Fluoroquinolones’ Biological Activities against Laboratory Microbes and Cancer Cell Lines

Development of novel derivatives to rein in and fight bacteria have never been more demanding, as microbial resistance strains are alarmingly increasing. A multitude of new fluoroquinolones derivatives with an improved spectrum of activity and/or enhanced pharmacokinetics parameters have been widely explored. Reporting novel antimicrobial agents entails comparing their potential activity to their parent drugs; hence, parent fluoroquinolones have been used in research as positive controls. Given that these fluoroquinolones possess variable activities according to their generation, it is necessary to include parent compounds and market available antibiotics of the same class when investigating antimicrobial activity. Herein, we provide a detailed guide on the in vitro biological activity of fluoroquinolones based on experimental results published in the last years. This work permits researchers to compare and analyze potential fluoroquinolones as positive control agents and to evaluate changes occurring in their activities. More importantly, the selection of fluoroquinolones as positive controls by medicinal chemists when investigating novel FQs analogs must be correlated to the laboratory pathogen inquest for reliable results.

Additionally, literature reviews pointed out FQs' potential activities as anticancer, antitumor, antiviral, and antifungal agents in addition to their antibacterial activity where the latter is attributed to their ability to selectively inhibit bacterial type II topoisomerases, DNA gyrase, and/or topoisomerase IV [12][13][14][15].
Currently, FQs are one of the most widely used antimicrobial drugs, with a wide range of indications, covering respiratory infections, urinary tract infections (UTIs), gastrointestinal infections, and gynecologic infections [16]. Moreover, FQs are indicated as a prophylactic treatment in immune-compromised neutropenic patients [17]. FQs are usually classified into four generations with enhanced efficacy and spectrum of activity, along with enhanced safety and pharmacokinetic characteristics ( Figure 2) [18,19]. Ciprofloxacin is the most prosperous derivative, both economically and clinically [20], and the newer generations such as levofloxacin, gemifloxacin, and moxifloxacin offer enhanced activity against aerobic Gram-negative bacilli and Gram-positive bacteria over ciprofloxacin, e.g., against Streptococcus pneumoniae (S. pneumoniae) and S. aureus [20]. Ciprofloxacin and moxifloxacin retain enhanced in vitro activity against P. aeruginosa [21]. In terms of potency, moxifloxacin is more potent against Gram-positive and anaerobes than ciprofloxacin and levofloxacin. Newer generations displayed potent activity against penicillin-resistant and multidrug-resistant (MDR) pneumococcus and anaerobic bacteria. Recently, delafloxacin was granted approval in 2017 for the systemic treatment of acute bacterial skin infections [22]. Additionally, literature reviews pointed out FQs' potential activities as anticancer, antitumor, antiviral, and antifungal agents in addition to their antibacterial activity where the latter is attributed to their ability to selectively inhibit bacterial type II topoisomerases, DNA gyrase, and/or topoisomerase IV [12][13][14][15].
Currently, FQs are one of the most widely used antimicrobial drugs, with a wide range of indications, covering respiratory infections, urinary tract infections (UTIs), gastrointestinal infections, and gynecologic infections [16]. Moreover, FQs are indicated as a prophylactic treatment in immune-compromised neutropenic patients [17].
FQs are usually classified into four generations with enhanced efficacy and spectrum of activity, along with enhanced safety and pharmacokinetic characteristics ( Figure 2) [18,19]. Ciprofloxacin is the most prosperous derivative, both economically and clinically [20], and the newer generations such as levofloxacin, gemifloxacin, and moxifloxacin offer enhanced activity against aerobic Gram-negative bacilli and Gram-positive bacteria over ciprofloxacin, e.g., against Streptococcus pneumoniae (S. pneumoniae) and S. aureus [20]. Ciprofloxacin and moxifloxacin retain enhanced in vitro activity against P. aeruginosa [21]. In terms of potency, moxifloxacin is more potent against Gram-positive and anaerobes than ciprofloxacin and levofloxacin. Newer generations displayed potent activity against penicillin-resistant and multidrug-resistant (MDR) pneumococcus and anaerobic bacteria. Recently, delafloxacin was granted approval in 2017 for the systemic treatment of acute bacterial skin infections [22].

Secound generation FQs
•Improved gram-negative coverage compared with first generation FQs •Limited Gram-positive coverage •Coverage of atypical pathogens, enterobacteriaceae, Pseudomonas aeruginosa •Oral and intravenous administration

Third generation FQs
•Similar to second-generation but with modest streptococcal coverage •Enterobacteriaceae, atypical pathogens, streptococci, Pneumoccoci •Oral and intravenous administration

Fourth generation FQs
•Similar to third-generation, but with improved gram-positive and anaerobic coverages •Enterobacteriaceae, P. aeruginosa, atypical pathogens, MSSA, streptococci, anaerobes, Pneumoccoci •Oral and intravenous administration Widening of the antibacterial activity of fluoroquinolones in relation to their generation. Reproduced/adapted from ref. [13].
Appraisal of the newer FQs' derivatives should be, in part, based on the relevant references. Herein, commonly employed FQ acting as positive controls in antimicrobial bioassays of up-to-date papers were reviewed. These results were reported in a constructive and comparative manner to facilitate the process of developing novel FQs' analogues. The chemical structures and key physical properties of the frequently adopted standard FQs, namely norfloxacin 1, ciprofloxacin 2, levofloxacin 3, and moxifloxacin 4 are summarized in Table 1. This should provide a facile referral guide to recent research areas concerning FQs derivatives antibacterial inhibitory effect, the adopted testing protocols, and generationsbased comparison between different FQs to be applied in innovative research. Choosing standard FQs will not only affect the assessment of the new counterparts, but also provide a more comprehensive and efficient performance in assays. Table 1. Most adopted standard fluoroquinolones, their chemical structures, and key physical properties.

Fluoroquinolone
Structure Generation Physical Properties References

Norfloxacin
Appraisal of the newer FQs' derivatives should be, in part, based on the relevant references. Herein, commonly employed FQ acting as positive controls in antimicrobial bioassays of up-to-date papers were reviewed. These results were reported in a constructive and comparative manner to facilitate the process of developing novel FQs' analogues. The chemical structures and key physical properties of the frequently adopted standard FQs, namely norfloxacin 1, ciprofloxacin 2, levofloxacin 3, and moxifloxacin 4 are summarized in Table 1. This should provide a facile referral guide to recent research areas concerning FQs derivatives antibacterial inhibitory effect, the adopted testing protocols, and generations-based comparison between different FQs to be applied in innovative research. Choosing standard FQs will not only affect the assessment of the new counterparts, but also provide a more comprehensive and efficient performance in assays.  [23,24] Ciprofloxacin Appraisal of the newer FQs' derivatives should be, in part, based on the relevant references. Herein, commonly employed FQ acting as positive controls in antimicrobial bioassays of up-to-date papers were reviewed. These results were reported in a constructive and comparative manner to facilitate the process of developing novel FQs' analogues. The chemical structures and key physical properties of the frequently adopted standard FQs, namely norfloxacin 1, ciprofloxacin 2, levofloxacin 3, and moxifloxacin 4 are summarized in Table 1. This should provide a facile referral guide to recent research areas concerning FQs derivatives antibacterial inhibitory effect, the adopted testing protocols, and generations-based comparison between different FQs to be applied in innovative research. Choosing standard FQs will not only affect the assessment of the new counterparts, but also provide a more comprehensive and efficient performance in assays.

Levofloxacin
Appraisal of the newer FQs' derivatives should be, in part, based on the relevant references. Herein, commonly employed FQ acting as positive controls in antimicrobial bioassays of up-to-date papers were reviewed. These results were reported in a constructive and comparative manner to facilitate the process of developing novel FQs' analogues. The chemical structures and key physical properties of the frequently adopted standard FQs, namely norfloxacin 1, ciprofloxacin 2, levofloxacin 3, and moxifloxacin 4 are summarized in Table 1. This should provide a facile referral guide to recent research areas concerning FQs derivatives antibacterial inhibitory effect, the adopted testing protocols, and generations-based comparison between different FQs to be applied in innovative research. Choosing standard FQs will not only affect the assessment of the new counterparts, but also provide a more comprehensive and efficient performance in assays.

Moxifloxacin
Appraisal of the newer FQs' derivatives should be, in part, based on the relevant references. Herein, commonly employed FQ acting as positive controls in antimicrobial bioassays of up-to-date papers were reviewed. These results were reported in a constructive and comparative manner to facilitate the process of developing novel FQs' analogues. The chemical structures and key physical properties of the frequently adopted standard FQs, namely norfloxacin 1, ciprofloxacin 2, levofloxacin 3, and moxifloxacin 4 are summarized in Table 1. This should provide a facile referral guide to recent research areas concerning FQs derivatives antibacterial inhibitory effect, the adopted testing protocols, and generations-based comparison between different FQs to be applied in innovative research. Choosing standard FQs will not only affect the assessment of the new counterparts, but also provide a more comprehensive and efficient performance in assays.

Comparison of the In Vitro Antimicrobial Assays
A variety of methods and tactics could be adopted to evaluate the antibacterial activity of potential agents, and to draw constructive conclusions. In this regard, choosing and performing these assays varies according to the antimicrobial agents, availability of equipment, and cost-related reasons. The most known and basic standard methods are disk-diffusion [29] and broth or agar dilution methods [30]. The advantages and disadvantages of these assays are summarized in Table 2 and reviewed elsewhere [31,32], being apart from the scope of this article. In brief, standardized antimicrobial bioassays (antimicrobial susceptibility testing) are nowadays published and approved by the Clinical and Laboratory Standards Institute (CLSI) for bacteria and yeasts testing [33], herein the most commonly reported bioassays and the antimicrobial values of various FQs analogues are reported.
Dilution methods afford quantitative evaluation of the in vitro antimicrobial activity, which are usually expressed as minimum inhibitory concentration (MIC) values and represent the lowest concentration of the tested antimicrobial agent that inhibits the visible growth of tested microorganism. A number of approved guidelines for dilution antimicrobial susceptibility testing of fastidious or non-fastidious bacteria, yeast, and filamentous fungi are reported [30].
On the other hand, agar disk-diffusion method is the standard qualitative method for routine antimicrobial susceptibility testing. This method provides qualitative results by categorizing bacteria as susceptible, intermediate, or resistant based on the obtained growth zones of inhibition (ZOI) diameters. However, important parameters, including the growth media, temperature, period of incubation, and the required inoculum size should be optimized to fulfil CLSI standards [22].
Differently, measuring the inhibition of supercoiling activity (catalytic activity) of DNA gyrase or the concentration of compounds required for inhibiting 50% of gyrase supercoiling activity (IC 50 ) has been widely reported as an alternative assay to test the antibacterial activity of different FQs derivatives, particularly if the mechanistic and catalytical activity of the developed analogues are of concern [34,35].

FQ's Antibacterial Activity against Gram-Positive Bacteria
According to the reviewed literature in the past five years, and for the sake of including up-to-date activities on the most common FQs applied as golden antimicrobial positive controls in laboratories, herein, standard FQs and their antimicrobial activity against a panel of laboratory microbes are reported ( Table 3).

FQs Antibacterial Activity against Gram-Negative Bacteria
A summary of common laboratory tested Gram-negative bacteria and standard fluoroquinolones antibiotics are presented in Table 4. It is noticeable that ciprofloxacin has potential antibacterial activity against Gram-negative bacteria as P. aeruginosa and E. coli. [28,48]. Moreover, ciprofloxacin had prospective growth inhibitory activity against H. pylori NCTC 11916 and 12 more H. pylori clinical isolates as reported by Abu-Sini et al. [72]. Ciprofloxacin broad spectrum of activity against aerobic and anaerobic Gram-negative bacteria is shown in Table 4.
Nevertheless, Gorityala et al. [56] reported that ciprofloxacin potency against P. aeruginosa were superior compared to moxifloxacin. This pattern was also noticed in results published by Türe et al. and Garza et al., [28,50].
A similar pattern of the wide range of MIC values against the same strain was observed, where the MIC of norfloxacin against E. coli ATCC-25922 ranged from <0.094 µM [24] to 117.433 µM [45].

FQs' Antifungal, Antiparasitic, and Anticancer Activity
Apart from their antibacterial activity, FQs were also tested for their antifungal activity with little effect on most fungi. Since the late 1980s, studies revealed anti-trypanosomal activity for the quinolones prototype, nalidixic, and oxolonic acid derivatives [14]. Other studies illustrated the antiparasitic activity of norfloxacin against Plasmodium falciparum and the inhibitory effect of other fluoroquinolones against Plasmodium family [14,83,84]. Today, quinolone-amides related derivatives were used to design anti-trypanosomal compounds with many of them presenting potential in vivo activity [85].

FQs Inhibitory Effect as Anti-Viral Agaents against SARS-CoV-2 and HIV-1
As researchers investigate several approaches to combat COVID-19 infection, there is a wide interest in fluoroquinolones. Ciprofloxacin and Moxifloxacin were tested through in silico molecular docking and showed the potential binding capacity to SARS-CoV-2 main protease (M pro ) and low binding energy. Moreover, a recent study evaluated the potency and cellular toxicity of selected FQs (enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin) against SARS-CoV-2 and MERS-COV. This study showed that a high concentration of the tested FQs should be employed to prevent viral replication with enoxacin being the superior (EC50 of 126.4) against SARS-CoV-2 [14,83,84]. Other studies evaluated FQs anti-HIV-1 activities. However, FQs standards activity were not presented [65].

Recommendations
Based on recently published research where FQs were used as positive controls against several microorganisms and cancer cells, it is recommended to use the most active FQ in future studies in addition to the parent drugs to compare the benefits and to have an accurate insight when reporting results.
The difference perceived in FQs' potency according to different research articles is challenging and could be attributed to several factors, including the different testing protocols implemented by each research group, solvents or broth used in bacterial culturing, incubation time, bacterial concentration tested, bacterial growth phase, reader instrument sensitivity, etc.
Ciprofloxacin is recommended to be used as a control against Gram-negative bacteria whether resistant or susceptible. If mainly Gram-positive activity is concerned, levofloxacin or moxifloxacin might be the best choices. The wide-spectrum and potent newer generations should be compared with, when broader comparison is desired. Choose moxifloxacin if the development of newer FQs derivatives is not a biologically-based design. This should provide a proper perspective when reporting novel FQs and their activities. Working against Mycobacterium stains, moxifloxacin was found to be more active compared to the other FQs, thus it is advisable to consider it as a positive control.
Moreover, the authors spur adopting preliminary activity testing of the chosen strains before commencing biological evaluation of interest as some of the stains might not be susceptible to the reference drugs. Lastly, given that some stains exhibited varied MIC values against the same drug, we recommend revising the adopted protocols beforehand to get more accurate comparable results of the reference drug, which will be then more reliable to base the conclusions upon.