High Carriage Rate of the Multiple Resistant Plasmids Harboring Quinolone Resistance Genes in Enterobacter spp. Isolated from Healthy Individuals

Antimicrobial-resistant bacteria causing intractable and even fatal infections are a major health concern. Resistant bacteria residing in the intestinal tract of healthy individuals present a silent threat because of frequent transmission via conjugation and transposition. Plasmids harboring quinolone resistance genes are increasingly detected in clinical isolates worldwide. Here, we investigated the molecular epidemiology of plasmid-mediated quinolone resistance (PMQR) in Gram-negative bacteria from healthy service trade workers. From 157 rectal swab samples, 125 ciprofloxacin-resistant strains, including 112 Escherichia coli, 10 Klebsiella pneumoniae, two Proteus mirabilis, and one Citrobacter braakii, were isolated. Multiplex PCR screening identified 39 strains harboring the PMQR genes (including 17 qnr,19 aac(6′)-Ib-cr, and 22 oqxA/oqxB). The genome and plasmid sequences of 39 and 31 strains, respectively, were obtained by short- and long-read sequencing. PMQR genes mainly resided in the IncFIB, IncFII, and IncR plasmids, and coexisted with 3–11 other resistance genes. The high PMQR gene carriage rate among Gram-negative bacteria isolated from healthy individuals suggests the high-frequency transmission of these genes via plasmids, along with other resistance genes. Thus, healthy individuals may spread antibiotic-resistant bacterial, highlighting the need for improved monitoring and control of the spread of antibiotic-resistant bacteria and genes in healthy individuals.


Introduction
Quinolones, a group of broad-spectrum antimicrobial agents, are widely used for treating infections caused by Gram-negative bacteria and are routinely used in animal breeding. In China, total antibiotic usage is estimated at approximately 162,000 tons/year [1], with human consumption accounting for approximately 48% and the remainder attributed to consumption by livestock and other domesticated animals [2]. Fluoroquinolone usage accounts for 17% of this total [3]. The acquisition of quinolone resistance may be caused by (i) chromosomal mutations in the bacterial genes encoding the protein targets of quinolones or by mutations that cause reduced drug accumulation via decreased uptake or increased efflux, or (ii) via the plasmid-located genes associated with quinolone resistance [4]. To date, three plasmid-mediated quinolone resistance (PMQR) mechanisms have been described: the first involves Qnr proteins, the second involves aac(6 )-Ib-cr genes, and the third involves oqxA, oqxB, and qepA plasmid-mediated efflux pumps [5,6]. Since the first PMQR gene was identified in a Klebsiella pneumoniae isolate [7], horizontal gene transfer has been considered the main route of quinolone resistance dissemination [8].
Numerous studies on the prevalence of PMQR genes in clinical settings and in animals have been reported worldwide [9][10][11][12]. However, few studies have investigated the prevalence of PMQR genes in healthy people. To ensure food safety, it is a regulatory requirement in China for healthy individuals in food-related industries to undergo health examinations to determine whether they harbor transferable drug resistance genes. In this study, we selected healthy individuals from the catering industry to investigate the prevalence of PMQR genes, and to estimate the potential threat that mobile quinolone resistance genes pose to the food industry.

Drug Resistance Caused by Gene Mutations
Gene mutations in the DNA topoisomerase IV and gyrase encoding genes were found in all 26 E. coli strains (100%, 26/26). Ser at position 83 of the E. coli gyrase subunit A (gyrA) gene was mutated to Leu, and Asp at position 87 was mutated to Asn or Tyr. Ala at position 56 of the DNA topoisomerase IV, subunit A (parC) gene was mutated to Thr, Ser at position 80 was mutated to Ile, and Glu at position 84 was mutated to Val. Ser at position 458 of the topoisomerase IV, subunit B (parE) gene was mutated to Ala and Ile at position 529 was mutated to Leu. Among these, the parC and gyrA gene mutations were the most frequent.

Discussion
Antibiotic resistance is recognized as a 'One Health' challenge because of the rapid emergence and dissemination of resistant bacteria and genes that not only exists in humans (both in healthy individuals and patients), but also in wildlife, companion animals, agriculture (food-producing animals, fruits, and vegetables), and the environment (water and soil) on a global scale. Enterobacter spp. resistant to quinolones frequently arise in animals, being easily disseminated through the food-chain [13]. In rectal swab samples from some wildlife (non-human primates, mice) rectal swab samples, different variants of the qnrB gene were detected [14]. In the rectal swab samples selected from the companion animals, Gibson et al. reported that the qnrA1 or qnrB2 gene was often detected long with aac(6 )-1b-cr [15]. Approximately 4.1% (15/363) of strains isolated from the food-producing animals, such as swine, poultry, rabbit, and cattle, carried the qnrB2, qnrB19, and qnrS1 genes, simultaneously [13]. In addition, fruits are usually consumed without cooking or processing, making them a potentially high-risk source in humans [16]. As mediators for the dissemination of antimicrobial-resistant E. coli, raw salads vegetable present a great risk to public health, and Nayme et al. detected PMQR determinants were detected in 52% of isolates [17]. In a study of wastewater samples from the swine feedlot, qnrD, qepA, oqxB, qnrS, and oqxA genes were detected [18]. Vaz-Moreira et al. found that the resistance strains could be disseminated from hospital effluent to aquatic environments, and that quinolone-resistant Gram-negative bacteria carried one or more of the PMQR genes [19]. Among 200 faecal samples randomly collected from chickens and pigs originating from different farms at the time of slaughter in Ibadan and Nigeria, PMQR genes were detected in 18 E. coli strains, which suggesting that the food animals may represent an important reservoir of PMQR in this region of Africa, and that antibiotic-resistant bacteria carried by animals can enter the human food chain through the consumption of meat or by direct contact [20]. PMQR genes (including aac(6 )-1b-cr, qnrB, and qnrS) were frequently detected in the herbs originating from Thailand (Water morning glory, Acacia and Betel leaf), Vietnam (Parsley, Asian pennywort, Houttuynia leaf, and Mint) and Malaysia (Holy basil and Parsley), suggesting that fresh culinary herbs from Southeast Asia are a potential source of contamination of food with quinolones resistant bacteria [21]. Because these herbs are generally consumed without appropriate heating, the resistant bacteria may be readily transferred to humans. Therefore, AMR Gram-negative bacteria can transfer from human-to-human directly or indirectly.
In this study, the isolation rate for Gram-negative strains with CIP resistance was alarmingly high at 79.6%. Almost all of the CIP-resistant strains were also resistant to moxifloxacin, norfloxacin, and levofloxacin. This may be related to the fact that these antibiotics are commonly used to treat gastroenteritis in adults [22]. Furthermore, low-dose antibiotics are added to animal feed as growth promoters and, while providing economic benefits, this accelerates the emergence of drug-resistant bacteria [13]. These factors contribute to the high prevalence of quinolone-resistant bacteria in healthy people. Multidrug resistance to different antibiotic categories was found in most isolates, particularly to commonly used antimicrobials such as TET, FEP, AXO, and cefepime. The minimum inhibitory concentration 50 (MIC 50 ) and MIC 90 for TET exceeded 16 µg/mL. Resistance to some commonly used clinical antibiotics, especially third-and fourth-generation cephalosporins, is concerning because this may lead to clinical treatment failure. To the best of our knowledge, few prevalence studies on PMQR genes in healthy people have been conducted. In this study, the PMQR gene detection rate was 31.2% and PMQR genes were identified in E. coli, K. pneumoniae, P. mirabilis, and C. braakii isolates. The PMQR gene carriage rate for E. coli in health practitioners was shown to be higher than that in healthy volunteers (14.3%) [23], and the reported detection rate for PMQR genes in animal breeders (55.9%) and the carriage rate in patients (51.4%) [24] were both higher than in our study [12]. The reason for these differences could be the high use of these antibiotics in clinical treatment and animal breeding. We found that the PMQR gene carriage rate for K. pneumoniae was higher than that for E. coli, which was consistent with previous studies showing carriage rates for K. pneumoniae and E. coli of 60.0-65.3% and 12.6-22.3%, respectively [25,26].
The most prevalent PMQR genes were oqxB and oqxA, followed by aac(6 )-Ib-cr and qnr. The detection rates for the aac(6 )-Ib-cr and qnr genes in E. coli were 9.8% and 6.25%, respectively, which were higher than the 9.0% and 0.85% rates recorded in Sweden and Norway [27]; these differences might be related to the scope and degree of antibiotic use in the different countries.
The prevalence rates for aac(6 )-Ib-cr, oqxB, and oqxA were slightly higher than those reported previously in healthy populations, but the prevalence of qnr was lower than that reported previously [28]. Some studies have shown that oqxB and oqxA are widely distributed among E. coli strains isolated from animal husbandry, with detection rates of 58.5% and 33.8%, respectively [29,30]. The oqxB and oqxA genes are also prevalent among K. pneumoniae isolates [31]. Of the 125 isolates we tested, two or more PMQR genes were present in 27 (21.5%) of the isolates. Some of the E. coli isolates (8.9%) only carried a single PMQR gene, whereas all of the K. pneumoniae isolates carried three or more PMQR genes, a result similar to previous findings [32].
According to the rules and regulations of the Public Places Sanitation Administration, the participants who work in the food processing and service trades must undertake a preventive health examination each year. In this study, we just focused on the individuals who work in the food processing and service trades; however, in future studies, we will expand our research to include all healthy individuals as the sample sources to represent the general healthy population.
In the present study, 39 PMQR-positive strains carried six to 16 types of acquired resistance genes, with most of the resistance genes co-existing on the same plasmid. The tet(A) and bla CTX-M-55 genes were spread throughout the population, which may have public health significance. K. pneumoniae isolates also have a tendency to acquire resistance [33]. In this study, we found that the sul1, floR, and tet(A) genes were more common, which may also have public health significance. The most prevalent plasmid replicon types were IncFIB and IncFII in E. coli and IncFIB, IncR, and IncHI2 in K. pneumoniae. Other studies have also reported that these plasmids are the main types [31]. aac(6 )-Ib-cr and qnr were found to be chromosomally located in E. coli, which is in agreement with previous reports [34,35]. We found that oqxA and oqxB were chromosomally located in all strains except for one in which these genes were not detected. Previous studies have also reported that oqxA and oqxB are chromosomally located in K. pneumoniae [36]. In addition, two strains showed resistance to polymyxin, with mcr-1.1 located on plasmids IncI2 and IncX4. Previous studies have reported that variant mcr-1.6 was present in Salmonella isolated from healthy people [37]. Plasmids that carry mcr-1.1 are a concern for healthy people because the frequent exchange of antibiotic resistance genes among E. coli, K. pneumoniae, and Salmonella enterica serovars could lead to the spread of polymyxin resistance [38].

Samples and Bacterial Strains
From May to August 2015, approximately 40 rectal swab samples were collected once a month; 157 rectal swab samples were collected at the Nanchang Center for Disease Control and Prevention (China) from healthy participants (55 males, 102 females; age range: 18-57 years), who received preventive health examination because of their occupation in food processing and other service trades, according to the rules of the administration for market regulation. None of the healthy individuals included in this study had been exposed to antibiotics or a hospital environment during the 3 months prior to sample collection. The swab samples were screened using ciprofloxacin (CIP) (4 µg/mL)-containing LB agar plates incubated at 37 • C for 18 h. For each sample, one transparent and smooth colony was selected, followed by identification using the API20E test (bioMerieux, France).

Genome and Plasmid Sequencing
DNA was extracted from 39 PMQR gene-positive isolates using the Wizard Genomic DNA Extraction Kit (Promega, Madison, WI, USA), followed by sequencing (HiSeq sequencer; Illumina, California, USA). Plasmid DNA was purified from 100 mL of liquid culture of the strain using the Qiafilter Plasmid max kit (Qiagen, Dusseldorf, German) as per the protocol for low-copy plasmids, and then sequenced using MinION (Oxford Nanopore, Oxford, UK) sequencers. MinION libraries of all plasmid DNAs were prepared using the SQK-LSK108 nanopore sequencing kit, R9 version (Oxford Nanopore, Oxford, UK). 1D base-calling was performed on a local computer in real-time by MinKNOW. We assembled each genome using a combination of short-and long-reads using the Unicycler hybrid assembler [41]. Plasmid replicons and drug resistance genes were identified in silico using online tools (http://www.genomicepidemiology.org/, accessed on 21 June 2021).

Conclusions
In this study, we investigated the prevalence of quinolone-resistant, Gram-negative bacteria in healthy people. The prevalence rate of PMQR genes in healthy participants was relatively high, and the co-existence of resistance genes on plasmids is an important finding. Bacterial drug resistance in healthy people is a serious issue because it may unknowingly lead to the spread of antibiotic-resistant bacteria, which poses a serious public health threat. These findings highlight the necessity for improved monitoring of antibiotic-resistant bacteria in healthy individuals.