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In Vitro Inhibition of Klebsiella pneumoniae by Lactobacillus delbrueckii Subsp. delbrueckii LDD01 (DSM 22106)

An Innovative Strategy to Possibly Counteract Such Infections in Humans?

Mogna, Luca PhD; Deidda, Francesca BS; Nicola, Stefania PhD; Amoruso, Angela PhD; Del Piano, Mario MD; Mogna, Giovanni BS

doi: 10.1097/MCG.0000000000000680

Goals: To determine the in vitro antimicrobial activity of selected Lactobacillus strains isolated from the feces of healthy humans against Klebsiella pneumoniae.

Background: Klebsiella is ubiquitous in nature and may colonize the skin, the pharynx, or the gastrointestinal tract of humans. Despite the widespread use of antibiotic molecules with a broad spectrum in hospitalized patients, an increased overall load of klebsiellae as well as the subsequent development of multidrug-resistant strains able to synthesize extended-spectrum beta-lactamase have been registered. These strains are particularly virulent, express capsular-type K55, and have a considerable ability to propagate.

Study: The 4 strains Lactobacillus paracasei LPC01 (CNCM I-1390), Lactobacillus rhamnosus LR04 (DSM 16605), Bifidobacterium longum B2274 (DSM 24707), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106) were tested. The analysis was performed using both a disc-diffusion assay and the broth-dilution procedure, also including an evaluation of the supernatants obtained from a fresh broth culture of each bacterium.

Results: L. delbrueckii subsp. delbrueckii LDD01 demonstrated the best inhibitory results among all the tested strains. The antibacterial activity of the supernatant was retained even after treatment with α-amylase and neutralization with NaOH 1N, thus suggesting the protein structure of the inhibitory molecule. In contrast, it was completely lost after treatment with proteinase K.

Conclusions: Overall results suggest that the inhibitory effect of L. delbrueckii subsp. delbrueckii LDD01 should be attributed to the production of a bacteriocin. This strain may be prospectively useful for strengthening probiotic formulations and possibly counteract infections by K. pneumoniae in humans.

*Biolab Research Ltd

Probiotical SpA, Novara

Gastroenterology Department, Santa Rita Hospital—Policlinico di Monza, Vercelli, Italy

Supported by Probiotical S.p.A.

L.M., F.D., S.N., and A.A. are employees of Biolab Research Ltd. G.M. is the General Manager of Probiotical S.p.A. M.D.P. is the Chief of Gastroenterology Department, Santa Rita Hospital—Policlinico di Monza.

Address correspondence to: Luca Mogna, PhD, Research & Development, Biolab Research Ltd, Via E. Mattei, 3, Novara 28100, Italy (e-mail:

The genus Klebsiella belongs to the family Enterobacteriaceae.1 The bacteria are named after Edwin Klebs, a 19th century German microbiologist. Klebsiellae are rod-shaped, nonmotile, gram-negative bacteria with a noticeable polysaccharide capsule coating the whole cell surface. This capsule is responsible for the large appearance of the bacterium on Gram stain and their resistance toward various defense mechanisms of the host.2

The genus Klebsiella was formerly divided into 3 main species on the basis of its biochemical reactions. At present, 7 species with demonstrated similarities in genomic sequence are known,3 namely Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica. K. pneumoniae is the most important species of the group from a medical point of view, whereas K. oxytoca and K. rhinoscleromatis have also been recovered from human clinical specimens. In recent years, klebsiellae have acquired a steadily growing significance in nosocomial (hospital-acquired) infections.4

Klebsiellae are ubiquitous in nature. In humans, this microbe may colonize the skin, the pharynx, or the gastrointestinal tract5 as well as sterile wounds and urine. The bacterial load varies with different studies. Klebsiellae may be considered as a part of the normal microbiota in many segments of the colon and the biliary ducts.6 Oropharyngeal foci of infection have been related to compromised host defenses, endotracheal intubation, and the use of antimicrobials.

The range of clinical syndromes includes pneumonia, urinary tract infection, thrombophlebitis, diarrhea, bacteremia, wounds infection, cholecystitis, upper respiratory tract infection, osteomyelitis, and meningitis. The presence of invasive devices, the use of urinary catheters and antibiotics, and the contamination of respiratory support equipment increase the likelihood of nosocomial infections sustained by Klebsiella species. Sepsis and septic shock may be caused by the diffusion of bacteria into the blood from a focal source.7–9

The widespread use of antibiotic molecules characterized by a broad spectrum in hospitalized patients has steered to an undesirable increase in the overall load of klebsiellae and, subsequently, to the development of multidrug-resistant strains able to synthesize extended-spectrum beta-lactamase.10 They efficiently hydrolyze penicillins, cephalosporins, carbapenems, monobactams, and even β-lactamase inhibitors. These strains are particularly virulent, express capsular-type K55, and have a considerable ability to propagate.

The bacteria escape the innate immunity of the host through several means, mainly due to the polysaccharide capsule, which is the main determinant of their pathogenicity.

In some parts of the world, K. pneumoniae is a significant cause of community-acquired pneumonia in the elderly. Studies conducted in Japan and Malaysia reported an incidence rate of 15% to 40%, which is equal, if not superior, to that of Haemophilus influenzae.11,12 In the United States, however, the most relevant risk involves persons with alcoholism as they constitute up to 66% of the people affected by this disease. The associated mortality rate is around 50%, even if in persons with alcoholism and bacteremia could approach 100%.13

Klebsiellae also account for roughly 8% of all hospital-acquired infections among the pediatric populations. In the United States, they represent one of the top 8 pathogens in hospitals. Klebsiellae could be regarded as the second reason of gram-negative sepsis after Escherichia coli and are involved in around 14% of the cases of primary bacteremia.14,15

In this context, it may be useful to find one or more probiotic bacteria with a high inhibitory activity, at least on an in vitro basis, against klebsiellae, with particular reference to the species K. pneumoniae.

Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” The actual mechanism of action of probiotics is subject to constant, increasingly in-depth studies, with regard to their beneficial effects manifested at the gut level and also in other parts of the body, including the respiratory tract. The beneficial effects exerted by probiotics on the host include the inhibition of harmful or pathogenic bacteria growth. A previous study by Savino et al16 showed that 2 of 27 strains of Lactobacillus examined possessed an antimicrobial effect against 6 species of gas-forming coliforms isolated from colicky infants, including K. pneumoniae CG 23a.

The aim of the present study was to determine the in vitro antimicrobial activity of selected Lactobacillus strains isolated from the feces of healthy humans against K. pneumoniae.

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Bacterial Strains and Growth Conditions

The strains Lactobacillus paracasei LPC01 (CNCM I-1390), Lactobacillus rhamnosus LR04 (DSM 16605), Bifidobacterium longum B2274 (DSM 24707), and Lactobacillus delbrueckii subsp. delbrueckii LDD01 (DSM 22106), all isolated from the human feces of healthy individuals, were classified on the basis of their phenotypic and genotypic characteristics. Furthermore, L. rhamnosus GG (ATCC 53103) was used as the control strain.

Two K. pneumoniae strains from the American Type Culture Collection (ATCC) were used to assess the inhibitory activity of lactobacilli. In particular, the 2 bacteria ATCC 10031 and ATCC 13883 (type strain) were used.

All the probiotic strains were cultured in de Man, Rogosa and Sharpe (MRS) medium (BD Difco, Milan, Italy) at 37°C under anaerobic conditions (GasPak system) with Anaerocult A kits (Merck Millipore, Vimodrone (MI), Italy) and stored at −80°C in spent MRS broth, supplemented with 20% (vol/vol) glycerol. MRS broth (BD Difco) was used in all experiments.

Each K. pneumoniae was cultured using MacConkey broth (Merck Millipore) and incubated at 37°C for 24 hours.

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Assessment of the Inhibitory Activity of Probiotic Strains

The screening of the possible production of inhibitory molecules, such as bacteriocins, was performed by the disc-diffusion method carried out according to the standard method described by Bauer et al.17

A Klebsiella pneumoniae culture, after adjustment to 0.5 McFarland standard, was used to seed MacConkey agar plates evenly using a sterile swab. The plates were dried for 15 minutes and then used for the sensitivity test.

Culture supernatants of the 5 probiotics tested (LGG, LR04, LPC01, LDD01, B2274) were prepared as follows: an overnight culture of each isolate was centrifuged at 5000g. The resulting supernatant was neutralized at pH 6.5 with NaOH 1N (Carlo Erba, Cornaredo (MI), Italy), sterilized by filtration through syringe filters (Minisart pore size 0.2 μm) (Sigma-Aldrich, Milano, Italy), and assayed for the presence of any inhibitory molecule in the broth.

Disks, after being soaked with 0.1 mL of a supernatant, were placed on the MacConkey agar surface, and plates were incubated overnight at 37°C. At the end of the incubation, the plates were examined for the appearance of clear zones showing antagonistic activity. The halos were then measured using calipers and recorded. This test was repeated 3 times for each strain to ensure reliability and reproducibility.

Results were expressed as the diameter of inhibition halos (mm). The cutoff for a result to be regarded as significant was 2 mm. The quantification of inhibitory activities of probiotics was performed at Biolab Research Pathogens Laboratory of the Mofin Alce Group in Novara, Italy.

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Cocultivation Assay With L. delbrueckii Subsp. delbrueckii LDD01

In the cocultivation experiments, 105 to 106 Klebsiella pneumoniae cells were cocultivated with 105 to 106L. delbrueckii subsp. delbrueckii LDD01 cells in MRS broth.18 The incubation was continued for 6 and 16 hours at 37°C. The K. pneumoniae strain was then enumerated using suitable decimal dilutions with a sterile saline. About 1 mL of each appropriate dilution was plated on the MacConkey agar selective medium (Sigma-Aldrich).

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Proteinase K Sensitivity

A cell-free supernatant was harvested starting from 1 mL of overnight culture of L. delbrueckii subsp. delbrueckii LDD01 and added with proteinase K (Sigma-Aldrich) to achieve a final concentration of 50 mg/mL, and then incubated for 2 hours at 37°C.19

At the end of the incubation, the reaction mixture was boiled for 10 minutes to inactivate the hydrolytic enzyme and the residual antibacterial activity was measured using the disc-diffusion method as described above. The proteinase K-treated sample and untreated cell-free supernatant were used as controls in the experiment.

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Inhibitory Activity of Probiotic Strains

Diameters of the clear zones measured at the end of incubation of agar plates are reported in the histogram in Figure 1. The results obtained with K. pneumoniae ATCC 10031 only are shown as very similar numbers were achieved with the other biotype. L. delbrueckii subsp. delbrueckii LDD01 showed the utmost antagonistic effect, with an inhibition halo close to 1 cm and a very good reproducibility of the results. The other 4 strains tested, L. paracasei LPC01, L. rhamnosus LR04, B. longum B2274, and L. rhamnosus GG, had a slight to moderate inhibition feature, with L. paracasei LPC01 rated as the second most effective bacterium.



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Cocultivation Assay With L. delbrueckii Subsp. delbrueckii LDD01

The cocultivation of L. delbrueckii subsp. delbrueckii LDD01 with a K. pneumoniae strain induced a significant inhibition of the growth extent of the pathogen starting from 4 hours after the beginning of incubation (Fig. 2). After 16 hours of incubation, a >5-log inhibition was recorded in all the 3 independent experiments compared with the positive control represented by Klebsiella grown alone. As for Figure 1, only results obtained with K. pneumoniae ATCC 10031 are shown.



The treatment of the LDD01 supernatant with proteinase K showed a complete disappearance of the inhibitory activity (data not shown).

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Some selected probiotic bacteria can hinder the growth of definite microbial groups within the gut microbiota using various metabolic pathways, either specific or nonspecific. The former are predominantly mediated by the synthesis of defined metabolites, of which hydrogen peroxide, extracellular proteins, and bacteriocins deserve particular emphasis, whereas the latter involves the acidification of the surrounding microenvironment by organic acids, with a specific mention of lactic and acetic acids as well as short-chain fatty acids.20–22

During the last few years, a substantial body of scientific evidence has accumulated suggestions that certain surface-associated and extracellular components produced by probiotic bacteria could be involved in various mechanisms of action. These bacterial components are able to directly interact with the host mucosal cells and include exopolysaccharides, bacteriocins, lipoteichoic acids, and surface-associated and extracellular proteins. About 25% to 30% of the bacterial proteins function in the cell envelop or outside of the cell.23

The present study demonstrates the in vitro ability of 4 specific lactobacilli and bifidobacteria, first of all L. delbrueckii subsp. delbrueckii LDD01 (DSM 22106), to strongly antagonize K. pneumoniae growth. This feature was confirmed both using the sterilized supernatant at neutral pH of an overnight culture of the lactobacillus and during a cocultivation test.

It is also interesting to note that treatment of the cell-free supernatant with proteinase K induced a complete disappearance of the antagonistic activity, therefore suggesting the protein structure of the molecules responsible for the experimental data observed.

The overall microbiological results relevantly involve specific metabolites underlying the in vitro inhibitory activity observed in this study, with particular reference to bacteriocins in the case of L. delbrueckii subsp. delbrueckii LDD01. For what concerns the other 4 bacteria, extracellular proteins or hydrogen peroxide may also be involved, even if some additional experiments would be needed to further investigate the nature of the inhibition recorded with the disc-diffusion test.

In any case, the present data are appropriate to claim the contribution of more precise pathways other than acidity alone, detectable only in a restricted number of beneficial bacteria with specific properties and mainly in the view of characterizing and optimizing innovative probiotic-based products.

This research could disclose new perspectives for the application of this probiotic to offer an acute and possible long-term protection against K. pneumoniae strains, well known to be responsible for widespread nosocomial infections and relatively frequent outbreaks. In vivo studies will be needed to confirm and extend these preliminary indications.

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Klebsiella pneumoniae; probiotics; antibiotic-resistance; nosocomial infections; antimicrobial activity

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