In vitro activity of antimicrobial agents with and without sulbactam, EDTA and sulbactam-EDTA on ESBLs-producing Escherichia coli isolated from healthy Tibetan yaks

The spread of ESBLs-producing bacteria has been strikingly rapid in many regions of the world and it causes therapeutic diculties in everyday practice. The aims of this study were to investigate the prevalence and susceptibilities of ESBLs-producing Escherichia coli isolates from healthy Tibetan yaks in China, to evaluate the activity of drug combinations on ESBLs-producing E. coli isolates. From July 2018 to August 2019, a total of 750 nasal swab samples were tested for the presence of E. coli and ESBLs-producing strains. The MICs of 11 antimicrobial agents alone and combinations with sulbactam, EDTA or sulbactam-EDTA against 240 ESBLs-producing E.coli strains were determined by the broth microdilution method.

0, 0 and 0% to 50 ~ 87.5, 4.2 and 100% respectively. The addition of EDTA to uoroquinolones, doxycycline, orfenicol, amikacin and fosfomycin, showed improved activities and resulted in low MICs, increasing the level of susceptibility from 0, 0, 8.3, 0 and 0% to 4.2 ~ 29.2, 33.3, 33.3, 66.7 and 45.8%, respectively. All other antibacterials (except uoroquinolones, doxycycline and orfenicol), when combined with sulbactam-EDTA, were found to be more active than combinations only with sulbactam or with EDTA against most of isolates, with lower MIC 50 s and MIC 90 s.

Conclusion
In conclusion, ESBLs-producing E. coli isolates were widespread in healthy Tibetan yaks in China. ESBLsproducing E. coli isolates exhibited varying degrees of multidrug resistance. This study these ndings suggested that sulbactam can enhance activity of β-lactams and some non-β-lactams of antimicrobial agents and had a synergistic effects with EDTA in improving activities of some families of antimicrobials.

Background
Nowadays, the growing frequency of antibiotic resistances is a universal problem. The emergence of multidrug resistant (MDR) strains poses several challenges to the clinical facilities [1]. The production of β-lactamases is still the main mechanism for resistance of gram-negative bacilli to β-lactams [2].
Resistance to third generation cephalosporins is mediated by extended-spectrum β-lactamases (ESBLs), which are derivatives of narrow spectrum TEM and SHV β-lactamases [3]. Such enzymes are most commonly found in E.coli isolates from chickens and have been recently detected at high frequency in China chicken farms [4][5][6]. A typical characteristic of ESBLs is their ability to hydrolyze cephalosporins and aztreonam while being inhibited by β-lactamases inhibitors [7]. β-lactamase inhibitor sulbactam can greatly enhance the antibacterial activity of β-lactams [8], but MICs of their combinations against ESBLs producers were still higher than MICs against non-ESBLs-producing strains [5,6]. Additionally, MICs of combinations on induced ESBLs producers were higher than MICs on strains before induction [9,10].
These data suggested that β-lactam combinations with β-lactamase inhibitors could not completely resolve the problem of MICs rise that caused by ESBLs-producing and ESBLs-producing was not the only reason for MICs rise. There may be other reasons, such as AmpC β-lactamases production, membrane permeability droping, the role of active e ux, and so on. The suicide inhibitor sulbactam and cephalosporins combination has been assessed in vitro and in vivo on ESBLs-producing bacteria [8,[11][12][13]. Studies about effects of membrane permeability enhancers ethylenediamine tetraacetic acid (EDTA) on activity of the antibacterial drugs have also been described [14,15]. However, there is paucity of data on effects of sulbactam on activity of non-β-lactams and whether there is interaction between sulbactam and EDTA in affecting activity of the antibacterial drugs is unknown.
Because these antimicrobials are a signi cant part of drug therapy to some human bacterial infections, resistance to these drugs could ultimately pose a signi cant threat to human health [16]. The spread of ESBLs-producing strains has been strikingly rapid in many regions of the world and it causes therapeutic di culties in everyday practice, therefore it would thus seem necessary to nd new therapeutic weapons against this growing threat [3]. Because of the close relationships between humans and animals, thus, investigating prevalence and researching drug combinations of ESBLs-producing E. coli isolates in healthy Tibetan yaks is paramount for establishing guidelines for veterinary and human clinical medication use, it would be of particular signi cance for medical science.
This present study aimed at evaluating the activity of 11 antibacterial agents alone and in combination with sulbactam, EDTA and sulbactam-EDTA against ESBLs-producing E.coli strains respectively, analyzing the interactions among β-lactamase inhibitor sulbactam, membrane permeability enhancers EDTA and antimicrobial agents from phenotype, and providing theory basis for designing more effective antibiotic combinations for treating ESBLs-producing strain infections.

Results
Isolation and identi cation of E. coli A total of 449 (59.87%) E. coli isolates were recovered from 750 nasal swab samples in healthy Tibetan yaks between 2018 and 2019.

Prevalence of ESBLs
In screening for ESBLs, 282 E. coli isolates were identi ed as suspected ESBLs producers. The detection results showed that 240 (53.45%) of 449 E. coli isolates were con rmed to be ESBLs-producing. Their zones produced by the discs with clavulanic acid were ≥5 mm larger than those without inhibitor in ceftazidime, ceftazidime/clavulanic acid or cefotaxime, cefotaxime/clavulanic acid. Thus, according to the CLSI criterion, 240 E. coli isolates produced ESBLs.
As shown in table 2, the addition of β-lactamase inhibitors sulbactam to the third generation cephalosporins resulted in low MICs for all ESBLs-producing isolates, increasing the level of susceptibility from 0 to 50-87.5% respectively, showed that sulbactam may improve much activity of third generation cephaloporins against ESBLs-producing E.coli isolates in vitro. However, some among ESBLs-producing E.coli isolates was still found to be intrmediated (50% to Ceftriaxone, 8.3% to Cefotaxime) or resistance(4.2% to Cefotaxime). We have examined the effect of sulbactam to activity of non-β-lactam antibacterials against ESBLs-producing E.coli strains. To our knowledge, this is the rst time that antimicromial activity of non-β-lactam antimicromials against ESBLs-producing E.coli of animal origin, when combined with sulbactam, have been determined. Our studies revealed that, depending on the classes of non-β-lactam antimicrobials tested, sulbactam can have synergy or no effect when it is used in combination with non-β-lactam antibacterials. Amikacin, fosfomycin and orfenicol were found to be more active against ESBLs-producing E.coli isolates with some exceptions, when combined with sulbactam, whereas uoroquinolones, doxycycline was found to have similar activies. The addition of sulbactam to amikacin, or fosfomycin resulted in low MICs for all ESBLs-producing isolates, increasing the level of susceptibility from 0 and 0% to 4.2 and 100% respectively. Although the addition of sulbactam to orfenicol also had the effect of lowering the MICs in all but very few strains on which orfenicol alone and in combinations with sulbactam were found to yield the same MIC, the susceptibility showed improved activities and resulted in low MICs for some isolates, the susceptibility level remained unchanged.
As shown in table 4, in the present study, we also investigated the activity of sulbactam-EDTA based combinations against ESBLs-producing E.coli strains. All these antibacterial agents, when combined with sulbactam-EDTA, were more active than combinations only with sulbactam against most of the tested isolates, with lower MIC 50 s and MIC 90 s. With the exception of uoroquinolones, doxycycline and orfenicol, all other drugs (the third generation cephaloporins, amikacin and fosfomycin) when combined with sulbactam-EDTA were also found to be more active than combinations only with EDTA against most of the tested isolates, with lower MIC 50 s and MIC 90 s. Among the various combinations containing EDTA, susceptibility to uoroquinolones, doxycycline and orfenicol combinations with sulbactam-EDTA was greater than that to respective combination only with EDTA (the susceptibility level increased of from 4.

Discussion
This is the rst study to investigate the prevalence and drug combinations on ESBLs-producing E. coli isolated from healthy Tibetan yaks. As shown in table 1, the emergence of multidrug resistant (MDR) strains, these nding is in accordance with our previous studies [4][5][6]. This MDR is due to the coexistence of genes encoding drug resistance to other antibiotics on the plasmids which encode ESBLs [17]. In our study, varying range of resistance, from 83.3 to 100% for third generation cephaloporins has been observed. This resistance is due to the hydrolysis of β-lactam ring of lactam antibiotics by the action of ESBLs. Other mechanism of drug resistance to β-lactam group of antibiotics are loss of outer membrane protein, active e ux action and AmpC β-lactamase production. Amongst the mechanisms of resistance to third generation cephaloporins in gram-negative bacilli, production of ESBLs and AmpC β-lactamase are the most common [18,19]. In general, ESBLs resulted in very high levels of extended spectrum cephaloporins resistance, whereas hyperproduction of AmpC β-lactamase and/or impermeability/e ux function gave lower levels resistance [20].
The effect of β-lactam/β-lactamase inhibitor combinations varied depending on the subtype of ESBLs present [17]. Some TEM-derived β-lactamases which are resistant to β-lactamase inhibitor (inhibitorresistant TEMs or IRTs) have beeen described [21,22]. Also, there is limited clinical experience with use βlactam/β-lactamase inhibitor in treating animal infection with ESBLs-producing organisms. Because of these variables, β-lactam/β-lactamase inhibitor combinations should not be considered as rst line of therap and it would seem necessary to nd new therapeutic weapons against this growing threat of MDR in ESBLs-producing isolates. The antibacterial activies of sulbactam was not determined in the present study and its intrinsic activies against ESBLs-producing E.coli isolates should not be excluded. The intrinsic activies of β-lactamase inhibitors, has already been described for some important human pathogens [23,24], with sulbactam being the most effective, but there is still paucity of data on animal pathogens. The phenomenon of sulbactam's enhancing activity of some non-β-lactam antimicromial agents was particularly interesting from a scienti c perspective, as it may indicate a basis for designing more effective antibiotic combinations for treating ESBLs-producing bacterial infections. Further investigations, such as measuring the fractional inhibitory concentration (FIC) index of each combination by checkerboard testing and the killing curve test, should be carried out to understand interactions between sulbactam and non-β-lactam antimicromials and to explore potential clinical implications in veterinary medicine.
In the present study, synergism between EDTA and many antimicrobials have been widely reported against P. aeruginosa and E. coli [14], but activities of the combinations with EDTA against ESBLproducing organisms and their clinical experience is still limited [15]. Our results showed that EDTA may be involved in improved susceptibility of ESBL-producing isolates to antimicrobial agents and MICs reductions are because of the loss of the barrier function of the outer membrane. There are many hydrophilic protein channels located throughout the cytoderm and cell membrane of bacteria, which are nonselective for hydrophilic substances through the cell. It is very important for the hydrophilic drugs (e.g. levo oxacin and doxycycline) to cross into or out of the cell. Moreover, the declination of the outer membrane permeability and the increase of the drug e ux pump have been involved in the mechanism of the MDR [25]. Meanwhile, EDTA can chelate the divalent cations (e.g. Ca 2+ Mg 2+ ), which are the essential component to maintain the structure and function of cell membrane, to result in the increase of the cell mobility and permeability, this explains the mechanism of its anti-multiple drug-resistance. The work suggested combinations of antimicrobials with EDTA may be bene cial in the treatment of infections caused by ESBLs-producing E.coli strains. Further clinical studies or animal models of infection are needed to con rm e cacy and safety of antimicrobials-EDTA combinations.
In this study, these ndings suggested that sulbactam and EDTA had synergism in improved activities of antimicrobials mentioned above (cephaloporins, amikacin and fosfomycin). Meanwhile, these results presented here also implied that there was synergy between speci c resistant mechanism (producing βlactamase) and nonspeci c resistant mechanism (the declination of the outer membrane permeability). This synergy is worth further investigation to elucidate the precise mechanism responsible for this effect and to explore its therapeutic potential in veterinary clinical.

Conclusion
This study presents the rst insight into the prevalence, antimicrobial resistance, and drug combinations on ESBLs-producing E. coli isolated from healthy Tibetan yaks. The high prevalence and ESBLsproducing of E. coli highlights the importance of effective animal hygiene measures to prevent the further spread of E. coli. It is important to consider the prevalence of E. coli in yaks and the risk of its transmission through the food chain. In a word, we have demonstrated that sulbactam can enhance activity of β-lactams and some non-β-lactams of antimicrobial agents (amikacin and fosfomycin) and had a synergistic effects with EDTA in improving activities of the third generation cephaloporins, amikacin and fosfomycin. Future epidemiological investigations should be conducted with larger number of strains and samples.

Collection of samples
From July 2018 to August 2019, a total of 750 nasal swab samples from healthy Tibetan yaks in eight large-scale farms were collected. The samples were immediately transported to the laboratory under required preservation conditions (in a cooler with ice) within 6 h of collection, and processed within 2 h for samples to test the presence of E. coli [16].

Isolation and identi cation of E. coli
Isolation and identi cation of E. coli were performed by enrichment and sequential plating onto selective plates, as previously described [16]. The samples were incubated in LB Broth (Beijing Land Bridge Technology Co., Ltd, Beijing, China) at 37 °C overnight for 12 h, and draw the line on MacConkey agar plateafter dipping the culture the next day. All presumptive E. coli colonies were identi ed using VITEK 2 compact automated identi cation system (BioMérieux, Marcy-I'Etoile, France). Reference strains E. coli ATCC 25922, K. pneumoniae ATCC 25923 and K. pneumoniae ATCC 700603 were used as quality control strains. For all the strains, 3 µl bacteral culture incubated at 37 ℃ for 12 h were diluted in Mueller-Hinton broth to obtain a starting inoculum at 10 5 cfu/mL.

Detection of ESBLs-producing strains
ESBLs were detected by the reference double-disc diffusion test method by the Clinical and Laboratory Standards Institute (CLSI) [26], using discs of cefotaxime (30 µg) and ceftazidime (30 µg) and discs of cefotaxime plus clavulanic acid (30 and 10 µg) and ceftazidime plus clavulanic acid and discs of cefotaxime plus clavulanic acid (30 and 10 µg) and ceftazidime plus clavulanic acid (0.5 McFarland inoculum size) of suspected ESBLs producing clinical isolates on Mueller-Hinton Agar (MHA). E. coli ATCC 25922 was used as the negative control and K.pneumoniae ATCC 700603 was used as the ESBLs positive control. ESBLs production was inferred if the inhibition zone increased by 5 mm towards the cefotaxime plus clavulanic acid disc or ceftazidime plus clavulanic acid disc in comparison to the third generation cephalosporin disc alone.

Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of 11 antimicrobial agents against the 240 ESBLsproducing E. coli strains, were determined by the broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) guidelines [26]. The stock solutions of all antimicrobial compounds were prepared to a nal concentration of 5120 µg/mL. Each antimicrobial solution was sterilized by ltration using 0.2 µm-pore size lters. MICs for gati oxacin, levo oxacin, enro oxacin, cipro oxacin, ceftriaxone, cefotaxime, cefepime, doxycycline, amikacin, orfenicol, fosfomycin alone and their respective combinations with either sulbactam or EDTA or sulbactam-EDTA were determined. Sulbactam was added to these antimicrobial drugs at a xed ratio of 2:1 and EDTA at a xed concentration of 2560 µg/mL which was a twofold concentration below the respective MIC for all isolates.