Reference Lactobacillus strains as Potential Antimicrobial Agents Against Priority Pathogens with Different Levels of Drug Resistance

doi:10.21203/rs.3.rs-3771802/v1

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Reference Lactobacillus strains as Potential Antimicrobial Agents Against Priority Pathogens with Different Levels of Drug Resistance

https://doi.org/10.21203/rs.3.rs-3771802/v1

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Probiotics are beneficial microorganisms that can exert antibacterial activity against pathogenic bacteria. In this study, we evaluated the antibacterial activity of probiotics reference strains of, Lactobacillus acidophilus(LA, NCDC Accession No. 13), Lactobacillus casei(LC, NCDC Accession No. 17), and Lactobacillus plantarum(LP, NCDC Accession No. 20) against twelve pathogens, some of which are classified as High priority by the World Health Organization (WHO) due to their extensive antibiotic resistance. The probiotics were obtained from the National Collection of Dairy Cultures (NCDC); National Dairy Research Institute (NDRI), Karnal; India as reference strains. The pathogens were isolated from clinical samples and identified by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry, while their antibiotic resistance profiles were identified using VITEK®2. The antibacterial activity of the probiotics was measured by two standard methods (agar well diffusion and agar slab method). The zone of inhibition and the antagonistic activity of the probiotics were recorded and compared. All three probiotics exhibited antibacterial activity against all 12 pathogens, with varying degrees of inhibition and antagonism. The study demonstrated that lactobacilli strains from NDRI had shown to be effective in inhibiting the growth of WHO-priority pathogens.

Biological sciences/Biotechnology

Health sciences/Health care

probiotics

antibacterial activity

WHO priority pathogens

agar slab method

reference Lactobacillus strains

Antibiotic resistance is a serious global health threat that undermines the effectiveness of antimicrobial medicines in treating infections caused by bacteria, viruses, fungi, and parasites¹. Rising Antimicrobial Resistance (AMR) substantially threatens public health and the economy. Antibiotics become less effective due to ever-rising numbers of Multidrug-Resistant (MDR) pathogenic strains. AMR infections are causing more than 700,000 deaths worldwide each year, a number expected to surge to 10 million by 2050 ¹. At the same time, research is increasingly showing that genes are being exchanged between harmful pathogens and what were considered harmless or helpful bacteria. Current data indicates a crude mortality rate attributable to infectious etiologies in India at approximately 417 incidences per 100,000 individuals^2,3.Notably, antibiotic resistance is documented in an array of bacterial taxa, encompassing both Gram-positive and Gram-negative categories. Comprehensive meta-analyses of susceptibilities across diverse Indian microbiological research facilities have elucidated a trend of escalating AMR, particularly against critical pathogens including Staphylococcus spp., Vibrio spp., Salmonella spp., Shigella spp., Neisseria spp., Mycobacterium tuberculosis, and HIV^2,3. Recent observations have also reported the emergence of MDR 'superbugs' with resistance to advanced-spectrum antibiotics such as carbapenems and colistin, which are conventionally considered agents of last resort^2,3.To address the challenge of the complexity of antibiotic resistance, World Health Organization (WHO) has published a list of antibiotic-resistant “priority pathogens” that pose the greatest threat to human health and require urgent research and development of new antibiotics. The list includes twelve families of bacteria that are classified into three categories namely Critical (e.g., Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacteriaceae, Klebsiella, E. coli, Serratia, and Proteus), High (e.g., Enterococcus faecium, Staphylococcus aureus) and Medium (e.g., Streptococcus pneumoniae) priority ⁴. These bacteria are also known as ESKAPE pathogens because they can escape the action of antibiotics and are responsible for a large proportion of hospital-acquired infections ⁵.

Traditionally, effective treatment of infections has relied on antibiotics, used alone or in combination. However, the growing number of ineffective antibiotics against "ESKAPE" pathogens paints a stark picture of a future where these lifesaving drugs might fail us. To combat this evolving threat, scientists are exploring diverse alternatives currently in practice or under development. These promising new avenues include: antibiotics in combination, atimicrobial peptides, photodynamic therapy, bacteriophage therapy, antibacterial antibodies, phytochemicals and nanoparticles^6,7.

Another possible strategy to combat antibiotic resistance is to introduce live microorganisms that confer health benefits to the host when administered in adequate amounts like probiotics. Lactic Acid Bacteria (LAB) are a group of probiotics, which are live microorganisms that have beneficial effects on the gut microbiota. These bacteria have Generally Recognized As Safe (GRAS) status and Qualified Presumption of Safety (QPS) status. They are often added to foods for specific health benefits^8,9. Substantial evidence-led benefits of probiotics have spurred massive interest in their characterization and application for nutrition and health ¹⁰.Probiotics can modulate the host immune system, enhance the intestinal barrier function and inhibit the growth and survival of pathogenic bacteria by producing various antimicrobial substances ¹¹. Lactobacillus is a genus of probiotic bacteria that have been shown to have antimicrobial activity that interferes with pathogen’s DNA replication or metabolism¹².

Lactobacillus strains could help fight antibiotic resistance as they can combat pathogens that the WHO is concerned about, possibly decreasing the reliance on standard antibiotics. It was reported that Lactobacillus strains produce organic acids, hydrogen peroxide, bacteriocins, and other compounds that lower the pH, generate reactive oxygen species, disrupt the cell membrane and interfere with the metabolism of pathogenic bacteria ^13,14. Therefore, Lactobacillus can be used as a potential candidate for preventing and treating infections caused by priority pathogens and reducing the need for antibiotics¹⁵. This approach aligns with the global strategy to diversify the arsenal against infectious diseases and curb the alarming trend of rising antimicrobial resistance. Nonetheless, to implement this strategy effectively, it is essential to invest in comprehensive research to identify the most effective probiotic strains and determine their appropriate doses, formulations, and administration methods for various infections and patient groups.

The current study evaluates the inhibitory potentials of three Lactobacillus strains—Lactobacillus acidophilus (LA) NCDC Accession No. 13, Lactobacillus casei (LC) NCDC Accession No. 17, and Lactobacillus plantarum (LP) NCDC Accession No. 20using agar slab and agar well diffusion techniques. This assessment was aimed at determining their effects on twelve pathogens categorized under the WHO's priority list using standardized protocols. Furthermore, we deliberated on the potential consequences of our results for the application of probiotics as either alternative or adjunctive treatments for infections attributed to high-priority pathogens.

Procurement of Probiotic Strains and Their Maintenance

Probiotic strains namely, Lactobacillus acidophilus (Moro 1900) Hansen and Mocquot 1970 (NDRI III, NCDC Accession No. 13); Lactobacillus casei ssp. Casei, Hansen and Lessel 1971 (NDRI strain RTS, NCDC Accession No. 17); Lactobacillus plantarum, Bergey’s et al. 1934 (NDRI strain 184, NCDC Accession no. 20) were procured from NDRI, Karnal; India in freeze dried condition. The basic growth media for LAB were Man-Rogosa-Sharpe (MRS; Hi-Media, Mumbai, India) agar, broth and Reconstituted Skimmed Milk (RSM − 11 g skimmed milk powder in 100 mL distilled water, pasteurized at 90ºC for 15 min and cooled to 40ºC).

The sterilized skimmed milk bottles were inoculated with freeze-dried cultures and incubated at 37˚C for 8h thereafter stored at 2˚C. The propagation of stock cultures (37˚C) was done once in 15 days to maintain their activity, whereas working cultures were freshly prepared in RSM as and when needed. Cultures were stored at 2˚C between the transfers as per the standard procedure ¹⁶.

Isolation and Characterization of Pathogenic Strains

In this study, we selected twelve pathogens that had previously been isolated from clinical samples. Initial identification was based on phenotypic and morphological analysis, followed by confirmation using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry. Their antibiotic resistance patterns were additionally determined using the VITEK®2 system. Based on their antibiotic resistance profiles, we classified the pathogens as AMR, MDR, Extensively Drug-Resistant (XDR) and Pandrug-Resistant (PDR) using the guidelines of WHO and Global Antimicrobial Resistance and Use Surveillance System (GLASS)¹⁷. The pathogens we selected were Acinetobacter baumannii, Elizabethkingiameningoseptica, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus mitis.

Biochemical Tests for Lactobacillus Strains

The colony morphology of the Lactobacillus strain was examined as per the standard procedures outlined by ^18–20. Observation of the colony morphology was facilitated through the use of a compound light microscope, while cell shape was determined through microscopic examination. Biochemical characterization of the strains was conducted using catalase, oxidase, urease, motility, and nitrate reduction tests as described by ²¹. Fermentation assays for carbohydrates were performed in MRS fermentation broth containing 0.004% bromocresol purple and 2% of the sugars under investigation, as per the methodology proposed in the literature²².

Hemolytic Activity

An investigation was conducted to assess the Red Blood Cell (RBC) lytic activity of Lactobacillus strains using a blood agar medium. The medium, comprised of a base medium supplemented with 7% defibrinated blood, was inoculated with the lactobacilli strains in discrete spots. Following incubation at 37°C for a 24-hour period, the presence of clear zones surrounding the microbial colonies was evaluated. Hemolytic zones were classified as: clear(-hemolysis), greenish (-hemolysis), or none (-hemolysis) ²³.

Antibiotic Susceptibility Test for Lactobacillus under study

The antibiotic susceptibility test was performed following disc diffusion method ²⁴, as described before by Halder and Mandal ²⁵, using MRS agar (Hi-Media, Mumbai, India), and approximately 10⁸ CFU inocula from the three lactobacilli strains ²⁶ were inoculated. The antibiotic discs (Hi-Media, Mumbai, India) used were Amoxicillin (30µg), Ceftriaxone (30µg), Levofloxacin (5µg), Gentamicin (10µg), Aztreonam (30µg), Meropenem (10µg), Erythromycin (15µg), Linezolid (30µg), Tetracycline (30µg), Amoxiclav (30µg), Co-Trimoxazole (25µg).The ZDI values obtained were interpreted according to the criteria mentioned in literature ^27,28; the lactobacilli were grouped into resistant (ZDI: ≤15 mm), sensitive (ZDI: ≥21 mm), or intermediately susceptible (ZDI: 16–20 mm).

Antagonistic Activity Testing of LactobacillusAgainst Pathogens

The antagonistic activity of three lactobacilli strains procured (designated LA, LC and LP) was determined by agar slab and agar-well diffusion method, against the pathogenic strains; Acinetobacter baumannii, Elizabethkingiameningoseptica, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Staphylococcus aureus, Streptococcus pyogens and Streptococcus mitis.

Agar-Well Diffusion Assay

The agar-well diffusion method was performed according to Tagg and McGiven²⁹. On the surface of Muller-Hilton (MH) plate poured with indicator bacterial broth culture of test pathogen, wells (of 8 mm diameter) were prepared using cork borer, and culture filtrates (75 µL/well) of each the lactobacilli (cultivated in both MRS-Broth and RSM) were loaded in the wells marked properly with the isolates’ names. Following 24h incubation at 37ºC (in presence of atmospheric CO₂), Zone Diameter of Inhibition (ZDI) values in mm were recorded. The tests were performed thrice, and the data were represented with mean ± SD. The antagonistic activity of the test lactobacilli in arbitrary unit per mL (AU/mL) was calculated from the calculated values of mean ± standard deviation (SD) as a measure of production of bioactive components using the formula mentioned elsewhere³⁰.

$$\text{A}\text{U}/\text{m}\text{L} =\left(ZDI x 1000\right)/\text{V}\text{o}\text{l}\text{u}\text{m}\text{e} \text{t}\text{a}\text{k}\text{e}\text{n} \text{i}\text{n} \text{t}\text{h}\text{e} \text{w}\text{e}\text{l}\text{l} \left({\mu }\text{L}\right)$$

Where, ZDI denotes “zone diameter of inhibition”

Agar-Slab Assay

The three lactobacilli inoculum, exhibiting a density of 1.8 x 10⁹ Colony Forming Units per milliliter (CFU/mL) and equivalent to a McFarland Standard of 6.0, was aseptically inoculated onto petri dishes separately containing MRS agar substrates. Subsequent to inoculation, the cultures were incubated at a controlled temperature of 37°C for a duration of 24 hours. Then, three slabs of 8 mm diameter each were cut from the solid medium using a sterile cork borer and transferred to MH agar medium containing pathogens at a density of 6.0 x 10⁸ CFU/mL (2.0 according to McFarland Standard) inoculated by pour plate method. The zones of growth inhibition represented as ZDI around the disks were measured after 24h of incubation at 37°C ³¹. The tests were performed thrice, and the data were represented with mean ± SD.

Statistical Analysis

The results of antibacterial activity of lactobacilli strains (grown in both MRS-Broth and RSM) against the indicator pathogens following agar well diffusion method and agar slab method (grown in MRS-Broth) were expressed in terms of ZDI and AU/ml. The two-way Analysis of Variance (ANOVA) and post hoc (Duncan’stest) analysis was done to compare the antagonistic activity of lactobacilli strains against pathogens. All tests were conducted using SPSS software version 27 (Chicago, USA). P-values less than 0.05 were considered statistically significant.

Morphological, Biochemical characteristics and Antibiotic resistance profile of Lactobacillus under study

In this study, we assessed the antibacterial activity of three Lactobacillus strains – LA, LC, and LP – against twelve bacterial pathogens. These strains, sourced from NCDC, NDRI, India, were characterized both morphologically, as shown in Fig. 1, and biochemically, as detailed in Table 1.The colonies of LA were cream-white, circular, and smooth; LC's were white to very light yellow, smooth, with lens-shaped or rhomboid forms; and LP's were creamy-white, circular, smooth, and low convex (Fig. 1). Biochemically, all the three Lactobacillus species were characterized as non-motile, gram-positive, non-spore forming, and negative for catalase, oxidase, urease, motility, and nitrate reduction, with distinct profiles in carbohydrate fermentation and enzymatic reactions. (Table 1). Hemolytic activity demonstrates that there is no clear or greenish zone observed around spot of lactobacilli strains. The hemolytic activity test was significant in demonstrating that none of the lactobacilli strain exhibited harmful hemolytic effects, thus emphasizing their safety profile (Table 1).

The antibiotic susceptibility of three different Lactobacillus strains; LA, LC and LC which are represented in table 2. All three lactobacilli strains show resistance to a variety of antibiotics. For antibiotic Aztreonam, a notable difference in susceptibility was observed. LC exhibited intermediate susceptibility with a 20 mm zone of inhibition, while both LA and LP were categorized as resistant, indicated by the absence of an inhibition zone.Tetracycline showed a range of activity from sensitive to intermediate, with inhibition zones ranging from 19 to 30 mm.Co-Trimoxazole was largely ineffective, as evidenced by the absence of an inhibition zone in LC and LP, and a marginal 8 mm zone in LA, indicating a high level of resistance. Interestingly, Linezolid exhibited consistent intermediate susceptibility against all three LAB strains, with inhibition zones between 19 to 20 mm. The beta-lactam antibiotics Amoxicillin and Amoxiclav demonstrated a range of intermediate susceptibility across the strains, with zones of inhibition from 15 to 23 mm. Ceftriaxone exhibited intermediate susceptibility for all the LAB strains, as evidenced by their zonesof inhibition from 13–15 mm respectively. Lastly, all LAB strains were resistant to Levofloxacin with zones of inhibition from 8 to 10 mm.

Antibiotic Resistance Profiling of Pathogens and categorizing their priority levels as per WHO

A comprehensive overview of the antimicrobial resistance (AMR) patterns exhibited by a range of pathogenic bacterial species presented in table 3. Species like Escherichia coli and Klebsiella pneumoniae have a high priority and are tagged as possible XDR, emphasizing their potential threat to public health. Interestingly, Elizabethkingiameningoseptica is the only strain marked as 'No Priority', despite being categorized as possible XDR. Streptococcus mitis and Streptococcus pyogens have low priority but are nonetheless listed as AMR, indicating that while they are resistant to some drugs, they are not as severe a public health concern as others. Acinetobacter baumannii shows resistance against a wide range of antibiotics, including last-resort options like Colistin, but is still sensitive to Minocycline.

Antagonistic Activity of Lactobacillus using Agar-Well Diffusion Assay (MRS-Broth)

The antibacterial activity of three lactobacilli strains against twelve bacterial pathogens is represented in Fig. 2 and the quantitate measures of ZDIs are represented in table 4. In evaluating the antibacterial activity through the Agar Well Diffusion method, strains of LA, LC, and LP grown in MRS-Broth were collectively tested against a range of pathogens (Table 4). The combined antagonistic effect of these lactobacilli strains varied notably among the pathogens. LA, LC, and LP were most effective against Elizabethkingiameningoseptica, which showed the highest sensitivity, with inhibition zones up to 22.5 ± 0.71 mm. On the opposite end of the spectrum, Klebsiella pneumoniae proved to be the most resistant, presenting the least susceptibility with inhibition zones only as large as 12.0 ± 1.41 mm for LC. Other pathogens displayed intermediate responses; for instance, Acinetobacter baumannii was inhibited with a zone size ranging from 13.0 ± 1.41 mm for LA and LC, to 15.0 ± 1.41 mm for LP. Similarly, Enterococcusfaecium's growth was inhibited by a zone of 17.0 ± 1.41 mm for LC, contrasting with a slightly larger zone of 20.5 ± 2.12 mm for LA. These disparities highlight the specific antagonistic potential of lactobacilli against different pathogens, indicating a spectrum of susceptibility within these bacteria, with Elizabethkingiameningoseptica being the most sensitive and Klebsiellapneumoniae the most resistant to the lactobacilli under study.

The assessment of growth inhibitory activity in terms of AU/ml of lactobacilli strains against range of bacterial pathogens was conducted using MRS-broth as a culture medium is shown in table 5. Strain LA effectively inhibited Pseudomonas aeruginosa (286.67 ± 9.43AU/ml) and Enterococcusfaecium (273.33 ± 28.28AU/ml). Strain LC was most inhibitory against Streptococcus mitis (293.33 ± 18.86 AU/ml) and Salmonellatyphi (240 ± 18.86 AU/ml). Strain LP demonstrated the highest activity against Elizabethkingiameningoseptica (300 ± 9.43 AU/ml) and a slightly higher effect on Streptococcus mitis (280 ± 18.86 AU/ml) compared to other strains. The ANOVA analysis revealed that the Lactobacillus strains do not differ significantly in antibacterial activity (p > 0.05), whereas the pathogens exhibit significant variability in susceptibility (p < 0.05) to these probiotics.

Antagonistic Activity of Lactobacillus using Agar-Well Diffusion Assay (RSM)

The results of the agar well diffusion technique, which was used to test the antibacterial activity of three Lactobacillus strains grown in RSM against twelve bacterial pathogens were represented in Fig. 3 and their quantitate measures of ZDIs shown in table 6. In the investigation of antibacterial properties using the Agar Well Diffusion method, the strains LA, LC, and LP grown in RSM demonstrated comparable results across the board in their effectiveness against different pathogens. The zones of inhibition were more reflective of the pathogen's susceptibility rather than the specific LAB strain used. For instance, all three LAB strains—LA, LC, and LP—produced inhibition zones against Acinetobacter baumannii that were closely aligned, measuring 8.5 ± 0.71 mm for LA and LC, and slightly less for LP at 7.5 ± 0.71 mm. This pattern of similar antibacterial activity was also noted against Streptococcus mitis, with inhibition zones of 8.5 ± 0.71 mm for both LA and LC, and again a marginally smaller zone for LP at 7.5 ± 0.71 mm. The variability in the size of the inhibition zones was more notably attributed to the type of pathogen rather than the lactobacilli strain. Elizabethkingiameningoseptica, for example, was consistently more susceptible, with the highest zone of inhibition at 9.5 ± 0.71 mm for LC, indicative of its general sensitivity to LAB. In contrast, Klebsiella pneumoniae was uniformly more resistant across all three LAB strains, with the smallest recorded inhibition at 6 ± 0.00 mm for LC. These findings underscore the pathogen-dependent nature of the antibacterial activity of LAB strains, suggesting that the effectiveness of LA, LC, and LP is broadly similar against different pathogens, with the degree of inhibition varying more significantly with the type of pathogen rather than the lactobacilli strain employed, same is seen from the statistical analysis. The of two-way ANOVA where internalcomparison Lactobacillus strainsagainst pathogens being non-significant,while significance being observed amongst pathogens tested.

Further, when measured in Antagonistic Units per milliliter (AU/ml). The results uniformly suggest that the difference in growth inhibition is largely influenced by the type of bacterial pathogen rather than the specific LAB strain (Table 7). All three LAB strains showed similar inhibitory effects on Acinetobacter baumannii, with LA and LC registering an activity of 113.33 ± 9.43 AU/ml, and LP closely following at 100 ± 9.43 AU/ml. All the pathogens all exhibited varying degrees of susceptibility to the LAB strains, yet the differences in the inhibitory activity were subtle among LA, LC, and LP.For instance, Elizabethkingiameningoseptica was inhibited to a similar degree by all three strains, with a slightly higher activity observed for LA at 98.33 ± 11.56 AU/ml compared to 87 ± 12.45 AU/ml for LP and 84.53 ± 13.52 AU/ml for LC. Similarly, Enterococcus faecium was inhibited by LA at 106.67 ± 18.86 AU/ml and by LP at 113.33 ± 9.43 AU/ml, with LC showing an activity of 120 ± 18.86 AU/ml, again reflecting minimal variation among the LAB strains.The consistent trend across the data is that the variability in the growth inhibitory activity is more reflective of the intrinsic resistance or susceptibility of the bacterial pathogens, rather than the antagonistic potency of the individual LAB strains which is further validated by two-way ANOVA.

Antagonistic Activity of Lactobacillus using Agar-Slab Assay

The antibacterial activity of three Lactobacillus strains: LA, LC and LP against a variety of bacterial pathogens delineates by Fig. 4 and their quantitate measures of ZDIs shown in table 8. LA and LC Lactobacilliexhibited the strongest antibacterial activity with the highest ZDI observed against Streptococcus pyogenes (21 ± 1.41 mm, 23 ± 2.83 mm and 21.5 ± 2.12 mm respectively for LA, LC and LP) and least effective ZDI against Serratia marcescens (10 ± 1.41 mm, 10.5 ± 2.12 mm and 11 ± 1.41 mm respectively for LA, LC and LP). These results suggest that Streptococcus pyogeneis uniformly the most sensitive to the antibacterial action of the Lactobacillus strains. Subsequent analysis using ANOVA and post hoc (Duncan’s test) indicated the antagonistic activities of the different Lactobacillus strains do not differ significantly (p˂0.05)while the comparison of ZDI values among various pathogens yielded a significant difference (p˃0.05).

The alarming issue of AMR is characterized by the ability of microorganisms to neutralize the efficacy of antimicrobials, posing a significant threat to global public health³². Guidelines for combating AMR are provided by WHO, which also supports research and collaborates with member states for the development of national action plans³³. The classification of antibiotics into distinct groups such as “Access, Watch, and Reserve” is facilitated by WHO to guide proper antibiotic usage. A priority list of antibiotic-resistant bacteria has been developed by WHO, categorizing pathogens into “Critical, High, and Medium” priority based on various factors such as resistance levels, mortality rates, and prevalence³⁴. Efforts by governments and organizations are intended to be guided by this list to focus on the most dangerous pathogens. The term “ESKAPE” pathogens, an acronym for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterales, is used to describe a group of microorganisms known for their resistance to multiple antibiotics and their ability to cause hospital-acquired infections. The urgency of this issue is underscored by these pathogens’ capability to “escape” the action of antibiotics, necessitating ongoing surveillance, research, and global cooperation to address this escalating crisis effectively ^5,35.

The selection of pathogens for this study was meticulously designed to encompass a wide range of antibiotic resistance profiles, including AMR, MDR, XDR and PDR. This approach aligns well with the World Health Organization’s priority list of antibiotic-resistant bacteria, thus ensuring that the study focuses on pathogens that are of high to medium priority in terms of public health risk ³⁶. Additionally, the chosen pathogens include both Gram-negative and Gram-positive bacteria, some of which are also part of the alarming “ESKAPE” pathogens list known for their propensity for hospital-acquired infections and multiple antibiotic resistances. In the context of the research article the comprehensive coverage of various resistance profiles allows for a robust evaluation of Lactobacillus strains as potential probiotic agents. By focusing on these priority pathogens, the study aims to address some of the most pressing challenges in antimicrobial resistance today.

Under current study, the utilization of these well-characterized reference strains ensures a standardized baseline for evaluating their antagonistic efficacy against the chosen antibiotic-resistant pathogens, facilitating comparability across different studies and meta-analyses ^37–39. In terms of approaches for controlling antibiotic-resistant pathogens, a spectrum of strategies is being investigated, from novel antibiotic development to phage therapy. However, there has been increasing attention on the utilization of probiotics, notably Lactobacillus strains, in this context ^15,40,41. This focus is substantiated by the generally safe profile of Lactobacillus, its minimal side-effects, and its capacity to modulate both microbial antagonism and host immune response ¹¹. The emphasis on probiotics like Lactobacillus is further justified by the urgent need for alternative strategies to combat the growing public health crisis of antimicrobial resistance. These reference strains of Lactobacillus offer a unique advantage in this regard, as they have been previously established to exert antagonistic effects against a variety of pathogens ^42,43.

In the broader scientific community, several notable studies have focused on the antagonistic activity of Lactobacillus strains against pathogenic bacteria. For instance, the work by Fábio M. Carvalho and colleagues investigated the efficacy of Lactobacillus plantarum and Lactobacillus rhamnosus in combating Escherichia coli and Staphylococcus aureus biofilms on urinary tract devices ⁴⁴. Similarly, the research by Debashis Halder and colleagues concentrated on the antagonistic activity of indigenous Lactobacillus isolates against human pathogenic bacteria like Escherichia coli and Acinetobacter baumannii⁴⁵. However, these studies did not specifically focus on pathogens characterized for their antibiotic resistance profiles through sophisticated techniques like VITEK® 2, nor did they classify these pathogens as MDR, PDR, or XDR, based on WHO priority lists.

In contrast, the current study elevates the existing research paradigm by employing well-characterized reference strains of Lactobacillus, sourced from the NCDC; NDRI, India to assess their efficacy against pathogens that have been meticulously characterized for their antibiotic resistance profiles. These pathogens were not only identified through advanced methods like MALDI-TOF but were also classified based on the WHO priority list into high-priority pathogens by performing their AMR profiling using VITEK® 2. Furthermore, while other studies like those by Tambekar and Bhutad evaluated the probiotic potential of Lactobacillus sp. from milk of domestic animals ⁴⁶, or Aleksandra Leska and colleagues investigated the antagonistic activity of various Lactobacillus strains against honeybee pathogens ³¹, they did not make use of any reference LAB strains.

Overall, the methodologies selected for this study, although simple, are in perfect alignment with scientific best practices. Additionally, the use of MH agar medium for pathogen culture and MRS agar for Lactobacilli strains adheres to established guidelines, ensuring that the results are compatible and consistent across various studies. Importantly, the use of RSM alongside MRS broth in agar-well diffusion assays adds another layer of robustness to the study. The inclusion of RSM, a medium more closely mimicking natural conditions, allows for a more realistic assessment of the antibacterial potential of Lactobacillus strains. Further in current study, when the strains were cultured in MRS-Broth, LA showed a ZDI of 11 ± 2.83 mm against Acinetobacter baumannii, while in RSM, the ZDI reduced to 8.5 ± 0.71mm. Such variations were consistently observed across all tested pathogens, which resonates with existing studies that have emphasized the strain-specific and pathogen-specific nature of antibacterial activities ^45,47–49.When considering antagonistic activities, AU/mL values ranging from 173.33 ± 37.71 to 300 ± 9.43 was observed, depending on the Lactobacillus strain and the target pathogen. These values align with the previously reported activities ^31,44,45, indicating that while the strains are effective, they may not be as potent as some of those previously studied. The ZDI values in MRS-Broth ranged from 11 ± 2.83 mm (for Acinetobacter baumannii by LA) to 23.0 ± 2.83 mm (for Streptococcus pyogens by LC), whereas in RSM, the ZDIs were generally lower, ranging from 6 ± 0.00 mm (for Klebsiella pneumoniae by LC) to 9.5 ± 0.71 mm (for Serratia marcescens and Salmonella typhi by LC). This underlines the importance of the growth medium in influencing the antibacterial potential of lactobacilli, a crucial aspect often overlooked in probiotic research.

Further, the tests for antibiotic susceptibility and hemolytic activity were conducted to enrich the characterization of the Lactobacillus strains. The disc diffusion method was judiciously chosen for its proven efficacy in determining antibiotic resistance profiles ^24,25,45. We observe a notable correlation regarding the antibiotic susceptibility profiles of Lactobacillus strains. The existing literature on the sensitivity of Lactobacillus species, including L. acidophilus, L. fermentum, and L. plantarum, to a range of antibiotics such as ampicillin, gentamicin, erythromycin, and tetracycline, while also noting an intrinsic resistance to vancomycin is reported^45,50,51. Our data corroborate with these findings to some extent, demonstrating that LA exhibits sensitivity to amoxicillin, which is structurally and functionally similar to ampicillin, with a moderate inhibition zone of 15 mm. This supports the notion that Lactobacillus strains, including LA, maintain their susceptibility to beta-lactam antibiotics.Furthermore, our results reveal a varied response to gentamicin, with LP showing a larger zone of inhibition (14 mm) compared to LA (7 mm), which may indicate a strain-specific variation in sensitivity within the Lactobacillus genus. This is in line with the literature^45,50,51, which suggests a general sensitivity to gentamicin but does not preclude variability among individual strains.With respect to erythromycin, both the literature and our findings show a consistent pattern of susceptibility across the Lactobacillus strains, reinforcing the potential of these probiotics to be used safely without the risk of harboring resistance to commonly used antibiotics.However, our study identified a discrepancy in the susceptibility pattern to co-trimoxazole, where LC and LP showed to be not inhibited by this antibiotic, which contrasts with the broad sensitivity reported in the literature.Hemolytic activity was also not found for lactobacilliunder study corroborating the results in the literature²³. Future research could consider integrating genomic or proteomic approaches for a deeper mechanistic understanding of the interactions between these probiotics and pathogens. Nonetheless, the current study provides a robust foundation for further investigations into the antagonistic potentials of lactobacilli strains against antibiotic-resistant pathogens.

In conclusion, this study confirms the efficacy of Lactobacillus strains Lactobacillus acidophilus (LA), Lactobacillus casei (LC) and Lactobacillus plantarum (LP) in exerting antagonistic effects against a variety of bacterial pathogens that are resistant to multiple drugs, including those with MDR, XDR, and PDR profiles as classified by the WHO. The probiotic strains, characterized by their non-pathogenic biochemical properties and resistance to certain antibiotics, demonstrated inhibitory activities, against multiple priority pathogens. These results underscore the potential of Lactobacillus strains as a novel approach in the fight against the growing threat of antibiotic-resistant bacteria, thereby justifying the study's focus as encapsulated in its title.

This research did not receive any specific grant from funding agencies of public, commercial or of non-profit sectors. The research did not involve human or animal participants.

Acknowledgements

Author, VS is thankful to ScHemeof Developing High quality research (SHODH), Education department, Government of Gujarat, India for providing student support fellowship.Authors acknowledge the support provided by Department of Life science, Food science & Nutrition, School of Sciences, Gujarat university for performing experiments.

Author contributions

VS wrote the manuscript; VS performed experiments, prepared figures, and tables; MD performed experiments; RS conceptualized the idea and supervised the work; RS critically proofread the manuscript; DG conceptualized the work and supervised; RJ supervised the work.

Authors Approval

All listed authors have seen and approved the manuscript, including the names and order of authors.

Competing interest

Authors declare no competing interest.

Data availability

All data generated or analyzed during this study are included in this published article.

Additional Information

Correspondence and request for the material should be addressed to RS.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for profit sectors.

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Table 1 to 8 are available in the Supplementary Files section.

No competing interests reported.

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Reference Lactobacillus strains as Potential Antimicrobial Agents Against Priority Pathogens with Different Levels of Drug Resistance

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Abstract

Figures

Introduction

Materials and Methods

Procurement of Probiotic Strains and Their Maintenance

Isolation and Characterization of Pathogenic Strains

Hemolytic Activity

Agar-Well Diffusion Assay

Agar-Slab Assay

Statistical Analysis

Results

Antibiotic Resistance Profiling of Pathogens and categorizing their priority levels as per WHO

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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