Concomitant Therapy of Inactivated Enterococcus faecalis CECT7121 with Fluoroquinolones in a Salmonella Enteritidis Murine Sepsis Model

Abstract

Optimization of existing antimicrobial therapies is a strategy proposed for extending antimicrobial activity and delaying resistance development. This study aimed to assess the effect of inactivated E. faecalis CECT7121 (I-EFCECT7121) in a combined therapy with Enrofloxacin or Ciprofloxacin in a S. Enteritidis murine sepsis model. Firstly, dose titration studies were performed to set up: (a) Salmonella Enteritidis (SE) Lethal dose 99 (LD99) and (b) safety of I-EFCECT7121 (c) I-EFCECT7121 dosage scheme. Then, I-EFCECT7121 in combined therapy with 4 doses (5 mg/kg) per 12 h of CFX and EFX in an LD99 BALB/c infection, were evaluated. Survival rate was monitored for 20 days in order to estimate the treatment success. The efficacy of both drugs was improved by combining them with the inactivated bacteria. However, only a significant increase (p < 0.05) was observed after I-EFCECT7121 and CFX combined treatment (40% of survival rate). By contrast, each drug alone achieved a 10% of survival rate. These outcomes showed a potential therapeutic synergism when I-EFCECT7121 was concomitantly given with ciprofloxacin.

Introduction

Non-typhoid Salmonella enterica (NTSE) is a zoonotic agent widely distributed in nature being commensals or pathogens in the gastrointestinal tract of animals. Salmonella enterica serotype Enteritidis and Typhimurium constitute the most frequent serovars worldwide and are considered a public health threat [1].

Salmonella isolates from human [2] and animal sources [3] have shown alarming levels of antimicrobial resistance (AMR) for clinically important antimicrobial groups including Fluoroquinolones.

The emergence and widespread of AMR and the lack of new antibiotic development have narrowed the options for treating bacterial infections and led to the need for searching tools to work on the existing antimicrobials to make them useful [4]. Probiotics use has been widely explored over the past 20 years and it has arisen as a promising intervention to target infectious diseases [5]. However, the use of these non-pathogenic microorganisms could carry some risk in certain circumstances [6], but this limitation may be overcome by the use of inactivated probiotics strains (postbiotics) that could preserve the health-promoting effects to the hosts [7].

Enterococcus faecalis CECT7121 (EFCECT7121) is a non-pathogenic strain isolated from natural corn silage that showed immunomodulatory properties in vitro and in vivo [8, 9], while heat-killed EFCECT7121 has proven to induce the production of IL-12, IL-6, TNF-α and IL-10 [9].

Fluoroquinolones are bactericidal antimicrobials that highly concentrate inside phagocytic cells [10]. Enrofloxacin (EFX) is used systemically for the treatment of many infectious diseases in veterinary medicine, while Ciprofloxacin (CFX), the active metabolite of EFX, is mainly used in human medicine [11].

The main goal of this study was to assess the effect of inactivated E. faecalis CECT7121 (I-EFCECT7121) in combination with EFX or CFX in a S. Enteritidis murine sepsis model. Additionally, the S. Enteritidis sepsis model in mice was validated and safety and dosage regimen of I-EFCECT7121 was addressed.

EFCECT7121 was grown in brain heart infusion broth (BHI, Laboratorio Britania, Argentina) for 18 h at 35ºC in an ordinary atmosphere. After incubation, the culture was harvested by centrifugation at 5800×g for 20 min and washed and killed by heating at 70 °C for 20 min. Bacterial suspensions were prepared in sterile saline at approx. 3.0 × 10⁸ CFU/mL. Samples were plated onto BHI agar to verify bacterial death [8].

S. Enteritidis HS182 (SE) was isolated from blood cultures from a patient with an invasive infection at Ramón Santamarina Hospital (Tandil, Argentina).

This strain showed susceptibility in vitro to CFX (MIC = 0.03 μg/mL) and EFX (MIC = 0.06 μg/mL) according to the Clinical and Laboratory Standards Institute [12]. Viable cell counts were validated through the determination of CFU/mL in nutritive agar [8].

Pure reference standards solutions of EFX (Vallée, Campo Grande, Brazil) and CFX hydrochloride (Labyes, Buenos Aires, Argentina) were utilized for orogastric gavage treatments at doses of 5 mg/kg each.

One hundred and seventy BALB/c male and female mice weighing 25–30 g were used for the trials. Male and female mice were assigned in equal proportion to each experimental group. The animals were housed in a temperature-controlled (21 ± 2 °C), light-cycled (12-h light/dark cycle) room. Food and water were provided ad libitum. Mice were daily monitored for assessing clinical and behavioural signs.

First, the Lethal Dose 99 (LD99) of SE was validated using fifty BALB/c mice divided in 5 groups (n = 10). The dose of SE according to Castro et al. [8] was modified and all groups received orogastric inoculums by oral gavage as follows: group 1: 300 µl of sterile saline solution (SSS); group 2: 2 doses per 24 h of 300 µl SE 5 × 10⁶ CFU/mL; group 3: 3 doses per 24 h of 300 µl 5 × 10⁶ CFU/mL; group 4: 2 doses per 24 h of 300 µl SE 5 × 10⁷ CFU/mL; group 5: 3 doses per 24 h of 300 µl 5 × 10⁷ CFU/mL. Mice of Group 4 and group 5 reached 100% of lethality on day 11 post-challenge. However, to reduce mice manipulation and SE dosing, the protocol selected for validating the septic shock model- LD99 was the one used in group 4 (Fig. 1).

Fig. 1.

Lethality curves: determination of LD99 in mice challenged with S. enterica serotype Enteritidis HS182 (SE) according to the different protocols of orogastric administration. Group 1: 300 µl of SSS; group 2: two doses per 24 h of 300 µl SE 5 × 10⁶ CFU/mL; group 3: three doses per 24 h of 300 µl 5 × 10⁶ CFU/mL; group 4: two doses per 24 h of 300 µl SE 5 × 10⁷ CFU/mL; group 5: three doses per 24 h of 300 µl 5 × 10⁷ CFU/mL

Then, fifty BALB/c mice were used to estimate the safety of I-EFCECT7121 and a preliminary dosage scheme of I-EFCECT7121 in the SE septic shock model (Table 1). The scheme used in group 5, giving seven doses of I-EFCECT7121 post-challenge with SE, achieved a higher efficacy on survival rate (20%) and was utilised as guidance for designing the combined treatment assay. However, to test a reduced length therapy, two doses per 24 h of 100 µl I-EFCECT7121 3 × 10⁸ CFU/mL post-challenge was selected for the combined treatment with Fluoroquinolones.

Table 1.

Experimental design for assessing the safety and efficacy of I-EFCECT7121 in a sepsis infection model

Groups	Days
	−3	−2	−1	0	1	2	3	4	5	6	20
Group 1 (n = 10)				□	□	○	○	○	○		Survival %
Group 2 (n = 10)	●	●	●	●	●	●	●	●			Survival %
Group 3 (n = 10)	●	●	●	□ ●	□ ●	●	●	●			Survival %
Group 4 (n = 10)				□ ●	□ ●	●	●	●			Survival %
Group 5 (n = 10)				□ ●	□ ●	●	●	●	●	●	Survival %

Infection challenge: (□) 300 µl Salmonella Enteritidis 5 × 10⁷ CFU/mL (LD99). Treatments: (○) 300 µl Sterile saline solution; (●) 100 µl Inactivated E. faecalis CECT7121 3 × 10⁸ CFU/mL

Finally, seventy BALB/c mice were divided into 7 groups (n = 10) in order to test a potential synergism between I-EFCECT7121 and Fluoroquinolones antimicrobials in the septic shock model. Mice were treated with I-EFCECT7121, EFX or CFX alone, or with the combination of the I-EFCECT7121 with one of the conventional antimicrobial agents (Table 2). The efficacy study was statistically analysed by 2 × 2 contingency tables as Fisher´s Exact Test and Chi² Test. A p-value < 0.05 was considered significant.

Table 2.

Experimental design for assessing the efficacy of combined I-EFCECT7121 and antimicrobials in a sepsis infection- model

Groups	Days
	0	1	2	3	20
Group 1 (n = 10)	□	□	○	○	Survival %
Group 2 (n = 10)	●	●			Survival %
Group 3 (n = 10)	□	□	∆∆	∆∆	Survival %
Group 4 (n = 10)	□	□	∇∇	∇∇	Survival %
Group 5 (n = 10)	□	□	●	●	Survival %
Group 6 (n = 10)	□	□	● ∆∆	● ∆∆	Survival %
Group 7 (n = 10)	□	□	● ∇∇	● ∇∇	Survival %

Infection challenge: (□) 300 µl Salmonella Enteritidis 5 × 10⁷ CFU/mL (LD99). Treatments: (○) 300 µl Sterile saline solution; (●) 100 µl Inactivated E. faecalis CECT7121 3 × 10⁸ CFU/mL; (∆) 300 µl of Ciprofloxacin (5 mg/kg); (∇) 300 µl of Enrofloxacin (5 mg/kg)

The combination of I-EFCECT7121 with CFX and EFX (groups 6 and 7) showed an increase of 40% and 20% in survival rate respectively when compared to group 1. However, only a statistical significance was observed with the combination I-EFCECT7121 + CFX (p < 0.05). Survival values obtained after the separated administration of CFX, EFX, and I-EFCECT7121 (group 3, 4, and 5 respectively) was 10% (Fig. 2). No differences were found between male and female mice within each treatment group.

Fig. 2.

Survival rate in mice challenged with LD99 SE HS182 and treated with I-EFCECT7121 combined with CFX and EFX according to the protocol previously established. Group 1: LD99 SE + two doses of 300 µl SSS per 24 h; group 2: two doses per 24 h of 100 µl I-EFCECT7121 3 × 10⁸ CFU/mL; group 3: LD99 SE + four doses of 300 µl of CFX (5 mg/kg) per 12 h; group 4: LD99 SE + four doses of 300 µl de EFX (5 mg/kg) per 12 h; group 5: LD99 SE + two doses per 24 h of 100 µl I-EFCECT7121 3 × 10⁸ CFU/mL; group 6: LD99 SE + two doses per 12 h of 100 µl I-EFCECT7121 3 × 10⁸ CFU/mL + four doses of 300 µl of CFX (5 mg/kg) per 12 h; group 7: LD99 SE + two doses per 12 h of 100 µl I-EFCECT7121 3 × 10⁸ CFU/mL + four doses of 300 µl of EFX (5 mg/kg) per 12 h

Inactivated lactic acid bacteria have proven to increase the survival rate of mice challenged with Salmonella [13]. In this research, a survival rate of 10% was observed after the administration of I-EFCECT7121 alone (Fig. 2). Although these findings were lower than other reports [13], it is worth pointing out that the challenge of SE utilized in all the efficacy studies was a high inoculum of 5 × 10⁷ CFU/mL SE.

The postulated mechanisms of action behind the beneficial effects of postbiotics include enhancement of epithelial barrier functions and modulation of the microbiota, the immune responses and the systemic metabolic responses [7]. Previous studies have shown the immunomodulatory effects of I-EFCECT [8, 9] and this outcome could be considered the main mechanism behind the protective effect observed in the present report. Non-viable microorganisms remove shelf-life issues and reduce the risks of microbial translocation and infection, especially in immunocompromised patients [6].

The optimisation of the conventional antimicrobial therapy for invasive salmonellosis with an immunomodulatory approach using I-EFCECT7121 was the most outstanding finding. The higher efficacy observed by combining CFX and I-EFCECT7121 could be explained with a pharmacological analysis as CFX is an active metabolite of EFX and usually shows higher potency than EFX [14]. The significant reduction in lethality rate observed with these combinations could be considered a potentiation phenomenon and we hypothesize that it could be explained by: 1- the positive balance in the immune response generated by the direct action of I-EFCECT7121; 2- an increased transport of the antimicrobial agents to the site of infection facilitated by the arrival of phagocytic cells produced by direct stimulus of I-EFCECT7121. It has been postulated that high liposoluble antimicrobials accumulate within phagocytic cells attaining greater concentration at infection sites [10].

An additional advantage of potentiating the effects of antimicrobial drugs with combined therapies with immunomodulators is the possibility of decreasing the time of exposure and the number of antimicrobials used in therapies [15].

The combination of I-EFCECT7121 with CFX showed an increase of 40% (p < 0.05) in survival rate when compared with the control group. Therefore the combination of I-EFCECT7121 with CFX showed a potential therapeutic synergism as a safety-promising strategy for treating invasive SE infections.

Randomized controlled trials, with a more profound study of the immunologic response, are needed to obtain more scientific evidence on the efficacy of the combination of postbiotics and conventional antimicrobial agents against invasive Salmonella (non-typhoidal) infections.

Author’s contributions

All the authors meet the ICMJE authorship criteria. AC, MS and SSB designed the study, contributed to the analysis and interpretation of data, and assisted in the preparation of the manuscript. LS, NGA and MGdY contributed to data collection, interpretation, and writing of the final manuscript. All authors approved the final version of the manuscript.

Funding

This work was financially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina, PICT 2016 nº 608 ANPCyT.

Availability of data and material

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Code availability

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Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Ethical approval

Animal procedures and management protocols were approved by the Ethics Committee according to the Animal Welfare Policy (act 087/02) of the Faculty of Veterinary Medicine, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, Argentina (http://www.vet.unicen.edu.ar).