Enhancement of capsular hypermucoviscosity in Klebsiella pneumoniae by Acanthamoeba

Background Acanthamoeba and Klebsiella pneumoniae are both environmental commensals. Recently, clinical harm caused by hypermucoviscous K. pneumoniae has been observed. However, the interaction between these microbes and the origin of hypermucoviscous K. pneumoniae have not been reported Methodology/Principal findings Here, we report that the bacterial capsule is enlarged when co-cultured with Acanthamoeba using India ink staining, and this effect depends on the number of parasites present. This interaction results in an enhancement of capsular polysaccharide production in the subsequent generations of K. pneumoniae, even without co-culturing with Acanthamoeba. The hypermucoviscosity of the capsule was examined using the sedimentation assay and string test. We also screened other K. pneumoniae serotypes, including K1, K2, K5, and K20, for interaction with Acanthamoeba using India ink staining, and found the same interaction effect Conclusions/Significance These findings suggest that the interaction between Acanthamoeba and K. pneumoniae could lead to harmful consequences in public health and nosocomial disease control, particularly hypermucoviscous K. pneumoniae infections.


Introduction
Klebsiella pneumoniae is a gram-negative bacterium that mainly attacks hosts with a weakened immune system, causing opportunistic and multiple infections [1].K. pneumoniae represents a high proportion of common pathogens in community pneumonia and nosocomial infection in Taiwan [2].K. pneumoniae is an environmental commensal rather than only a clinical pathogen.The environmental habitats of K. pneumoniae include the human mucosal surfaces, soil, and surface water [3].Recently, with the emergence of extremely aggressive K. pneumoniae strains, a new K. pneumoniae infection has been identified in Taiwan, which has been reported to have spread globally [4,5].These hypervirulent strains, which can occasionally cause pneumonia and lung abscesses, are the predominant isolates from patients with pyogenic liver abscesses.Based on the previous studies, these clinical stains of K. pneumoniae contain thickened capsule and increased mucoviscosity compared with those of classical strains (cKP) [6].However, although many studies have investigated the capsular formation mechanism of hypermucoviscous K. pneumoniae (hvKP), the process of transformation of cKP into hvKP is not clear.
Acanthamoeba, a free-living amoeba (FLA), have been isolated from soil, water, and air, and frequently form organic biofilms [7].They can growth private and public water systems, including those used by hospitals, homes, swimming pools, dental clinics, and cooling towers [8].Acanthamoeba feed on bacteria, fungi, and other protists for nutrition; however, many microbes escape digestion by Acanthamoeba [9,10].In the microenvironment, Acanthamoeba secrete various proteins and metabolites, and in recent years, these extracellular substances have been found to have positive and negative effects on the pathogenicity of co-localizing microorganisms [11].For instance, previous studies have revealed that certain secreted proteins, such as aminopeptidases and exosome-like vesicles, have been identified as virulence factors in pathogenic Acanthamoeba [11,12].These proteins, when released by Acanthamoeba, disrupt epithelial cells and induce apoptosis [13].The extracellular proteins secreted by Acanthamoeba not only affect the amoeba itself but also influence the microbiota in the microenvironment [14,15].For instance of its location in the rhizosphere, which is crucial for plantbacteria interactions and plant nutrient uptake [16].Amoeba treatment confirms the dominant role of bacterial grazers in shaping bacteria-plant interactions and promoting plant growth Some microorganisms have evolved adaptive mechanisms that confer resistance against protists.These microorganisms have developed strategies to either avoid internalization by amoebae or to survive, multiply, and ultimately exit from free-living amoebae following engulfment.Previous studies have demonstrated that Legionella pneumophila possesses the ability to undergo intracellular multiplication within Acanthamoeba [17,18].By successfully evading or surviving within protists, these microorganisms demonstrate their capacity to adapt and thrive in the presence of amoebal predators.So far, there are only few studies that reported the extracellular effect of the interaction between Acanthamoeba and co-localizing bacteria on human health.

Culture of Klebsiella pneumoniae
K. pneumoniae isolates were cultured in Luria-Bertani (LB) agar (solidified with 1.5% (w/v) agar) or LB broth.Clinical strains of K. pneumoniae were isolated from the corneal surface of hospitalized patients without any ocular disorder.

Klebsiella pneumoniae stimulation by Acanthamoeba
To evaluate the interaction between K. pneumoniae and Acanthamoeba, we modified the direct contact method described in a previous study [19].K. pneumoniae isolates were cultivated in LB broth at 37˚C until they reached the exponential phase.The cultures were then harvested by centrifugation at 3000 rpm for 5 minutes and resuspended in 200 μL of phosphate-buffered saline (PBS) to obtain the desired experimental concentrations (OD 600 = 0.2, 0.4, 0.8, 1.6), respectively.A calculated number of Acanthamoeba cells (4 × 10 4 , 5 × 10 4 , 8 × 10 4 , and 1.6 × 10 5 ) were uniformly mixed with the bacterial cells in 200 μL of PBS.The mixture containing both bacterial and Acanthamoeba cells was incubated in a 24-well plate and placed in a thermostatic incubator for overnight incubation at 28˚C.

India ink staining
The application of India ink stain reveals the presence of capsules surrounding the organisms, which are visually identifiable as a halo [20].Equal amounts of co-culture and India ink (Sigma-Aldrich, St. Louis, MO) were mixed on a glass slide and then covered with a coverslip.
An OLYMPUS BX51 microscope (Olympus, Tokyo, Japan) was used at a magnification of 1000 ×.

Size measurement of Klebsiella pneumoniae cells
After India ink staining, a total of 50 K.pneumoniae cells were screened under OLYMPUS BX51 microscope for measuring the diameter of dye-unpenetrated area.The GraphPad Prism 5.0 (La Jolla, CA, USA) was used to calculate and interpret statistical data using unpaired twotailed Student's t-test [21].

Phenotype passage test
K. pneumoniae cells were co-cultured with Acanthamoeba for 24 h.The broth containing Acanthamoeba co-cultured K. pneumoniae was subcultured in LB broth using bacteriological loop and then passaged two times overnight at 37˚C.The final passage bacterial colony was screened by India ink staining.Three independent experiments were performed, and images were captured.

Sedimentation assay
Being hypermucoviscous, K. pneumoniae capsules do not sediment well after centrifugation.We have modified a sedimentation assay from the previous study [22].Briefly, utilizing comparable bacterial culture quantities in 3 mL of LB broth, followed by a 10-min centrifugation at 3000 rpm.The optical density (OD) of the sedimentation assay's supernatant was measured at a wavelength of 600 nm.Student's t-test was used to determine statistical significance.

Phenol-sulfuric acid assay
K. pneumoniae capsules containing uronic acid were subjected to the phenol-sulfuric acid assay using previously described techniques [23,24].Extracts from similar amounts of overnight bacterial cultures were reconstituted in 0.1 mL of water, and mixed with 1.2 mL of 12.5 mM tetraborate in concentrated H 2 SO 4 .The liquid was vigorously vortexed before being allowed to boil for 5 min.After cooling, 20 mL of 0.15% 3-hydroxydiphenol (Sigma-Aldrich, St. Louis, MO) was added, and absorbance at 520 nm was measured.Student's t-test was used to determine statistical significance.

String test
K. pneumoniae isolates co-cultured with or without Acanthamoeba were assessed for the hypermucoviscous phenotype using the string test.On blood agar plates, each isolate was grown overnight at 37˚C before expansion of bacterial colonies using an inoculation loop.A positive test result is determined when bacterial colonies stretched on an agar plate with a bacteriology inoculation loop form a viscous string measuring more than 5 mm in length as previous described [25].

Identification of Klebsiella pneumoniae serotype
The serotyping was performed on the wzc sequencing according to a previous study [26].Briefly, after K. pneumoniae DNA extraction, two primer pairs were used to amplify the consensus sequence, including primer pair 1: KP-wza-CF1 and KP-wzc-CR1, and primer pair 2: KP-wza-CF2 and KP-wzc-CR2.The cycling program consisted of 96˚C for 3 min, followed by 30 temperature cycles at 96˚C for 30 s, 46˚C for 15 s, and 72˚C for 3 min.After DNA amplification, the products with the predicted size of 2.7 kb (primer pair 1) and 3.1 kb (primer pair 1)

PLOS NEGLECTED TROPICAL DISEASES
were obtained, and nBLAST tool and National Center for Biotechnology Information (NCBI) database were used.

Isolation of environmental Klebsiella pneumoniae
Samples of water were gathered from canals and rivers located within a 20-kilometer radius of Taichung City.Each sample was filtered through a 0.45 mm-diameter filter (Sartorius, Go ¨ttingen, Germany).The membranes were placed on Simmons citrate agar with 1% (w/v) inositol and incubated for 48 h at 37˚C.The recovery of Klebsiella is not inhibited but is very selective in this medium [27].Isolation of potential Klebsiella colonies was followed by identification using the biochemical tests, including fermentation of melezitose and L-sorbose, gas production from lactose at 44.5˚C, growth at 10˚C, pectate degradation, and utilization of mhydroxybenzoate and hydroxy-L-proline.

Results
To investigate the impact of the interaction between Acanthamoeba and Klebsiella pneumoniae, Acanthamoeba cells at different calculated numbers were co-cultured with clinical K. pneumoniae isolates adjusted to a consistent optical density.Simultaneously, K. pneumoniae cells, adjusted to various experimental densities, were co-cultured with Acanthamoeba cells at a fixed count.After overnight co-culturing, K. pneumoniae cells were harvested using India ink staining.K. pneumoniae cells containing capsule prevent large particles of dye to penetrate the cell and thus provide a negative background for analysis.Interestingly, K. pneumoniae capsules were visually enlarged after co-culturing with Acanthamoeba cells at a density of 8 × 10 4 and 1.6 × 10 5 but showed similar size with the cells at 4 × 10 4 compared with K. pneumoniae without Acanthamoeba as negative control (Fig 1A).However, Acanthamoeba cells at a density of 5 × 10 4 could not stimulate K. pneumoniae capsule to be enlarged at either of the OD 600 value of 0.2, 0.4, 0.8, or 1.6 (Fig 1B).India ink staining data showed that Acanthamoeba could affect K. pneumoniae in the microenvironment, resulting in enlargement of K. pneumoniae cells containing capsule depending on the numbers of Acanthamoeba cells but not on the density of K. pneumoniae cells.
Furthermore, the size of enlarged K. pneumoniae capsule was analyzed.Acanthamoeba cells at a density of 1.0 × 10 5 were added into the microenvironment containing K. pneumoniae cells adjusted to an OD 600 of 0.2 for 24, 72, and 168 h.After the end of experiment, K. pneumoniae cells incubated alone or co-cultured with Acanthamoeba were harvested for cell size measurement.The capsules of K. pneumoniae cells were then stained with India ink, and microscopic images of 50 individual cells were captured for size measurement.The size was measured as the diameter of dye-unpenetrated area of the cell containing the capsule surrounding the bacterial cell.K. pneumoniae cells cultured alone had an average capsular size of 2.827, 3.511, and 2.798 μm after 24, 72, and 168 h, respectively.By contrast, K. pneumoniae cells co-cultured with Acanthamoeba had an average capsular size of 3.346, 4.086, and 3.454 μm after 24, 72, and 168 h, respectively, indicating significant increase at all time intervals (Fig 2).The data suggested that Acanthamoeba could continually stimulate enlargement of the capsule size around K. pneumoniae cells.
However, whether the impact of Acanthamoeba presence on the capsule size of K. pneumoniae cells was brief or persistent would have importance in infection control.Hence, Acanthamoeba co-cultured K. pneumoniae cells were harvested and then passaged for two times without Acanthamoeba.The passaged K. pneumoniae cells were compared for the capsule size using India ink staining.Surprisingly, Acanthamoeba co-cultured K. pneumoniae cells passaged two times showed enlarged capsule compared with K. pneumoniae cultured alone (Fig 3).The presence of enlarged capsule of K. pneumoniae in cells passaged two times without Acanthamoeba indicated the outcome of microorganism's interaction was critical for the follow up problem in public health.
Based on significant enlargement in capsule size of K. pneumoniae after stimulating by Acanthamoeba, we further validated the capsule containing K. pneumoniae cell using phenolsulfuric acid assay to quantify the capsular polysaccharide.The polysaccharide layer of K. pneumoniae capsule extends outside the cell envelope, and uronic acid-based acid complex carbohydrates make up a sizable component of the polysaccharide conjugates.Uronic acid was detectable as an orange compound at a wavelength of 520 nm following phenol treatment, which followed sulfuric acid's conversion of the polysaccharide to monosaccharide.As a result, Acanthamoeba co-cultured K. pneumoniae had a more complex chemical makeup and appeared cloudy during cell diameter measurement.The abundance of the capsular polysaccharide in Acanthamoeba co-cultured K. pneumoniae was shown by considerably higher absorption (OD 520 = 0.351) of the strain than that of K. pneumoniae cultured alone (OD 520 = 0.213) (Fig 4).The data from phenol-sulfuric acid assay confirmed that Acanthamoeba enhanced the capsular polysaccharide secretion in K. pneumoniae.
Furthermore, the enlarged capsule of K. pneumoniae cell containing more polysaccharide would be important for clinical treatment of K. pneumoniae infection.Also, bacterial viscosity of K. pneumoniae is often associated with pathogenicity.Therefore, sedimentation assay and string test were performed to quantify the capsule viscosity.Sedimentation assay revealed that the supernatant of Acanthamoeba co-cultured K. pneumoniae was viscous, and that the precipitation of bacterial cells was difficult.The supernatant of Acanthamoeba co-cultured K. pneumoniae (OD 600 = 1.307) had higher viscosity than that of K. pneumoniae cultured alone (OD 600 = 0.397) (Fig 5A).The string test was used to identify pathogenic characteristic of hypermucoviscous phenotype of K. pneumoniae, and it is a standard method to test the hypervirulent K. pneumoniae in clinical laboratory.When a loop is used to stretch the colony on an agar plate, a positive string test is one that shows the development of viscous strings >5 mm in length.Therefore, we used the string test to examine the bacterial colonies of K. pneumoniae cultured alone, live Acanthamoeba co-cultured K. pneumoniae, and heat-killed Acanthamoeba co-cultured K. pneumoniae.Live Acanthamoeba co-cultured K. pneumoniae exhibited a bacterial strings > 5 mm in length.However, bacterial string from the colony of either heat-killed Acanthamoeba co-cultured K. pneumoniae or K. pneumoniae cultured alone presented a length of <5 mm (Fig 5B).The viscosity data of K. pneumoniae from sedimentation assay and string test showed that Acanthamoeba induced hypermucoviscosity in K. pneumoniae cells, and clinical treatment should be developed based on these findings.
Finally, we used the wzc sequencing to identify the serotype of K. pneumoniae and collect several different serotypes of K. pneumoniae to validate the stimulation of capsule secretion by Acanthamoeba.According to the wzc sequencing, the original tested K. pneumoniae belong to the K81.Simultaneously, the serotypes K1, K2, K5, and K20 were also collected from the environment to test the microorganism's interaction with Acanthamoeba.Obviously, the effect of Acanthamoeba on K. pneumoniae capsule secretion was observed after treatment with Acanthamoeba for 24 h using India ink staining (Fig 6).India ink staining data of

PLOS NEGLECTED TROPICAL DISEASES
Acanthamoeba treatment of diverse serotypes of K. pneumoniae indicated that enhancement of K. pneumoniae capsule secretion by the interaction with Acanthamoeba in the microenvironment was extensive, and that would be crucial for public health and infection control.

Discussion
The existence of diverse endosymbiotic bacteria within Acanthamoeba hosts has been wellestablished for a significant duration [28].Previous study showed that clinical isolates of Acanthamoeba harbored several medically important bacteria including Legionella, Mycobacterium, Pseudomonas, Malassezia and Chlamydia [29,30].It is noteworthy that these bacteria enable them to utilize Acanthamoeba as a training ground to further evade the challenges of the microenvironment, which in turn impacts human infections.For instance, L. pneumophila cells cultivated in amoebae demonstrated a minimum of 100-fold higher invasiveness towards epithelial cells and a 10-fold higher invasiveness towards macrophages and Acanthamoeba [31].Furthermore, it has recently been discovered that Campylobacter jejuni, which is sensitive to environmental stresses, has the ability to invade amoebae, and the invaded cells still undergo encystation [32].These studies collectively indicate that the interaction between Acanthamoeba and bacteria is an important and significant research topic concerning human health hazards.
In this study, we revealed that Acanthamoeba could influence K. pneumoniae capsular polysaccharide secretion in the microenvironment.While the quantity of Acanthamoeba tested in this study may not necessarily be highly prevalent in aquatic environments, the proportional calculation based on the amount of co-cultured K. pneumoniae suggests that a significant proportion of Acanthamoeba populations is still likely to occur in various water bodies, such as rivers, farmlands water, and water towers [33].If we neglect its presence, it could potentially lead to an increase in its population.It is important to note that population densities of Acanthamoeba can vary greatly across different habitats, including freshwater bodies, soil, and man-made environments.Factors such as nutrient availability, temperature, and the presence of suitable hosts can influence the abundance of Acanthamoeba in the wild [34].Therefore, the range mentioned in our study serves as a representative estimate rather than an absolute value applicable to all natural settings.
The enhancement of K. pneumoniae capsular polysaccharide production was dependent on the number of Acanthamoeba cells, suggesting that the effect was as a result of extracellular stimulation by Acanthamoeba.Previous studies have reported that several enzymes or metal ions are released from Acanthamoeba to the microenvironment.The function of potassium ion transporters in the sensory perception of the Acanthamoeba T4 genotype was discovered by Siddiqui et al [35].Also, proton pump, sodium and calcium transporters, and others have all been shown to have a role in the amoeba's differentiation [36].Besides, to survive in the environment and infect the host, Acanthamoeba releases a number of metal-containing enzymes.Iron superoxide dismutase (Fe-SOD) released by Acanthamoeba assist amoeba cells in preventing the environmental oxygen stress.Moreover, a 38-kDa copper-zinc SOD (Cu-Zn-SOD) was isolated from Acanthamoeba to demonstrate its antioxidant and anti-inflammatory properties.Hence, the concentration of microenvironmental metal ions change based on Acanthamoeba's survival, which might accidentally interfere with the phenotype of surrounding microorganisms.Recently, a hypermucoviscous strain of K. pneumoniae (hvKP) has spread across the world.The community can contract illnesses from hvKP as it is more virulent than that of cKP and frequently affects healthy individuals.
Owing to one of the most frequent causes of hospital acquired infections (HAI) and rise in antibiotic-resistant strains, K. pneumoniae has become a significant public health concern.This opportunistic pathogen typically colonizes the gut, throat, and nasal passages without spreading disease, but it can sometimes spread infections to the lungs, skin, urinary system, and bloodstream [37].The hvKP is now recognized for its propensity to cause a number of illnesses.It was first shown to be a cause of pyogenic liver abscesses in Asia [38].However, uncertainty regarding the origin of hvKP is a major problem in investigations.Coincidentally, the opportunistic pathogen in HAI was also thought to be free-living Acanthamoeba.They have been identified in approximately 10.5% of amoeba-associated nosocomial illnesses and have been detected in water samples from 68.9% of hospital faucets [39,40].Moreover, Acanthamoeba was present in 26% of catheter urine samples from a critical care unit in a Brazilian hospital [41].Therefore, the interaction between K. pneumoniae and Acanthamoeba can occur not only in the community but also in the nosocomial environment.
According to the data in this study, the polysaccharide level in K. pneumoniae capsule was elevated, resulting in hypermucoviscous K. pneumoniae with four distinct types of virulence factors, all of which have been extensively studied.Notably, hypervirulent bacteria are particularly hyperactive in terms of capsule production, siderophores, lipopolysaccharide (LPS), and fimbriae/pili.The structure known as the capsule encloses the bacteria and conforms to the polysaccharide to increase pathogenicity of K. pneumoniae [42].The hypermucoviscous capsule had greater immunological resistance than that of the conventional capsule because of the presence of exopolysaccharides with high mucoviscosity.Several important genes for capsule formation are present in the operon cps on the bacterial chromosome of both conventional and high pathogenic K. pneumoniae strains [43].The important capsule biosynthesis genes wzi, wza, wzb, wzc, gnd, wca, cpsB, cpsG, and galF are located at the cps gene cluster [26].Furthermore, multiple studies have demonstrated that upregulating the expression of two plasmid-encoded transcriptional regulators, namely rmpA and rmpA2, or manipulating the RCS two-component system genes, rcsA and rcsB, can enhance the biosynthesis of the hypermucoviscous K. pneumoniae capsule [44,45].Hence, whether the extracellular stress or secreted substances from Acanthamoeba directly or indirectly stimulates the capsule biosynthesis genes of K. pneumoniae needs further investigation.
In conclusion, cKP was transformed into hvKP by interacting with Acanthamoeba in the microenvironment.The phenotypic changes in K. pneumoniae was not dependent on the number of bacterial cells but that of the parasite.Also, the bacterial capsule was enlarged with the time of co-culturing with Acanthamoeba.Acanthamoeba-stimulated K. pneumoniae could continually present hypermucoviscous capsule containing abundant polysaccharides.The parasite could interfere with the K. pneumoniae serotypes K1, K2, K5, K20, and K81, indicating an extensive microorganism's interaction in the community or nosocomial environment.According to a previous study [46], we can utilize in silico molecular modeling investigations, molecular docking, molecular dynamics simulations, and computational toxicity assessments to understand the molecular mechanism underlying the interaction between the parasite and bacteria, taking advantage of advancements in computational approaches.In the future studies, we will constantly investigate the mechanisms underlying the interaction between K. pneumoniae and Acanthamoeba, and its impact on the public health and infection control.