiota of honeybees and use of streptomycin in crop farming in the United States of America (Ludvigsen et al., 2018). In this study, strB gene was detected in 35 out of 38 honey samples (Supplementary Table S3); such high detection rate of strB gene in our samples may also be related to the application of streptomycin in cultivated plants. As in the United States of America, streptomycin had ever been used in China for a long time to prevent and control plant pathogens, such as Athelia rolfsii causing southern blight on Helianthus tuberosus (Zhong et al., 2018), and Stagonosporopsis cucurbitacearum, a pathogenic plant fungus that causes gummy stem blight in crops of the Cucurbitaceae family (Zhao et al., 2018). Now, the use of streptomycin in plant agriculture is banned, and it will help reduce the level of streptomycin residue and thereby the spread of streptomycin resistance genes in the environment.

The bla _CTX-M-5 had the highest detection rate among bla _{CTX-M-1/2/3/4/5/6} in honey samples in this study (Supplementary Table S3). Notably, bla _CTX-M-5 confers resistance to cephalosporins, including the third- and fourth-generation cephalosporins, which are critically important for human medicine. bla _CTX-M-5 was initially found in a clinical isolate of Salmonella Typhimurium in Latvia (Bradford et al., 1998). Compared with other bla _CTX-M genes, which have now been spread worldwide and reported as the most prevalent extended-spectrum β-lactamases (ESBLs) in important pathogens (Zhao et al., 2013), bla _CTX-M-5 occurs infrequently because it has been suggested to be located on the chromosome or a small nonself-transmissible plasmid (Sjölund-Karlsson et al., 2011; Kozyreva et al., 2014). However, bla _CTX-M-5 harbored on nonconjugative plasmids can be mobilized by coexisting plasmid (Sjölund-Karlsson et al., 2011; Kozyreva et al., 2014). Notably, Punyadi et al. (2021) recently identified bla _CTX-M-5 harbored on IncA/C conjugative plasmid from blowfly in Northern Thailand, implicating that bla _CTX-M-5 may spread more rapidly. Therefore, to clarify the reason for the relatively high detection rate of bla _CTX-M-5 in bee honey, further investigation on the gene localization in bacteria from honey is needed.

Quinolone is a family of pure synthetic antibiotics, and we thus investigated PMQR genes in our samples. There are three types of PMQR genes: pentapeptide-encoding qnr genes (qnrA, qnrB, qnrC, qnrD, and qnrS), the aac(6′)-Ib-cr [variant of aac(6′)-Ib] gene coding for an aminoglycoside acetyltransferase, and efflux pump-encoding genes (qepA and oqxAB) (Strahilevitz et al., 2009). In this study, qnrD and qnrS were detected in almost all samples (Fig. 4A and Supplementary Table S3), indicating that PMQR genes are widely spread in Chinese honey. Increasing studies reported the presence of PMQR genes in the Enterobacteriaceae (Strahilevitz et al., 2009; Li et al., 2016; Azargun et al., 2019) and found a relationship between the presence of PMQR and ESBL genes among Escherichia coli isolates in clinics (Wu et al., 2007; Yang et al., 2008). However, we did not find any significant link between PMQR with high detection rate and ESBL resistance genes (Supplementary Table S4), indicating that the PMQR genes do not coexist with ESBL genes in honey samples.

Transposases catalyze the movement of mobile genetic elements (MGEs) that contribute to the occurrence and dispersal of ARGs (Stokes et al., 2011). The protein encoded by TnpA can catalyze the synthesis of cointegrase, which is a classic motif of replicative transposition in horizontal gene transfer (HGT) (Reed, 1981), and confers a higher probability of HGT occurrence (Partridge et al., 2018). In this study, eight out of the nine transposase genes were detected in honey samples, with tnpA-4 and tnpA-5 being the two most frequent (Fig. 5). Veress et al. (2020) found that the genome of Acinetobacter lwoffii strain M2a isolated from Transylvanian honey is strikingly crowded with MGEs. As mentioned above, microbes in honey are partly from the environment. In China, the tnpA genes have been found to be highly expressed in amended soils, manures, and waterbodies (Xie et al., 2016; Xu et al., 2016; Li et al., 2017). Moreover, the abundance of transposase genes in honey was associated with genes conferring resistance to tetracyclines, MLSB, FCA, and aminoglycosides (Supplementary Table S4). This agrees with a report by Liu et al. (2018) stating that tnpA-4 was highly correlated with aminoglycosides, FCA, MLSB, sulfonamides, tetracyclines, vancomycin, and multidrug resistance genes in waterbodies in China, as well as with another report by Yan et al. (2018) detailing that the integron IntI1 was discovered at a frequency of 100% in all water samples from the Three Gorges Reservoir and positively correlated with the tetracycline resistance genes, tetB, tetC, tetG, implying a relationship between ARGs in honey and environment in China. The high detection rate of transposase genes and positive correlation with ARG abundance in honey suggests that HGT may have contributed to the enrichment of ARGs. Therefore, when considering the ARGs in honey, it is necessary to investigate the abundances of transposase genes and other MGEs.

Conclusion

This study has revealed the diversity and abundance of ARGs in Chinese honey. Genes conferring resistance to FCA and aminoglycosides were the most dominant types of ARGs. Tetracyclines, MLSB, FCA, and aminoglycoside resistance genes were significantly associated with these transposase genes, indicating that HGT may have contributed to the enrichment of ARGs. Therefore, it is recommended to detect ARGs in more honey samples to make a complete assessment of the impacts of ARGs on honey safety.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work is financially supported by Shanghai Agriculture Applied Technology Development Program, China (Grant No. 2018-02-08-00-10-F01548) and the National Key R&D Program of China (Grant No. 2019YFA0904000).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Figure S1

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Supplementary Material

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