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. Author manuscript; available in PMC: 2021 Apr 21.
Published in final edited form as: Int Forum Allergy Rhinol. 2020 Jan 10;10(4):444–449. doi: 10.1002/alr.22521

The impact of Lactococcus lactis (probiotic nasal rinse) co-culture on growth of patient-derived strains of Pseudomonas aeruginosa

Do-Yeon Cho 1,2, Daniel Skinner 1, Dong Jin Lim 1, John G Mclemore 1, Connor G Koch 1, Shaoyan Zhang 1, William E Swords 2,3,4, Ryan Hunter 5, David K Crossman 6, Michael R Crowley 6, Jessica W Grayson 1, Steven M Rowe 2,3,7,8, Bradford A Woodworth 1,2
PMCID: PMC8058912  NIHMSID: NIHMS1688608  PMID: 31922358

Abstract

Background:

The Lactococcus strain of bacteria has been introduced as a probiotic nasal rinse for alleged salubrious effects on the sinonasal bacterial microbiome. However, data regarding interactions with pathogenic bacteria within the sinuses are lacking. The purpose of this study is to assess the interaction between L. lactis and patient-derived Pseudomonas aeruginosa, an opportunistic pathogen in recalcitrant chronic rhinosinusitis (CRS).

Methods:

Commercially available probiotic suspension containing L. lactis W136 was grown in an anaerobic chamber and colonies were isolated. Colonies were co-cultured with patient-derived P. aeruginosa strains in the presence of porcine gastric mucin (mimicking human mucus) for 72 hours. P. aeruginosa cultures without L. lactis served as controls. Colony forming units (CFUs) were compared.

Results:

Six P. aeruginosa isolates collected from 5 CRS patients (3 isolates from cystic fibrosis [CF], 1 mucoid strain) and laboratory strain PAO1 were co-cultured with L. lactis. There was no statistical difference in CFUs of 5 P. aeruginosa isolates grown with L. lactis compared to CFUs without presence of L. lactis. CFU counts were much higher when the mucoid strain was co-cultured with L. lactis (CFU+L.lactis = 1.9 × 108 ± 1.44 × 107, CFU−L.lactis = 1.3 × 108 ± 8.9 × 106, p = 0.01, n = 7). L. lactis suppressed the growth of 1 P. aeruginosa strain (CFU+L.lactis = 2.15 × 108 ± 2.9 × 107, CFU−L.lactis = 3.95 × 108 ± 4.8 × 106, p = 0.03, n = 7).

Conclusion:

L. lactis suppressed the growth of 1 patient P. aeruginosa isolate and induced growth of another (a mucoid strain) in in vitro co-culture setting in the presence of mucin. Further experiments are required to assess the underlying interactions between L. lactis and P. aeruginosa.

Keywords: Lactococcus lactis, Pseudomonas, chronic rhinosinusitis, sinusitis, microbiome, probiotic, nasal rinse, sinus rinse, microarray, biofilm

Characterized by impaired mucociliary clearance (MCC) with ensuing compromised microbial elimination, chronic rhinosinusitis (CRS) is known as a multifactorial disease process in which bacterial infection or colonization play a role in the initiation or propagation of the inflammatory response.1 There is mounting evidence that dysbiosis of the sinus microbiota contributes to the pathogenesis of CRS.2 Recent human sample studies have revealed that the microbiome of CRS patients have discrete characteristics compared to healthy controls, and may promulgate chronic inflammation in the absence of infection.3 Disruption of commensal microbiota can lead to benign microbial communities becoming proinflammatory, invasive, or overgrown with pathogens, such as Clostridium difficile colitis.4

The use of topical probiotics is an emerging therapy due to increasing evidence supporting the mutualistic behavior of microbiota, especially in host immunological defense of the gastrointestinal tract.5,6 Therefore, probiotic treatment is now marketed as a natural therapy to offer effective, drug-free alternatives for the treatment of rhinosinusitis. Specifically, the Lactococcus strain has been introduced as a probiotic nasal rinse for alleged salubrious effects on the sinonasal bacterial microbiome. Recent work by Schwartz et al.5 identified Lactococcus probiotics as being well tolerated on nasal epithelium and imparted anti-inflammatory properties (high interleukin 10 [IL-10] secretion) in a proof of concept trial for therapeutic treatment of CRS.

Pseudomonas aeruginosa is an opportunistic pathogen associated with recalcitrant CRS and cystic fibrosis (CF).7 Respiratory infections with P. aeruginosa in CRS populations are particularly detrimental and current therapeutic strategies consist of nasal rinses, corticosteroids, and antibiotics. The pathogenicity of P. aeruginosa includes biofilm formation (decreasing the capability of antibiotic treatment to reach bacteria) and acquired antibiotic resistance, which complicates the strategy for eliminating infections and has encouraged the development of alternate treatment options.8–12 Alexandre et al.13 recently screened a variety of Lactobacilli cultures from milk and the oral cavity of healthy individuals and described 8 strains of the bacteria that were devoid of antibiotic resistance and able to decrease P. aeruginosa virulence factors (elastolytic activity and biofilm formation). In addition, the motilities of P. aeruginosa were markedly inhibited by the lactose fermentation induced by Lactococcus lactis.14

The airway mucus provides an abundant nutrient source for organisms living in the microenvironment and mucins are the major macromolecular constituents of mucus that provide a large reservoir of both carbon and nitrogen.15 Mucins have been known to be the main nutrient source for niche-specific microbiota of the gut and oral cavity.15 Anaerobes produce a variety of enzymes that liberate bioavailable carbohydrates from the glycoproteins within the airway mucin.16 Data is lacking regarding the interaction between the Lactococcus strain and sinus opportunistic pathogens in the presence of mucin in the upper airway. Therefore, the purpose of the present study is to assess the interaction between L. lactis (ProbioRinse™ commercially available probiotic nasal and sinus rinse; Probionase Therapies, Inc., Montreal, QC, Canada) and patient-derived P. aeruginosa strains in the presence of mucin.

Materials and methods

P. aeruginosa preparation

P. aeruginosa PAO-1 strain was obtained as a gift from the University of Pennsylvania (Noam Cohen, MD). Strains of P. aeruginosa were isolated from CRS patients with and without CF in the Rhinology clinic at the University of Alabama at Birmingham (UAB) after approval from the Institutional Review Board (IRB). Luria-Bertani (LB)-Miller broth was inoculated with frozen stock of each strain and incubated at 37°C on a shaker (200 rpm) overnight. Overnight cultures were streaked on LB-Miller agar plates and incubated at 37°C overnight. Isolated colonies were transferred to 10 mL LB-Miller broth and incubated again at 37°C on a shaker (200 rpm) overnight. Colony forming units per microliter (CFU/mL) were determined by serial dilution and plating on LB-Miller agar.

L. lactis preparation

L. lactis W136 strain was obtained by culturing ProbioRinse in Lactobacilli de Man, Rogosa & Sharpe (MRS) broth (Becton Dickinson Difco, Franklin Lakes, NJ) at 30°C for 24 hours under anaerobic conditions. Twenty-four–hour cultures were streaked on Lactobacilli MRS plates and incubated at 30°C for 24 hours under anaerobic conditions. Isolated colonies were transferred to Lactobacilli MRS broth and incubated at 30°C for 24 hours under anaerobic conditions. Values of CFU/mL were determined by serial dilution and plating on Lactobacilli MRS agar. L. lactis W136 strain was also cultured under aerobic conditions; however, isolated colonies were not identified (repeated ×10).

In vitro co-culturing

To assess the interaction between L. lactis and patient derived P. aeruginosa, L. lactis was co-cultured with P. aeruginosa in the presence of mucin (Fig. 1). A minimal mucin medium containing intact mucins was purified from porcine gastric mucin (PGM) (Sigma-Aldrich, St. Louis, MO) per protocol described by Flynn et al.15 PGM was first dialyzed and filtered to remove small metabolites that could potentially support growth of bacteria. L. lactis was introduced into 2 mL of mucin minimal medium in molten 1.0% agar at 50°C in a polystyrene culture tube (Fisher Scientific Company, Pittsburg, PA), under anaerobic conditions for a final concentration of 5 × 105 CFU/mL (L. lactis did not grow under aerobic condition) and washed twice with phosphate buffered saline (PBS). Tubes without L. lactis were used as a negative control. After solidification of the L. lactis fraction, 1 mL of molten minimal medium 0.7% agar without mucins was introduced and then inoculated with 1 of the P. aeruginosa strains, as described in P. aeruginosa preparation above, for a final concentration of 1 × 106 CFU/mL. After solidification, co-cultures were incubated at 37°C for 72 hours. The upper agar plugs were removed and homogenized in a 1 to 10 dilution with PBS. Values of CFU/mL were determined by serial dilution and plating on LB-Miller agar.

FIGURE 1.

FIGURE 1.

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In vitro co-culturing. To assess the interactions between L. lactis and patient derived P. aeruginosa strains, P. aeruginosa was co-cultured in the presence of porcine gastric mucin with and without L. lactis.

RNA extraction

Total RNA was extracted from selected patient-derived P. aeruginosa using the Qiagen All Prep DNA/RNA MicroKit (Qiagen, Valencia, CA) was used according to the manufacturer’s instructions, with extended proteinase K digestion. Prior to amplification, the quality and level of degradation of the extracted RNA was assessed with RNA integrity number (RIN) assigned by the Agilent 2100 Bioanalyzer instrument using the RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA). All tested samples were stored in nuclease-free tubes and stored at −80°C until shipment for analysis.

RNA sequencing

RNA samples were submitted to the Genomics Core Laboratory in the Heflin Center of Genomic Sciences at the University of Alabama at Birmingham for sample preparation and sequencing. The samples were first DNase-treated and assessed for total RNA quality using the Agilent 2100 Bioanalyzer. Because the bacterial RNA is not polyadenylated, a bacterial ribosome reduction strategy was chosen. RiboMinus kit (Thermo Fisher Scientific, Waltham, MA) was used based on the manufacturer’s protocol to remove the ribosomal RNA. The RNA-sequencing libraries were made with the remaining RNA using the NEBNext Stranded RNA-Sequencing kit according to the manufacturer’s instructions (New England BioLabs, Inc., Ipswich, MA). The resulting cDNA libraries were sequenced on the Illumina NextSeq 500 instrument (Illumina, San Diego, CA) using standard protocols. Finally, single-end 75-basepair (bp) sequencing for all bacterial transcriptomics was performed.

Gene expression analysis

Estimated degree of gene expression in prokaryotes (EDGE-pro; v.1.3.1; http://ccb.jhu.edu/software/EDGE-pro/) was used to align the raw RNA-Seq fastq reads to the P. aeruginosa PAO1 reference genome (ASM676v1).17 Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) used the aligned reads from EDGE-pro to assemble transcripts, estimate their abundances, and test for differential expression and regulation.18 Cuffmerge, which is part of Cufflinks, merged the assembled transcripts to the reference annotation and was capable of tracking Cufflinks transcripts across multiple experiments. Finally, Cuffdiff was used to find significant changes in transcript expression. The false discovery rate (FDR) q values were calculated by Cuffdiff from the raw p values, which are estimated using beta negative binomial tests of variance in read counts. Transcripts were considered to be differentially expressed if their expression values (log2) differed by a factor of 1.5 and FDR <0.05.

Statistical analysis

Statistical analyses were conducted using Excel 2016 (Microsoft Corp., Redmond, WA) and GraphPad Prism 6.0 software (GraphPad, La Jolla, CA) with significance set at p < 0.05. Statistical evaluation utilized unpaired Student t tests or analysis of variance (ANOVA) based on the characteristics of analysis. Data is expressed ± standard error of the mean.

Results

Six P. aeruginosa isolates were collected from 5 CRS patients and a total of 7 P. aeruginosa isolates (including PAO1 strain) were co-cultured with L. lactis in the presence of mucin. Patient characteristics are described in Table 1 (male:female = 2:4, mean age = 51.5 ± 8.8). Of those 7 strains, 3 were from CF patients and 1 from a patient with primary ciliary dyskinesia (PCD). One CF patient grew 2 strains of P. aeruginosa and 1 of those was a mucoid strain of P. aeruginosa. Staphylococcus aureus was co-cultured in 3 patients during the sinus culture. These S. aureus strains were isolated at the time of sinus culture but not included in our co-culture analysis.

TABLE 1.

Demographics

Strain Age (years) Gender Presence of CF (genotype) or PCD Mucoid Other bacteria Pulmonary phenotype
1–1 30 Female Yes (F508del/Q1411X) Yes Methicillin-sensitive S. aureus Bronchiectasis
1–2 No
2 39 Female Yes (F508del/F508del) No Methicillin-resistant S. aureus Bronchiectasis
3 76 Male No No Methicillin-sensitive S. aureus None
4 45 Female Yes No None Bronchiectasis
5 68 Male No No None None
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CF = cystic fibrosis, PCD = primary ciliary dyskinesia.

Gross observations of the cultures’ physical characteristics in the polystyrene culture tubes were performed after 72 hours of co-culture. Of the 7 strains of P. aeruginosa, prominent growth (thick band) of P. aeruginosa was detected in several isolates after 72 hours of co-culture, especially in isolates PA 1–1 (upper panel) and PA 2 (upper and lower panels) (Fig. 2). When counting CFUs after 72-hour of co-culture (Fig. 3), there was no statistical difference in CFUs of 5 P. aeruginosa isolates grown with L. lactis compared to CFUs incubated without L. lactis. However, CFU counts were significantly higher when the mucoid strain (isolate 1–1) was co-cultured with L. lactis (CFU+L.lactis = 1.93 × 108 ± 1.44 × 107, CFU−L.lactis = 1.3 × 108 ± 8.9 × 106, p = 0.01, n = 7). L. lactis suppressed the growth of 1 non-mucoid strain (CFU+L.lactis = 2.15 × 108 ± 2.9 × 107, CFU−L.lactis = 3.95 × 108 ± 4.8 × 106, p = 0.03, n = 7) (Fig. 3).

FIGURE 2.

FIGURE 2.

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Culture tubes after 72-hour of co-culture. PA co-cultured with LL in the presence of mucin in culture tubes (upper panel). PA co-cultured without LL in the presence of mucin (lower panel). Prominent growth (a thick band) of PA was identified in some isolates (marked *) after 72-hour of co-culture. LL = L. lactis; PA = P. aeruginosa.

FIGURE 3.

FIGURE 3.

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Colony forming units after 72 hours of co-culture. There was no statistical difference in CFUs of 5 P. aeruginosa isolates grown with L. lactis compared to CFUs without presence of L. lactis. However, CFU counts were significantly higher when the mucoid strain (isolate 1–1) was co-cultured with L. lactis (**p = 0.01). L. lactis suppressed the growth of one non-mucoid strain (*p = 0.03). CFU = colony forming units.

For P. aeruginosa strains (PA 1–1 and PA 2) that demonstrated significant differences in growth with and without L. lactis, the RNA sequencing approach (RNA-seq) was used to assess the associated gene expressions of P. aeruginosa. Bowtie2 overall alignment rates (repeated twice) are shown in Table 2. More than 98% of the reads from PA 1–1 and PAO1 strains mapped to reference genome. However, only 38% of PA 2 strains mapped to reference genome, meaning that the colonies from PA 2 could be contaminated or divergent from the reference strain. Further whole-genome sequencing analysis showed that the rest of 62% of the read from PA 2 strains were from Stenotrophomonas maltophilia. Therefore, the PA 2 strain was excluded when analyzing the gene expression patterns.

TABLE 2.

Bowtie2 overall alignment rate

Strain 1–1 2 PAO1
Condition PA + M + LL PA + M PA + M + LL PA + M PA + M + LL PA + M
Rate, % 98.47 98.12 36.96 38.08 98.73 98.46
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LL = L. lactis; M = mucin; PA = P. aeruginosa

Pseudomonas genes were differentially expressed ≥2-fold in mucin with L. lactis compared to mucin only media without L. lactis (Table 3). When the specific mucoid strain from CF patient 1 was incubated with L. lactis, genes responsible for drug resistance and motility (swarming) were upregulated (p < 0.05).

TABLE 3.

Pseudomonas genes differently expressed in mucin with L. lactis

Gene ID Protein product Fold changes mucin + L. lactis vs mucin alone
PA4223 Pyochelin biosynthesis (swarming) +2.76
PA1078 flgC Flagellar basal-body rod protein (motility) +2.12
PA 4527 pilC Type 4 fimbrial assembly protein (motility) +2.29
armR Anti-repressor (multi-drug efflux pumps) +2.02
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Discussion

Probiotics are defined as nonpathogenic bacteria that are allochthonous to the bacterial community of the digestive tract. Most bacterial probiotics are strains of the lactic acid bacteria (LAB) Lactobacillus.19 Fermentation products produced by lactic acid producing bacteria have been shown to inhibit the proliferation of pathogenic bacteria (eg, Pseudomonas) and to improve human gastrointestinal health. Even et al.20 previously showed that LAB inhibited the expression of several virulence genes of Staphylococcus aureus without impacting its growth. However, data regarding its interactions with pathogens cultured from the human sinus are lacking and there is no data demonstrating effectiveness of probiotics in the upper airway in the presence of mucin. Therefore, in the current study, we investigated the growth of 6 patient-derived and 1 laboratory strain of P. aeruginosa with and without presence of L. lactis, cultured from commercially available probiotic nasal rinse. To our knowledge, this is the first study to quantitatively assess the interaction between P. aeruginosa and L. lactis using a co-culture technique in the presence of mucin.

Based on previously published studies, L. lactis is thought to prevent the growth of P. aeruginosa through several mechanisms (eg, pH or inhibiting the motility of flagellated pathogens).14 However, in the current study, L. lactis had no observable effects on the growth of 5 of the P. aeruginosa strains and only suppressed the growth of 1 isolate, which was later determined to be contaminated with S. maltophilia. Importantly, co-culture actually induced the growth of the 1 mucoid strain included for analysis by upregulating genes responsible for motility and drug resistance. These experiments were replicated 7 times and identical results were observed in each experiment. We are currently exploring underlying mechanisms of these findings.

Mucins are a family of large glycoprotein polymers expressed both as cell membrane–tethered molecules and as a major component of the mucus gel when secreted by the goblet cells of the epithelium.21 Mucins represent an abundant source of both amino acids and sugars, and play a key role in shaping the microbial community structure.15 Porcine mucin has been used in multiple studies to assess the interaction between mucin and P. aeruginosa in vitro.15,22–24 Early studies of PGM concluded that its general structure and chemical composition are similar to those of human gastric and respiratory mucins.25–27 In the current study, porcine mucin was filtered and dialyzed, leaving only large intact glycoproteins (as described by Flynn et al.15).

Mucin-fermenting microbes (mostly anaerobes) are known to produce mixed acid metabolites (short chain fatty acids [SCFAs]), which have the potential to stimulate pathogen growth by offering a carbon source to nonfermenting sinus pathogens (eg, Pseudomonas, Moraxella, Stenotrophomonas) that cannot utilize sugar.15 Although Pseudomonas ineffectually utilizes mucins as a carbon source on its own,15 mucin fermentation by these anaerobic microbes can stimulate the growth of P. aeruginosa. Moreover, authors recently found that SCFAs were also abundant and available in CRS patients with and without P. aeruginosa.28 Studies have shown that there are higher levels of airway mucins (upregulation of MUC5AC and MUC5B) in CRS, compared to controls.29–31 Therefore, the high levels of utilizable metabolites present in sinus mucus may be derived from bacterial mucin degradation by anaerobes in the sinus cavity, which could contribute to the proliferation of sinus pathogens in CRS. Although the mechanism by which L. lactis induced growth in this co-culture setting is unclear, 1 possible explanation is related to L. lactis’s (anaerobic bacteria) capability to convert glucose (sugar in the mucin) into lactate.28 The lactate produced fermentatively by L. lactis and mucin may serve as an electron donor for aerobic respiration of Pseudomonas and cause proliferation of certain strains.32

Probiotics have been used safely in foods and dairy products for over 100 years and there has been increasing interest in their use to prevent, mitigate, or treat specific diseases.33 A multitude of clinical trials have investigated the use of probiotics for diseases ranging from necrotizing colitis in premature infants to hypertension and allergic rhinitis in adults.34–36 Theoretical risks have been described in case reports, clinical trial results, and experimental models, include systemic infections, deleterious metabolic activities, excessive immune stimulation in immunocompromised patients, and gastrointestinal adverse effects.33 A series of rigorously designed prospective clinical trials should be executed to evaluate any new agent for safety and effectiveness.

There are several limitations to this study. We used an in vitro culture model with filtered porcine mucin rather than in vivo subjects with human mucus, and this may not reflect the in vivo situation in the human sinus cavity. It also ignores contributions of other bacteria in the microbiome and postoperative sinus cavities that could be aerated well with oxygen, and which may not be supportive for the growth of probiotic bacteria (anaerobes). To further explore these findings, future studies will include in vivo experiments using animal models. P. aeruginosa and L. lactis could presumably be interacting with other microbes including commensals in vivo (as seen in patient 2 with S. maltophilia). Therefore, co-habitation properties of these microbes could be altered and in vivo findings may be different from the results of this in vitro study. In future experiments, we plan to quantitatively analyze the interaction between other strains of LAB and clinical isolates of P. aeruginosa.

Conclusion

L. lactis co-cultured with P. aeruginosa in the presence of mucin induced growth in 1 strain, inhibited growth in another, and had no observable impact on 5 other isolates. These effects were highly reproducible and consistent. Topical probiotic nasal rinse may not be universally provided as a “one-size-fits-all” supplement and further experiments are required to assess the underlying interactions between L. lactis and P. aeruginosa.

Acknowledgments

Funding sources for the study: NIH (National Heart, Lung, and Blood Institute [NHLBI] 1 R01 HL133006-04 to B.A.W.; National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK] 5P30DK072482-04, CF Research Center Pilot Award to B.A.W.; National Institutes of Allergy and Infectious Disease [NIAID] K08AI146220); John W. Kirklin Research and Education Foundation Fellowship Award; UAB Faculty Development Research Award; American Rhinologic Society New Investigator Award; and Cystic Fibrosis Foundation Research Development Pilot grant (ROWE15R0) to D.Y.C.

Footnotes

Potential conflict of interest: B.A.W. is a consultant for Cook Medical and Smith and Nephew; he also receives grant support from Cook Medical and Bionorica Inc. There are no disclosures from other authors.

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