Nifuratel reduces Salmonella survival in macrophages by extracellular and intracellular antibacterial activity

ABSTRACT Salmonella are intracellular bacterial pathogens for which, as with many of the other Enterobacteriaceae, antibiotic resistance is becoming an increasing problem. New antibiotics are being sought as recommended by the World Health Organization and other international institutions. These must be able to penetrate macrophages, and infect the major host cells and the Salmonella-containing vacuole. This study reports screening a small library of Food and Drug Administration (FDA)-approved drugs for their antibacterial effect in macrophages infected with a rapid-multiplying mutant of Salmonella Enteritidis. The most effective drug that was least toxic for macrophages was Nifuratel, a nitrofuran antibiotic already in use for parasitic infections. In mice, it provided 60% protection after oral infection with a lethal S. Enteritidis dose with reduced bacterial numbers in the tissues. It was effective against different serovars, including multidrug-resistant strains of Salmonella Typhimurium, and in macrophages from different host species and against Listeria monocytogenes and Shigella flexneri. It reduced IL-10 and STAT3 production in infected macrophages which should increase the inflammatory response against Salmonella. IMPORTANCE Salmonella can keep long-term persistence in host's macrophages to evade cellular immune defense and antibiotic attack and exit in some condition and reinfect to cause salmonellosis again. In addition to multidrug resistance, this infection circle causes Salmonella clearance difficult in the host, and so there is a great need for new antibacterial agents that reduce intramacrophage Salmonella survival to block endogenous Salmonella reinfection.

The manuscript titled "Nifuratel reduces Salmonella survival in macrophages by extracellular and intracellular antibacterial activity" authored by Xie et al., has addressed pertinent problem of inhibiting Salmonella inhabiting within macrophages.The authors also demonstrated repurposing antiparasitic drug, Nifuratel to inhibit MDR Salmonella.The manuscript warrants major revision, the English communication is extremely poor.It is very hard to understand what is written.The manuscript has been submitted with no basic formatting, no line numbers or page numbers.Table 2: There is no possibility of determining "MIC of different Salmonella serotypes" .MIC Stands for Minimum inhibitory concentration of antibiotics where no visible growth is observed.It could be MIC of compound (Nifuratel) against different strains of Salmonella.Please correct the table title.The authors pretend significance level calculation by stating p value in the text.The authors may explain how many biological replicates were considered in determining significance level.Fig. 2B does not have error bars.The following two statements are self contradictory: "As with the antibiotic sensitive strain SL1344, the two MDR strains of S. Typhimurium TX 2-7 and TZF 10 were not killed completely by Nifuratel at 100 μM (Fig. 6)" in the manuscript text at page # 4 and the Figure legend of the Fig. 6 " Figure 6.Nifuratel showed potent activity against intramacrophage antibiotic resistant Salmonella Typhimurium" .Please clarify.Page #: it is not Lactose dehydrogenase, it is lactate dehydrogenase in the sentence "Cytotoxicity measured as a percentage (Fig. 3A, middle panel) or by lactose dehydrogenase release (Fig. 3A, right panel)" Figure 3A Right panel: The OD570 seems to be identical at all the concentrations of Nifuratel as compared to control and DMSO.Are cells equally viable at all the concentration of Nifuratel?How significant your data are?Please clarify and state how Nifuratel has become less cytotoxic at 25 µM, even better than CIP, if the cells are all equally viable in all concentrations?What was the control taken in this experiment?Ciprofloxacin (CIP) might have missed out in this experiment as it was not plotted.Please clarify the sentence on page # 5: "The antibacterial effects were expressed in several macrophage cell lines, avian, murine and human" In Fig. 2B, F6 compound exhibited less cytotoxicity and more cell viability as compared to E7 (Nifuratel).However, the F6 has not been tested to determine intracellular bacterial load (Fig. 2A).How come authors infer that E7 could be potent candidate to serve as future therapeutic?What are the plausible reasons of ineffectiveness of Nifuratel on antibiotic resistant Salmonella?Can author clarify how an antiparasitic drug could be potent inhibitor of an enteric bacterial pathogen?Does the inhibitor bind and inhibit the function of paralogous enzyme(s) in both the organisms (parasite and bacteria) in the same fashion?It would be nice if authors justify this point as well in the discussion.
Reviewer #2 (Comments for the Author):

General comments:
The rationale behind these studies is strong; i.e., to identify agents with antimicrobial activity versus intra-macrophage Salmonella.Furthermore, the experiments appear to have been carefully done with appropriate methodology, and hence the data are solid.However, the clinical relevance of the study is weakened considerably by two considerations.First, as emphasized in the Introduction, the potential relevance of these studies would be to "typhoid and typhoid-like infections", but data presented is limited to non-typhoid strains, particularly Salmonella Enterititis.Second, activity of the most promising agent, nifuratel, was dissapointingly low versus all Salmonella strains except the mutant Enteritidis strain SE C50041ΔspiC.(Relatedly, clarification is needed as to what the ΔspiC mutation is and why this strain used as model.) With respect to manuscript preparation, the figure axis labels are inconsistent and/or incorrect, and the figure legends require more details.
A control experiment without continuous gentamicin would be useful to rule out the possibility that the compounds screened simply altered macrophage membrane permeability and hence allowed gentamicin entry.Specific comments (no line numbering, so text to be edited in quotations): Figure y axis labels inconsistent and incorrect; i.e., "Intracellular bacteria/CFU" in multiple figures when it should be "Intracellular bacteria (CFU)/macrophage" (or better yet, just "CFU/macrophage", with specifics including bacterial strain and macrophage cell line clearly noted in Figure legend).
Confusing, reword: "...antibiotic classes including aminoglycosides penetrate the eukaryote cells, including macrophages (17,18), poorly, the main cell involved as the host for multiplication of the Salmonella infections (19,20).""After the first-round screen of 66 compounds..." Insert "(Appendix)".(However, this should probably be "Supplementary Table 1" or the like rather than "Appendix") "...10 x 10E5 CFU, and one of these, E7, was selected in contrast to the drug-free control where 12.5 x 10E6 CFU..." These numbers do not appear to match those in Fig. 2A.Specifically, the no drug control yielded ca.125 CFU/5 x 10E5 RAW cells, so to increase this 10E5 times to 12.5 x 10E6 CFU would require 5 x 10E10 RAW, an unlikely high number.
"E7 showed no cytotoxicity at all at the concentration tested of [? insert number] μM." "S.Pullorum strain SPS06004 was killed completely by Nifuratel at 100μM whereas complete killing was not obtained at this concentration for the S. Typhimurium or S. Dublin strains".Dissapointedly low activity then with all but SE C50041ΔspiC."Subsequent experiments showed that Nifuratel was highly active against selected serovars of Salmonella enterica."As noted above, "highly active" was only observed for SE C50041ΔspiC "Antibacterial activity, highly likely to be bactericidal..." Speculative since not tested -delete.
"It should not be surprising that some new antibiotics and other less conventional chemotherapies are found to be more effective than older, standard antibiotics (27).showed that highly lytic bacteriophages could be more effective than single doses of streptomycin in E. coli-infected mice and equally effective to eight doses."Vague and overly general -delete.
"Nifuratel is a nitrofuran derivative...It has also been given vaginally."References needed for this entire paragraph.
"Nifuratel is a good potential drug for the first-line treatment of bacterial vaginosis (35,36).It is active in vitro against the pool of bacteria recognized to cause bacterial vaginosis and, at the same time, it does not affect the normal the flora of lactobacilli (35).It is also active against other bacterial species (37,38)."Redundant with text above -delete or reduce considerably.
"Our previous studies have shown that S. Enteritidis (SE) C50041ΔspiC had an increased ability to proliferate and persist in cultured macrophages in comparison with the wild-type strain (26).On this basis,we selected this strain for screening in RAW264.7"Move from Materials and Methods to beginning of Results."...replaced by 10 μg/mL gentamicin alone or combined with the compound to be screened..." A control experiment without this continuous gentamicin would be useful to rule out the possibility that the compounds screened simply altered macrophage membrane permeability and hence allowed gentamicin entry."...analysis for release of lactate dehydrogenase.Absorbance was measured at 490 nm after mixing 120 μL cell culture supernatant with 60 μl LDH assay working solution..." Reference, or reagent source, or more details needed.
"Twelve hours later Nifuratel (20 mg/kg) in DMSO was also administered by the same route to the mice in group 3" According to Results, this was 24 h later ("administered Nifuratel 24h later")

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Comment 9：Please clarify the sentence on page # 5: "The antibacterial effects were expressed in several macrophage cell lines, avian, murine and human" Response: The sentence has been changed to "The antibacterial effects were expressed in several macrophage cell lines came from avian, murine and human"; Comment 10: In Fig 2B, F6 compound exhibited less cytotoxicity and more cell viability as compared to E7 (Nifuratel).However, the F6 has not been tested to determine intracellular bacterial load (Fig. 2A).How come authors infer that E7 could be potent candidate to serve as future therapeutic?
Response: That's a good question, I choose E7 as one targeted drug based on the drug properties with low cytotoxicity, high cell viability and high antibacterial effect, and this drug was rarely studied against intracellualar Salmonella; but F6(Pefloxacin mesylate dihydrate, which is same with CIP as Quinolone antibiotic) has been studied.
Comment 11: What are the plausible reasons of ineffectiveness of Nifuratel on antibiotic resistant Salmonella?Can author clarify how an antiparasitic drug could be potent inhibitor of an enteric bacterial pathogen?Does the inhibitor bind and inhibit the function of paralogous enzyme(s) in both the organisms (parasite and bacteria) in the same fashion?It would be nice if authors justify this point as well in the discussion.
Response: I am sorry, Nifuratel is effective to antibiotic-resistant Salmonella, such as TX 2-7, TZF 10 need higher concentration.Due to the detailed mechanism, but it is a good question, we mainly focus on this phenotype, we also worked on this mechanism by inhibiting STAT3 phosphorylation to promote inflammatory response to kill intramacrophage Salmonella in the paper, we will study further in-depth.

Responses to Reviewer #2 General comments:
Comment 1: First, as emphasized in the Introduction, the potential relevance of these studies would be to "typhoid and typhoid-like infections", but data presented is limited to non-typhoid strains, particularly Salmonella Enterititis.
Response: Many Salmonella serotypes have been done, among them, S. Typhimurium SL1344, (STm) is typhoid strains, TX 2-7 and TZF 10 are S. Typhimurium.S. Pullorum is typhoid strain to chicken.Other strains such as SE C50041 and S. Dublin 189 (SD) are non-typhoid strains.We will test more strains in the future; Comment 2: Second, activity of the most promising agent, nifuratel, was dissapointingly low versus all Salmonella strains except the mutant Enteritidis strain SE C50041ΔspiC.(Relatedly, clarification is needed as to what the ΔspiC mutation is and why this strain used as model.) Response: At first, "SE C50041ΔspiC"(line 95,100) in the manuscript have been modified to "SE C50041".Second, Nifuratel is effective to tested Salmonella strains, but the degree is different.Our previous studies (Wang, et al, 2021) have shown that C50041ΔspiC keep persistence with increased ability to proliferate in cultured macrophages in comparison with the wild-type strain, and so this strain was selected for durg screen in RAW264.7 to facilitate the analysis of the effect at 20 h poi; Comment 4: A control experiment without continuous gentamicin would be useful to rule out the possibility that the compounds screened simply altered macrophage membrane permeability and hence allowed gentamicin entry.
Response: Operation procedure of blank control experiment has been added."...replaced by 10 μg/mL gentamicin alone or combined with the compound to be screened from the compound library at a concentration of 50 μM, at the same time, no drug treatment was used as blank control.

Specific comments：
Comment 1: "Intracellular bacteria/CFU" in multiple figures when it should be "Intracellular bacteria (CFU)/macrophage" (or better yet, just "CFU/macrophage", with specifics including bacterial strain and macrophage cell line clearly noted in Figure legend)； Response: Figures have been modified, and the details can be seen below; Comment 2: Confusing, reword: "...antibiotic classes including aminoglycosides penetrate the eukaryote cells, including macrophages (17,18), poorly, the main cell involved as the host for multiplication of the Salmonella infections (19,20)." Response: The sentence has been changed to "antibiotic classes including aminoglycosides poorly penetrate the eukaryote cells, including macrophages (17,18), which is the main cell involved as the host for multiplication of the Salmonella infections (19,20).";Comment 3: "After the first-round screen of 66 compounds..." Insert "(Appendix)".(However, this should probably be "Supplementary Table 1" or the like rather than "Appendix") Response: "Appendix" has been changed to "Supplementary Table 1" ; Comment 4: "13 showed the numbers of SE C50041ΔspiC recovered from the cells of less than 10 x 10 5 CFU, and one of these, E7, was selected in contrast to the drug-free control where 12.5 x 10 6 CFU were recovered (Fig. 2A)."These numbers do not appear to match those in Fig. 2A.Specifically, the no drug control yielded ca.125 CFU/5 x 10E5 RAW cells, so to increase this 10E5 times to 12.5 x 10E6 CFU would require 5 x 10E10 RAW, an unlikely high number.
Response: The sentence in the manuscript was modified to"13 compounds including E7 showed the numbers of SE C50041ΔspiC recovered from macrophages with less than 10×10 5 CFU, were selected in contrast to the drug-free control where 12.5×10 6 CFU were recovered (Fig. 2A).Considerable variation in the bacterial counts were observed with some compounds increasing the recovery of viable bacteria.";Fig. 2A(0 to 200) has been recount and redawn (see Comment 10 for details).
Comment 5: "E7 showed no cytotoxicity at all at the concentration tested of [? insert number] μM." Response: The sentence has been added to "E7 showed no cytotoxicity at all at the concentration tested of 25 μM." ; Comment 6: "S.Pullorum strain SPS06004 was killed completely by Nifuratel at 100μM whereas complete killing was not obtained at this concentration for the S. Typhimurium or S. Dublin strains".Dissapointedly low activity then with all but SE C50041ΔspiC.
Response: Comment6 and Comment7 were same question about drug effect of Nifuratel.There was "SE C50041", not "SE C50041ΔspiC", which has been modified in line 100.Nifuratel is effective to tested Salmonella strains, but the degree is different because of other unknown reasons, which need further study.Nifuratel is effective not only to intramacrophage Salmonella, but also to intramacrophage Shigella L9 and Listeria monocytogenes EGD-e; The legend of Fig. 4 and Fig. 5 has been changed to "The ability of NIF against intracellular..." Comment 8: "...mice killed 48 h after oral infection with the SE strain and administered Nifuratel..." Indicate dose.
Response: The sentencehas been changed to "The viable numbers of SE C50041 in the liver and spleen of 5 mice killed 48 h after oral infection with 1.0×10 7 CFU, the LD 100 , of SE strain and administered Nifuratel (20 mg/kg) in DMSO 12 h later are shown in Figure 8A and B";  Response: The legend of Fig. 5 has been changed to "The ability of NIF against intracellular different Salmonella serotypes.";and the "Salmonella strains" was inserted before SE C50041 in Fig. 4; Comment 17: "Subsequent experiments showed that Nifuratel was highly active against selected serovars of Salmonella enterica."As noted above, "highly active" was only observed for SE C50041ΔspiC； Response: The "SE C50041ΔspiC"(line 95,100) in this manuscript should be

Fig 2 .
Is it "Durg" or "Drug" Please clarify.Edit the typo.

Fig
Fig. 2B y axis label: Change to "Cell viability and cytotoxicity (%)" Fig. 2B x axis label: Correct spelling of "Drug" Fig. 3A y axis label for graph on left: Why is this axis (10E0 to 10E8) so different than in Fig. 2A (0 to 200)?Use consistent and correct label for this axis.

Fig
Fig. 3A legend: Descriptions needed for graphs in center and on right.

Fig. 4 ,
Fig. 4, Fig.5 y axis: Again, why this axis and Fig. 2A axis so different?Also again, correct y axis label and make it consistent with Fig. 2A and 3A.

Fig. 5
Fig.5legend: Specify which macrophage used.Insert "Salmonella strains" before SE C50041 Reference: 26.Wang Y, Liu G, Zhang J, Gu D, Hu M, Zhang Y, Pan Z, Geng S, Jiao X. 2021.WbaP is required for swarm motility and intramacrophage multiplication of Salmonella Enteritidis spiC mutant by glucose use ability.Microbiol Res 245:126686.https://doi:10.1016/j.micres.2020.126686.Comment 3: With respect to manuscript preparation, the figure axis labels are inconsistent and/or incorrect, and the figure legends require more details.Response: Figures have been modified, and the details can be seen below(Specific comments).

Comment 9 :
"The P values for each Nifuratel concentration and strain are shown."Not needed here; delete.Response: The sentence of "The P values for each Nifuratel concentration and strain are shown."has been deleted; Comment 10: Fig 2A y axis label: Add "RAW264.7"after "5 x 10E5" (to reduce length, "Intracellular bacterial load" can be deleted).Response: The y axis label of Fig. 2A (0 to 200) was the number of colonies diluted to 10 5 , which has been recount and changed to "Bacterial loads (CFU) in Macrophage".and Fig.2A has been redawn(A2、F5 and F6 were added) ; Comment 11: Fig. 2B y axis label: Change to "Cell viability and cytotoxicity (%)" Response: Fig. 2B has been redrawn and divided into two figures "Cell viability (%)" and "Cytotoxicity (%)"; And the legend has been changed as follows.Comment 12: Fig. 2B x axis label: Correct spelling of "Drug" ?Response: We are sorry for bothering you with this kind of mistakes that we should have avoided.In Fig 2B, "Durg" has been changed to "Drug"; Comment 13: Fig. 3A y axis label for graph on left: Why is this axis (10E0 to 10E8) so different than in Fig. 2A (0 to 200)?Use consistent and correct label for this axis.Response: In the Fig.3A(left), y axis label has been changed to "Bacterial loads

Figure 2 .
Figure 2. Screen of drugs against intracellular Salmonella.(A) Loads (CFU) of SE C50041ΔspiC in RAW264.7 after treatment with 66 drugs.(B) Viable cell ratio and (C) cytotoxicity after treatment with 28 drugs.