Antimicrobial and Transconjugants Characteristics of Sul3 Positive Escherichia Coli Isolated from Animals in Nanning, Guangxi Province

Background: Sulfonamides is the second most popular antibiotic in many countries, which leads to the widespread emergence of sulfonamides resistance. Sul3 is a late sulfanilamide resistance gene, whose research is relatively little. Result: 46 sul3 positive E. coli strains were separated. A total of 12 ST types were observed, and 1 of those was previously unknown type. The ST350 is the most numerous type. All isolates were multidrug-resistant E. coli, with high antimicrobial rates to penicillin, ceftriaxone sodium, streptomycin, tetracycline, ciprooxacin, gatioxacin and chloramphenicol (100%, 73.9%, 82.6%, 100%, 80.4%, 71.7% and 97.8%), and with at least 3 resistance genes in addition to sul3. The plasmids transfered from 3 sul3-positive isolates to C600, the most of which brought 7 antibiotic resistance and increased resistance genes to C600. The transferred sul3 gene and the plasmid that carries it could be stably inherited in the recipient bacteria for at least 20 days. Those plasmids had no effect on the growth of the recipient bacteria, but it would greatly reduce (at least 60 time) the in vitro competitiveness of the strains. Conclusions: In Nanning, these sul3-positive Escherichia coli have strong antimicrobial resistance, and the plasmid carrying sul3 has the ability to transfer multiple resistance genes, so long-term monitoring is necessary. Since the transferred plasmid will greatly reduce the in vitro competitiveness of the strain, we can consider limiting the spread of antimicrobial in this respect.


Background
The problem of bacterial resistance has a long history, and now it has become a medical problem which we cannot be ignored. Many resistances in bacteria is dominated by mobile genetic elements, including plasmids, integrons and transposons [1]. Escherichia coli is a common Gram-negative bacteria and one of the symbiotic bacteria in the intestines and environment of most livestock and poultry. But many studies [2,3] have also shown that E. coli can cause a variety of diseases in humans and animal. And antibiotics have been used to treat bacterial infections and even used as feed additives to promote the growth of livestock and poultry for a long time [1,4]. These used antibiotics are not completely absorbed or metabolized by the body [5]. After being discharged, these antibiotics can pollute and spread in the environment through a variety of ways, such as agricultural runoff, sewage discharge and nearby farm leaching [6]. As a result, many symbiotic bacteria such as Escherichia coli have to live in the environment which is containing antibiotics for a long time, and this kind of environment can provide appropriate selection pressure for the emergence and spread of multi-antibiotic-resistant bacteria and antibioticresistant genes.
Sulfonamide is an antibiotic which is not easy to degrade, have low soil adsorption rate and high mobility [7,8]. It can inhibit bacterial growth and reproduction by competing for binding sites of Paminobenzoic acid and dihydrofolate synthase (DHPS) [9]. Moreover, the sulfonamide has many advantages, such as wide range of use, low cost, a wide variety and etc. Since the rst sulfonamide was used in clinical practice in 1935, it has been one of the commonly used antimicrobials in the prevention and treatment of aquatic and livestock diseases [10,11].
Sulfonamide resistance genes (including oP and sul) can encode a kind of DHPS with low a nity to sulfonamides, which makes bacteria grow and reproduce normally in the environment containing sulfonamides [12,13]. At present, four kinds of sulfonamides resistant genes (sul1-4) have been found in plasmids. Sul1 and sul2 were discovered successively in 1985 [14,15]. Sul4 was recently found in swinery in swiss [16]and was also found in type I integron transmission genes observed in Indus River sediments [9], but it has not been reported in clinical isolates [9]. Sul3 is a sulfanilamide drug-resistant gene type discovered in 2003 [17]. Since its discovery, sul3 has been successively found in more and more regions, sources and strains [18][19][20][21], among which even have human-originated Escherichia coli [22] Nanning is the capital of Guangxi Province, which is located in the southwest of China. The breeding industry that in Nanning is mainly composed of retail investors. The unreasonable use of antibiotics for livestock and poultry diseases, coupled with the lack of effective management measures, perpetuate the problem of bacterial resistance. The purpose of this study is to detect the antimicrobial, multi-locus sequence typing (MLST) and antimicrobial gene characteristics of sul3 positive E. coli from animals in Nanning area. At the same time, to evaluate the in uence of sul3 positive bacteria on host bacteria after conjugation.

Transconjugants And Related Experiments
Three suspected transconjugants were successfully obtained through the conjugation experiment. After sul3 positive identi cation and ERIC-PCR (Fig. 1), the 3 suspected transconjugants were all the plasmid strains obtained from the recipient bacteria (C600) (named as EC027/T, EC035/T and EC038/T according to the donor bacteria name) In comparison with the recipient bacteria, the MIC of the maximum 7 antimicrobials in transconjugants (EC027/T) showed different degrees elevated, including penicillin, ceftazidime, streptomycin, amikacin, tetracycline, cipro oxacin and chloramphenicol (Table 4). According to the detection results of resistance genes, in addition to sul3 gene, E027/T was detected with 6 new resistance genes, while E025/T and E038/T were 2 (Table 5). However, compared with the sensitivity changes of antibacterial drugs, we found that no corresponding resistance genes were detected in the chloramphenicol of streptomycin and chloramphenicol. The reasons for this phenomenon will be explained in our discussion. The plasmid stability experiment showed that the plasmid could be stably and continuously passed for at least 40 generations with strong stability (Fig. 2).  The Adaptive Cost Of Plasmid C600 The growth curves of the 3 transconjugants and the recipient bacteria showed that the transconjugants and the recipient bacteria had minor changes only during the logarithmic growth period, and the changes were not obvious after entering the stable period at 8 hours. It is indicating that the transconjugants had little in uence on the growth of the recipient bacteria (Fig. 3).
n the competitive test, we observed that the competitive ability of the 3 transconjugants was signi cantly reduced compared with that of the recipient bacteria C600, among which the most obvious one was EC035/T (0.043), followed by EC027/T (0.058) and EC038/T (0.061) (Fig. 4).

Discussion
Nowday, sulphonamides are rarely used to treat bacterial infections in humans in many regions, but they are still widely used in aquaculture, animal husbandry and veterinary because of the lower price [20]. Massive use plus great potential for penetrate into the environment. For these reasons, the concentration of sulfonamides becomes a priority for sewage treatment, rivers and water sources [20]. Sulfonamineresistant genes (sul) have also spread widely for these reason. Analogously, with regard to the detection rate of sul, we compared several recent studies [23][24][25][26] found that the detection rate of sulfonamineresistant genes was high and that the detection rate of sul1 and sul2 in sulfamine-resistant genes was generally higher than that of sul3. It was suggested that there may be more sulfamine-resistant bacteria in Nanning, and we should pay more attention to them.
After analyzing the genetic environment of sul gene, Jang et al. [20] said that compared with the other two genes, the diversity of adjacent genetic transfer elements and the resistance genes of sul3 were lower, and some sul3 even existed on chromosomes, which affected the transmission of sul3. But the studies also showed that [20,47], sul3 is related to type I integron, and can replace sul1 to form atypical type I integron, which emphasize the potential for widespread in the future. This indicates that there is still a certain transmission problem of sul3 at present, but this problem may be solved over time. And in our study, the stability test showed that the transferred sul3 wild plasmid could inheritance in bacteria for a long time, it also indirectly re ects that sul3 has the potential for long-term transmission. In addition, studies have shown that [27,28], sul3, tetQ, tetO and other sulfonamides and tetracycline resistance genes are signi cantly positively correlated with the content of Cu, Zn and other heavy metals (P < 0.05 or P < 0.01, r = 0.882-0.992), which indicates that heavy metals can help sul3 appear and there may be some heavy metal pollution in the Nanning's farms.
The emergence of multidrug-resistant bacteria seriously affects the cure rate of bacterial infection diseases, becoming a potential threat to the health of human beings and livestock [29]. In the study, all the strains we tested showed multiple antibiotic resistance, which is possible because we tested the sul3positive E. coli. Here we need to pay attention to the pathogenicity of these strains. Compare antibiotic resistance and resistance genes. In isolates strains, only quinolones and aminoglycosides had differences in the detection rate of resistance genes and antimicrobial resistance rate, similarly, no resistance genes associated with streptomycin and chloramphenicol were detected in conjugates, it suggests that there may be other related genes mediating the tolerance of the above-mentioned antimicrobials, which may be the e ux pump or the resistance genes of the relevant antimicrobials. Regarding the plasmids in these isolates, we cannot determine the type and quantity of these transfer plasmids, what we can con rm is that after acquiring the plasmid, there are several (at least 4) antibiotics resistance changes to the strains, and corresponding to the resistance genes tested. It indicates that the transferred drug-resistant genes can be expressed by host cells, it may affect the effective use of antibiotics in Nanning.
And compared with other studies [29][30][31], the recombinant plasmid had no effect on the growth performance of the strain, just like the wild plasmid in this study, but the wild plasmids reduced the competitiveness of host bacteria in vitro to a greater extent. Although the types and quantities of drugresistant genes studied are different, this also indicates that the adaptation cost of wild plasmids will bring greater adaptive cost to the recipient bacteria due to multiple drug-resistant genes or other unknown genes.
In the test, the diversity of each sul3 positive strain is low, but there are still more common types in ST typing. ST23, ST156 and ST10 were reported to be related to human [32][33][34], Among them, ST10 is the most common pedigree in human urine Escherichia coli isolates [32], and these reports also pointed out that these 3 types were also found in other E.coli strains. Although these 3 types were rarely detected in this study, it is still necessary to pay attention to the transmission between human and livestock.  (Table 1). The PCR product were sent to the company for sequencing, and uploaded to NCBI for BLST con rmation of suspected isolates and sul3 carrier. The 30% glycerol sample (V/V) and DNA sample of sul3 positive E. coli were stored at -20 ℃.
A total of 46 strains of sul3 positive E. coli were detected, PCR ampli cation was conducted using 7 pairs of primers (adk, fumC, gyrB, icd, mdh, purA and recA) ( Table 6). The positive products were sent to the company for sequence determination, and the results were uploaded to the MLST website (https://pubmlst.org) to obtain the ST type. (NOTE: Table 6 is longer than a page of A4, so we put it at the end of this document. You can put Table 6 at the end of this paragraph)

Conjugative Experiment
The conjugative experiment was conducted by lter membrane method. the Escherichia coli C600, which did not produce acid and has rifampicin resistance, was used as the recipient bacteria. And sul3-positive isolates were used as the donor bacteria. The transconjugants were screened from McConkey medium with a concentration of 6000 µg/mL sulfamethazine and 3500 µg/mL rifampicin.
The suspected transconjugants were subjected to PCR and antibiotics sensitivity tests to con rm whether the plasmid transfer was successful, and then ERIC-PCR was used to determine the correlation between the transconjugants and C600, with the ERIC-primers as described previously[35] ( Table 1). It also detects whether other resistant genes are co-transmitted.

Growth Curve
We used absorbance method to observe the change of the growth status of transconjugants and C600, speci c as follows. After shaking culture at 37℃ overnight, the bacterial solution was added to fresh LB broth according to the ratio of 1:1000. A total of 16 time points, 3 ml was taken from each time point for OD 600 absorbance detection. The observation lasted for 24 hours and need repeated 3 times in parallel.

In Vitro Competitive Test
In vitro competition experiments refer to previous descriptions [36]. First, two kinds of bacteria were cultured to 0.5 McFarland, then took 100 µL for each mixed in a 1:1 ratio and added to 10 mL LB broth, and incubated for 16 h at 37℃, 220 r/min. After diluted 10 6 times, 100 µL bacterial solution was respectively coated with streptomycin 60 µg/mL LB agar and streptomycin free LB agar, and cultured overnight at 37℃. The total CFU and streptomycin resistant CFU were counted, and the competition index of without resistant CFU and streptomycin resistant CFU was calculated, and the parallel repetition was 3 times.

Plasmid Stability
According to the previous description of plasmid stability [37], the transconjugants was shaken in LB medium at 37℃ for 12 h, and then inoculated in new LB medium and shaken at 37℃ for 12 h again, repeat every 12 h. Each time was counted as one generation, and the procedure was repeated for 60 generations. Every 10 generations, part of the bacterial solution was diluted and coated with agar medium, 24 colonies of bacteria were randomly selected. DNA was extracted by boiling method. Then the PCR of sul3 was performed to determine the positive rate of sul3. Availability of data and materials The others datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. The ERIC-PCR result of 3 transconjugants and C600 Note: 1-3 for transconjugants EC027/T, EC035/T and EC038/T, 4 for C600, The band size and combination of the 3 transconjugants were consistent with that of C600, indicating that these transconjugants and C600 were homologous strains Stability test of sul3 positive wild plasmid The positive rate of sul3 remained above 70% when the transconjugants were passed on to the 20th day(40 generations), indicating that the sul3 plasmid could be inherited stably for a long time in the transconjugants