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Detection and co-occurrence of Acanthamoeba and Klebsiella pneumoniae in freshwater river systems of Taichung, Taiwan

BMC Microbiology volume 25, Article number: 143 (2025) Cite this article

Abstract

Background

This study focuses on the detection and co-occurrence of Acanthamoeba and Klebsiella pneumoniae in freshwater river systems. Both microorganisms are known for their pathogenic potential, with Acanthamoeba capable of causing infections such as amoebic keratitis, and K. pneumoniae being a common cause of community infections. Understanding their presence and distribution in natural aquatic environments can provide insights into potential public health risks, especially in regions with significant human-water interactions.

Method

Water samples were collected from five major hydrophilic environments in Taichung, Taiwan. Polymerase chain reaction assays were employed to detect the presence of Acanthamoeba and K. pneumoniae. In addition to microbial detection, environmental parameters, including temperature, pH, reactive oxygen species, and water hardness, were measured to assess their potential influence on microbial presence. Data analysis focused on identifying patterns of detection and co-occurrence between the two microorganisms.

Results

The study revealed variable detection rates of both Acanthamoeba and K. pneumoniae across the five sampling sites. Co-occurrence of the two microorganisms was observed at several locations, indicating their potential interaction in the environment. While water quality parameters were measured, no strong correlation was found between these factors and microbial detection rates. The widespread distribution of both organisms highlights their resilience in diverse freshwater environments. However, this study did not include virulence factor analysis, and the role of environmental conditions in modulating bacterial pathogenicity remains speculative.

Conclusion

This research demonstrates that Acanthamoeba and K. pneumoniae are both prevalent in the freshwater ecosystems of Taichung, Taiwan, with notable instances of co-occurrence. Although environmental parameters such as temperature and pH did not significantly influence detection rates, the presence of these organisms in frequently accessed water bodies may pose significant public health risks. Further research is necessary to explore the ecological interactions of these microorganisms and assess their potential impact on human health, particularly in regions with high human activity near freshwater sources.

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Background

Klebsiella pneumoniae is a Gram-negative bacterium that can cause a wide range of opportunistic infections in both animals and humans [1]. Similar to other members of the Enterobacteriaceae family, K. pneumoniae is a common commensal in aquatic microenvironment. Environmental reservoirs of K. pneumoniae have been confirmed in surface water, drinking water, soil, plants, sewage, and industrial wastewater [2, 3]. Among nosocomial and community-acquired infections, classical K. pneumoniae (cKp) is one of the most frequently isolated pathogens [4]. As a result, clinical management of urinary and respiratory tract infections caused by cKp is generally straightforward. However, in recent years, a new strain of K. pneumoniae has been identified in many patients with liver abscesses. Due to the challenges in treatment and poor prognosis associated with the infections it causes, this strain has been defined as hypervirulent K. pneumoniae (hvKp) [5].

Currently, infections caused by K. pneumoniae are known to primarily rely on four virulence factors: pili, capsule, lipopolysaccharide (LPS), and iron carriers [6]. Among these, the capsule is considered to play a crucial role in resisting immune system attacks and facilitating colonization within the host [7, 8]. From a diagnostic perspective, the high expression of the capsule can result in hypermucoviscosity on culture plates, which is one of the known characteristics of hvKp [9]. However, it remains unclear why hvKp, which was less reported in the past, has seen a rise in infection cases over recent decades, and its source is still not well understood. Identifying the origin of hvKp is therefore of great importance for public health control and prevention.

Acanthamoeba is a unicellular eukaryote that can thrive independently in various environments, including both natural and man-made water bodies such as lakes, ponds, swimming pools, and even treated water sources [10, 11], classifying it as a free-living amoeba. As an environmentally ubiquitous protist, Acanthamoeba is best known for contaminating contact lenses, leading to opportunistic corneal infections [12]. However, due to its free-living nature, it can influence neighboring microorganisms within its microenvironment either directly or indirectly for survival. Previous studies have shown that Acanthamoeba can act as a carrier for bacteria through direct phagocytosis, allowing the bacteria to evade human or environmental threats [13]. Additionally, like other protists, Acanthamoeba secretes various excretory-secretory proteins (ESPs), which can affect surrounding microbial communities [14]. Although infections caused by Acanthamoeba in humans are less common compared to infections by other microorganisms, its indirect effects play a significant role in bacterial infections and other microbial-related diseases.

In our previous study, we co-cultured K. pneumoniae with Acanthamoeba in vitro and, for the first time, reported that Acanthamoeba can stimulate and induce capsule enlargement in K. pneumoniae [15]. This bacterial cell alteration was dependent on the number of Acanthamoeba, highlighting the role of Acanthamoeba in this process. Through chemical and bacteriological analyses, we confirmed an increase in the uronic acid content, a major component of the K. pneumoniae capsule. Moreover, the capsule's viscosity reached a level characteristic of hypervirulent strains, which is one of the defining features of hvKp. Finally, this effect was shown to have broad impacts across multiple serotypes of K. pneumoniae. However, there is still no evidence to confirm whether K. pneumoniae and Acanthamoeba can co-exist in natural environments.

In terms of the geographical distribution of K. pneumoniae infections, it has been well-established that hvKp is more commonly observed in Asia. Environmental sampling has documented the presence of K. pneumoniae in various locations, including mangrove estuaries in Malaysia, urban rivers in the Philippines, and surface waters in China [16,17,18]. Similarly, evidence of Acanthamoeba has been found in Taiwan's reservoirs and Thailand's natural water sources [19, 20]. However, these studies have been conducted independently. Although there are many overlapping sampling locations for both K. pneumoniae and Acanthamoeba, further field investigations involving both microorganisms are necessary to verify their interactions and assess the public health implications, particularly in the context of hvKp infections.

Hence, in this study, we conducted field sampling in Taichung, a densely populated city in central Taiwan. The sampling locations focused on major river systems in urban areas with frequent human activity. Using water quality assessments and molecular probes, we analyzed the presence of K. pneumoniae and Acanthamoeba in the collected water samples. Furthermore, we examined the influence of water quality parameters on the detection of these microorganisms and explored the possibility of co-occurrence between the two species.

Methods

Sampling site designation and map localization

To ensure that this study targets water bodies accessible to the public, we chose the five primary rivers in Taichung City for analysis: Han River, Meichuan Canal, Midori River, Liuchuan Canal, and the Taichung Park Lake. To increase the sample numbers for enhanced reliability, three sampling locations with direct public access were selected within each of the five rivers. To maintain variable consistency, all samples were collected between 10:00 and 12:00, under similar ambient and water temperatures. The water samples were collected within 10 cm of the water surface. For each sampling location, 1 L of water sample was placed into sterile 1 L polypropylene bottles and stored at 4°C, which was transported to the laboratory for subsequent analyses within 24 h.

After specimen collecting, we used ArcGIS web map (https://www.arcgis.com/home/webmap/viewer.html) for mapping. The maps on this website contain a basemap, a set of data layers (many of which include interactive pop-ups with information about the data), an extent, a legend, and navigation tools to pan and zoom. With these tools, we recorded the coordinates and drew the five rivers to understand the relationships between different water bodies.

Target microorganisms detected by polymerase chain reaction

Each water sample (1L) was filtered through 200 μm mesh to remove larger impurities and particles, and subsequently filtered through a 0.22 μm pore-size polycarbonate filters. After that, shred the filters, and the DNA was extracted with the ZymoBIOMICS™ DNA Miniprep Kit. The suspension was analyzed for the presence of Acanthamoeba and Klebsiella pneumoniae specific genes by PCR assays. The PCR mixture had 10 μl Taq DNA Polymerase 2 × Master Mix RED (3 mM MgCl2, 0.4 mM of each dNTP, and Ampliqon Taq DNA polymerase), 1 μl each of the oligonucleotide primers, and 8 μl template DNA and ddH2O (each tube of sample or positive controls contained 2 ng DNA). The PCR assay genus-specific primers set JDP1 (5’-GGCCCAGATCGTTTACCGTGAA-3’) and JDP2 (5’-TCTCACAAGCTGCTAGGGAGTCA-3’) used in this study were designed for Acanthamoeba genotyping as previously described [21]. Besides, the PCR assay genus-specific primers set Pf (5’-ATTTGAAGAGGTTGCAAACGAT-3’) and Pr1(5’-TTCACTCTGAAGTTTTCTTGTGTTC-3’) used in this study were designed for K. pneumoniae genotyping as previously described [22]. Cycling conditions of Acanthamoeba were as follows: 95°C for 5 min for the initial denaturation step; followed by 40 cycles of 30 s at 95°C for denaturation, 30 s at 53°C for annealing, 1 min at 72°C for extension; and a final extension at 72°C for 7 min. Cycling conditions of K. pneumoniae were as follows: 94°C for 5 min for the initial denaturation step; followed by 30 cycles of 30 s at 94°C for denaturation, 30 s at 55°C for annealing, 50 s at 72°C for extension; and a final extension at 72°C for 5 min. PCR products were detected with gel electrophoresis on a 1.5% agarose gel performed with 10 μl of the reaction solution. DNA fragments were confirmed using 2 μl nucleic acid gel stain staining. A 100-bp DNA ladder was used as a DNA size marker.

Co-occurrence analysis of Acanthamoeba and Klebsiella pneumoniae

To assess the co-occurrence of Acanthamoeba and K. pneumoniae in the sampled water environments, molecular detection was carried out using PCR assays. Samples from five hydrophilic sites were analyzed for the presence of both microorganisms. A Venn diagram was constructed to visually represent the co-occurrence of these microorganisms across the sampling locations. The diagram depicted the number of sites where only Acanthamoeba or K. pneumoniae were detected, as well as the number of sites where both microorganisms were found. This analysis allowed for a clear understanding of the overlap in distribution between these two species in the studied aquatic environments.

Temperature measurement and analysis across sampling sites

Temperature data were collected from five sampling aquatic environments. A portable digital thermometer was used to measure the water temperature at each site, with three replicate readings taken at each location to ensure accuracy. The temperature data were then compiled and analyzed to assess variations between the sites. A box plot was generated to visualize the temperature distribution at each location, highlighting the median, interquartile range, and overall spread of the temperature data. The box plot allowed for a comparison of temperature ranges, identifying potential environmental differences between the sites.

pH vs. detection of Acanthamoeba and Klebsiella pneumoniae

Water samples were collected from five distinct hydrophilic environments. The pH of each sample was measured by using water quality test strips, with readings taken in triplicate to ensure precision. The detection of Acanthamoeba and K. pneumoniae in each sample was conducted via polymerase chain reaction (PCR) analysis. Detection results were recorded as binary values (1 = detected, 0 = not detected), and these values were plotted against the corresponding pH measurements. A scatter plot was generated to visualize the relationship between pH and the detection of both microorganisms, allowing for the identification of any potential correlations between environmental pH levels and microbial presence.

Water quality assessment for heatmap analysis

To visualize the water quality data, a heatmap was generated using Python programming language, specifically the seaborn data visualization library. The heatmap was constructed by organizing the collected data for each parameter (temperature, pH, ROS, and hardness) along with the corresponding sampling locations. Each cell in the heatmap represents the value of a specific water quality parameter at a given site, with the color intensity indicating the magnitude of the parameter. A color gradient was applied, ranging from light yellow (lower values) to dark blue (higher values), allowing for easy identification of patterns and variations across different locations. The heatmap effectively summarized the environmental conditions, providing insights into potential correlations between water quality factors and the presence of microorganisms.

Results

Spatial distribution of sampling sites in Taichung freshwater systems

Water samples were collected from five distinct freshwater systems in Taichung City: Meichuan Canal, Midori River, Han River, Liuchuan Canal, and Taichung Park Lake. Each site was selected to capture a range of environmental conditions, with three sub-locations per site to ensure comprehensive sampling (Fig. 1A). The Meichuan Canal (blue) and Liuchuan Canal (purple) were sampled at upstream, midstream, and downstream points to assess potential variations in microbial content due to changes in water flow and human activity. Similarly, the Midori River (red) was sampled at three locations, focusing on areas with different flow dynamics, including sections near the riverbank and central flow. The Han River (green), which passes through both industrial and residential areas, was sampled at three points to explore differences in microbial contamination along its course. Taichung Park Lake (yellow) was sampled at three points, including inflow zones and areas prone to water stagnation, to evaluate microbial load across the lake’s microenvironments (Fig. 1B). These sampling locations were chosen to represent a broad range of urban freshwater systems, each influenced by varying degrees of pollution, human activity, and water flow. This design provides a detailed examination of the spatial distribution and co-occurrence of Acanthamoeba and K. pneumoniae across Taichung’s urban aquatic environments.

Fig. 1
figure 1

Sampling locations in Taichung City freshwater systems. A The geographical locations of the five freshwater sampling sites in Taichung City of Taiwan. B Meichuan Canal (blue), Midori River (red), Han River (green), Liuchuan Canal (purple), and Taichung Park Lake (yellow). Each site has three sampling points (labeled 1, 2, 3), strategically placed to capture variations in environmental conditions and microbial presence across different parts of the water system

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Environmental parameters and detection of Acanthamoeba and K. pneumoniae in water samples

Temperature, pH, ROS, hardness, and the presence of Acanthamoeba and K. pneumoniae were measured in water samples. Water temperature ranged from 26°C to 31°C, with the highest temperatures at Han River and Taichung Park Lake (31°C) and the lowest at Meichuan Canal (26°C). pH values ranged between 7.2 and 8.4, with Liuchuan Canal showing the highest (8.4) and Han River and Taichung Park Lake showing the lowest (7.2). ROS levels remained stable across all sites at 0.5 ppm, while water hardness varied: 250 mg/L at Meichuan Canal, Midori River, Han River, and Liuchuan Canal, and 100 mg/L at Taichung Park Lake. Acanthamoeba was absent in Meichuan Canal but detected at all points in Midori River and Han River, while only one point in Liuchuan Canal and two in Taichung Park Lake showed its presence. K. pneumoniae was consistently found across all points in Meichuan Canal, Midori River, and Han River, but absent in both Liuchuan Canal and Taichung Park Lake (Table 1). These findings indicate that K. pneumoniae is widespread in freshwater systems affected by urban and industrial activities, especially in Meichuan Canal and Han River, while Acanthamoeba showed more variable distribution, with higher detection in river systems and lower in canal and lake environments. Environmental factors like temperature, pH, and hardness likely play a role in its distribution.

Table 1 Environmental parameters and detection of Acanthamoeba and K. pneumoniae in Taichung freshwater systems
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PCR detection of K. pneumoniae and Acanthamoeba in water samples

PCR results for the detection of K. pneumoniae (130 bp) and Acanthamoeba (467 bp) in water samples from Taichung City’s five freshwater systems are shown in Fig. 2. Positive controls (PC) and negative controls (NC) were included for comparison. K. pneumoniae was detected at all sampling points in Meichuan Canal (MC1–MC3), Midori River (MR1–MR3), Han River (HR1–HR3), and Liuchuan Canal (LC1–LC3), but was absent in Taichung Park Lake. For Acanthamoeba, bands at 467 bp were observed in all samples from Midori River, Han River, and Taichung Park Lake (MR1–MR3, HR1–HR3, TPL1–TPL3). No Acanthamoeba was detected in Meichuan Canal, and in Liuchuan Canal, it was found only at one sampling point (LC2). No amplification was observed in negative controls, confirming the results' validity. These findings highlight the widespread presence of K. pneumoniae in most river systems, particularly in urban canals, while Acanthamoeba showed a more variable distribution, with strong detection in rivers but lower presence in canals. The absence of K. pneumoniae in Taichung Park Lake, despite the consistent detection of Acanthamoeba, suggests environmental or ecological factors may favor one organism over the other.

Fig. 2
figure 2

PCR amplification results for K. pneumoniae and Acanthamoeba detection. A The PCR results for the detection of K. pneumoniae (130 bp) in water samples from Meichuan Canal, Midori River, Han River, Liuchuan Canal, and Taichung Park Lake. Positive amplification bands are present in samples from the first four sites, while no amplification is observed in Taichung Park Lake. B Showing the PCR results for Acanthamoeba (467 bp) in the same water samples, indicating positive detection in Midori River, Han River, Taichung Park Lake, and some points in Liuchuan Canal, but no detection in Meichuan Canal

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Detection rates and comparative analysis of Acanthamoeba and K. pneumoniae

The detection rates of Acanthamoeba and K. pneumoniae were analyzed to compare their prevalence across various freshwater systems in Taichung City, providing insights into their distribution and environmental influences. K. pneumoniae was consistently detected at nearly 100% across all sites, indicating its widespread occurrence and ability to thrive in a wide range of environmental conditions, likely due to its adaptation to pollution, water quality, and other anthropogenic factors. In contrast, Acanthamoeba showed lower and more variable detection rates, fluctuating significantly between locations, suggesting that specific environmental factors strongly influence its survival and propagation, particularly in areas with variable water quality or flow dynamics (Fig. 3A). K. pneumoniae was detected in Han River, Meichuan Canal, Midori River, and Liuchuan Canal, with an overall detection rate of 53.33%, while it was absent in Taichung Park Lake. Acanthamoeba was detected in flowing river systems like Midori River and Han River, with a lower overall detection rate of 33.33%, but was not found in Liuchuan Canal, Meichuan Canal, or Taichung Park Lake (Fig. 3B).This suggests that Acanthamoeba may prefer certain environmental conditions related to water flow, nutrient availability, or temperature. The Venn diagram illustrates the co-occurrence of Acanthamoeba and K. pneumoniae across eight sampling sites where K. pneumoniae was detected. Among these, Acanthamoeba was found in a subset of samples, all of which also tested positive for K. pneumoniae, suggesting a high degree of co-occurrence between the two microorganisms (Fig. 3C). Notably, Acanthamoeba was never detected alone in any sample, implying that its presence might be dependent on the availability of certain environmental commensals such as K. pneumoniae. This observation aligns with the ecological role of Acanthamoeba as a phagotrophic protist, requiring bacterial prey to sustain its growth. The absence of Acanthamoeba in some water bodies, despite the presence of K. pneumoniae, suggests that additional environmental factors, such as nutrient availability, water flow dynamics, or competition with other microbial communities, may influence its distribution. Further investigation into these potential ecological interactions and environmental drivers is needed to better understand the mechanisms underlying the co-occurrence of Acanthamoeba and K. pneumoniae in urban freshwater systems.

Fig. 3
figure 3

Detection rates of Acanthamoeba and K. pneumoniae in Taichung freshwater systems. Detection rates and co-occurrence of Acanthamoeba and K. pneumoniae across sampling sites. A Detection rates of Acanthamoeba and K. pneumoniae at individual sampling sites. K. pneumoniae exhibited a higher detection rate across most locations, with 100% detection at Midori River and Han River. In contrast, Acanthamoeba was detected at lower rates, primarily at Midori River and Han River. B Overall detection rates of Acanthamoeba and K. pneumoniae. While K. pneumoniae had a detection rate of 53.33% across all sampling locations, Acanthamoeba was detected at a lower rate of 33.33%. C Venn diagram showing the co-occurrence of Acanthamoeba and K. pneumoniae at sampling sites. Among the eight sampling locations where K. pneumoniae was detected (green), Acanthamoeba was concurrently detected at five locations (brown), highlighting a significant overlap in their presence

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Correlation between environmental parameters and microbial presence

The relationship between environmental factors and the detection of Acanthamoeba and K. pneumoniae was explored to understand how these variables influence microbial distribution. Water temperature appeared to influence the distribution of K. pneumoniae and Acanthamoeba. K. pneumoniae was detected in Meichuan Canal (26–27°C), Midori River (28–29°C), Han River (29–30°C), and Liuchuan Canal (29–30°C), but was absent in Taichung Park Lake (30–31°C), suggesting a broad temperature tolerance for this bacterium. In contrast, Acanthamoeba was only detected in Midori River (28–29°C) and Han River (29–30°C) (Fig. 4A), both of which had moderate temperatures similar to laboratory culture conditions for Acanthamoeba. This suggests that Acanthamoeba may have stricter environmental temperature requirements compared to K. pneumoniae. The data also showed that K. pneumoniae was detected across a pH range of 7.2 to 8.4, indicating its ability to persist in neutral to slightly alkaline environments. In contrast, Acanthamoeba was only detected in samples with pH levels in 8.0 and 8.4 (Fig. 4B), which is an interesting observation. Previous studies have shown that Acanthamoeba can tolerate a broader pH range, typically from 6 to 8 [23]. Therefore, we cannot rule out the influence of other environmental factors that may have affected its detection in our study. These findings suggest that temperature and pH are significant drivers of microbial presence, influencing the distribution and survival of these microorganisms. Further investigation into other environmental factors, such as nutrient availability and water flow, will be needed to fully understand their interactions and habitat preferences.

Fig. 4
figure 4

Correlation between environmental parameters and microbial presence. Illustartion of the correlation between environmental factors (temperature and pH) and the presence of Acanthamoeba and K. pneumoniae across the sampled sites. A Box plot of water temperature at different sampling sites. The temperature varied across sites of Meichuan Canal (26–27°C), Midori River (28–29°C), Han River (29–30°C), Liuchuan Canal (29–30°C) and Taichung Park Lake (30–31°C). Black outlines represent sites where neither K. pneumoniae nor Acanthamoeba were detected. Red outlines indicate sites where only K. pneumoniae was detected, while green outlines indicate sites where both K. pneumoniae and Acanthamoeba were detected. B Distribution of K. pneumoniae and Acanthamoeba across different sampling sites in relation to pH levels. Each symbol represents a different sampling point (● Sample 1, ■ Sample 2, ▲ Sample 3). Red markers indicate samples where only K. pneumoniae was detected, while green markers indicate co-detection of both Acanthamoeba and K. pneumoniae. Black markers represent samples where neither microorganism was detected

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Heatmap analysis of co-occurrence between Acanthamoeba and K. pneumoniae across sampling sites and water quality parameters

A heatmap analysis was performed to visualize the co-occurrence of Acanthamoeba and K. pneumoniae across different sampling sites. In areas like Midori River and Han River, where K. pneumoniae was frequently detected, Acanthamoeba was also present, indicating favorable environmental conditions for both organisms. Notably, all sampling points in Midori River tested positive for both microorganisms, suggesting a strong co-occurrence. In contrast, K. pneumoniae was detected in some but not all sampling sites in Meichuan Canal, with no concurrent detection of Acanthamoeba, indicating that K. pneumoniae may have a broader environmental tolerance. Taichung Park Lake showed no detection of either microorganism, suggesting that additional environmental factors, beyond those measured in this study, may influence their distribution. These co-occurrence patterns imply that while K. pneumoniae thrives across various aquatic systems, Acanthamoeba is more influenced by specific ecological factors or microbial competition (Fig. 5). These findings emphasize the need for further studies on the environmental drivers shaping microbial distribution and interactions in urban water systems.

Fig. 5
figure 5

Heatmap analysis of co-occurrence between Acanthamoeba and K. pneumoniae. The heatmap presents the co-occurrence patterns of Acanthamoeba and K. pneumoniae across the sampling sites. It highlights locations where both microorganisms were detected, as well as areas where only one was present. The heatmap allows for the identification of potential environmental or ecological factors that may influence the distribution of these organisms and their co-existence in freshwater systems

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Discussion

In this study, we aimed to investigate the co-occurrence and environmental factors influencing the presence of Acanthamoeba and K. pneumoniae in freshwater systems across Taichung City, Taiwan. The results revealed notable spatial variations in the distribution of these microorganisms. K. pneumoniae was consistently detected in urban river systems, particularly in the Meichuan Canal, Midori River, and Han River, while Acanthamoeba exhibited a more variable distribution, with higher detection in river environments and lower detection in canal and lake systems. These findings highlight the resilience of K. pneumoniae to a broad range of environmental conditions, including elevated water temperatures and varying pH levels. In contrast, Acanthamoeba appears to be more selective, with its presence influenced by factors such as temperature, pH, and potentially water flow dynamics.

Several previous studies have identified urban freshwater systems as critical reservoirs for pathogenic microorganisms. K. pneumoniae has been found to be a dominant species in river systems exposed to industrial runoff and sewage contamination, highlighting its ability to thrive in polluted environments [24]. Similarly, the prevalence of K. pneumoniae in river systems has been shown to increase significantly in warmer climates, which aligns with our findings of higher detection rates at sites with elevated water temperatures, such as Han River and Taichung Park Lake [25]. Moreover, the rise in environmental temperature may also lead to an increase in the pathogenicity of K. pneumoniae [26], which could represent another naturally induced public health crisis. The consistent detection of K. pneumoniae across varied environmental conditions in our study further supports its status as an environmental pathogen capable of surviving in diverse habitats, likely due to its metabolic flexibility and resistance to environmental stressors [27].

On the other hand, the sporadic distribution of Acanthamoeba may align with previous research findings. Acanthamoeba tends to thrive in aquatic environments with stable water flow [28], potentially promoting biofilm formation, which may facilitate Acanthamoeba colonization [29]. Acanthamoeba is sensitive to fluctuations in water quality, particularly pH and nutrient availability [30]. Hence, the variable detection rates observed in this study, with higher prevalence in river systems such as Midori River and Han River, suggest that the presence of Acanthamoeba is more dependent on specific environmental conditions. Our results indicate that Acanthamoeba may prefer environments with moderate temperatures and lower pH levels, such as those observed at Midori River.

Many protists can alternate between two or more morphological forms, with transitions between forms depending on changes in the external environment. As a result, this may impose stricter environmental requirements for protists' distribution compared to bacteria. As a common pathogen in aquatic environments, Giardia lamblia shares a similar proliferation mechanism with Acanthamoeba. However, when the environmental pH reaches 6, the proliferation of its trophozoites is completely abolished [31]. Additionally, similar to Acanthamoeba, Entamoeba moshkovskii, which can engulf prokaryotes in the environment for nutrition, has also been found to experience reduced phagocytosis under extreme culture conditions (pH between 6.2 and 6.8) [32]. This indicates that unfavorable conditions in natural environments may lead to the death or depletion of protists. In our study, we also observed that the isolation rate of protozoa was lower compared to bacteria (46.67% vs. 33.33%), which may be attributed to the higher environmental sensitivity of protozoa. Even if unfavorable conditions do not directly cause protozoan mortality, they may still reduce their proliferation, leading to lower detection rates.

Our findings on the co-occurrence of Acanthamoeba and K. pneumoniae in freshwater systems highlight the potential for protozoa-bacteria interactions to shape microbial ecology. While our previous in vitro study demonstrated that Acanthamoeba can enhance K. pneumoniae’s hypermucoviscosity and capsule production under controlled conditions ​[15], our current environmental study does not establish direct evidence of such an interaction in natural freshwater systems. In fact, previous studies, including our own 16S rRNA sequencing of Acanthamoeba endosymbionts, have not detected K. pneumoniae as an intracellular resident [13]. Additionally, experimental studies have shown that K. pneumoniae is not phagocytosed by Acanthamoeba as an endosymbiont, but rather both organisms can coexist without affecting each other's survival [33]. Instead, the observed co-occurrence in this study may suggest shared ecological niches or environmental factors that support both microorganisms. While Acanthamoeba has been shown in laboratory settings to enhance the hypermucoviscous phenotype of K. pneumoniae, our study does not establish direct evidence of this interaction occurring in freshwater environments. Instead, the widespread detection of K. pneumoniae across diverse environmental conditions suggests that multiple ecological factors contribute to its persistence in aquatic systems.

Although protozoa-bacteria interactions have been implicated in bacterial survival and potential virulence modulation, confirming such effects in natural freshwater environments would require detailed assessments of protozoan and bacterial densities, as well as comparisons between free-living bacterial populations and those internalized by protozoa. Further investigations are needed to determine whether co-occurrence in freshwater systems translates to altered bacterial phenotypes and increased virulence. Nonetheless, the presence of K. pneumoniae in urban rivers remains a public health concern, particularly due to the potential for waterborne transmission of hypervirulent strains in densely populated areas.

The public health implications of these findings are significant, especially given the increasing global prevalence of hvKp and its rising antibiotic resistance. Previous literature highlights the difficulty of treating infections caused by hvKp due to its hypermucoviscosity, which contributes to its resistance against phagocytosis and antimicrobial agents [34]. While protozoa, such as Acanthamoeba, have been implicated in modulating bacterial phenotypes in controlled laboratory settings, our current study does not provide direct evidence of Acanthamoeba-mediated stimulation of K. pneumoniae in natural freshwater environments. Instead, the observed co-occurrence suggests that environmental conditions may facilitate the persistence of both organisms. Future studies incorporating endosymbiont detection and sequencing of Acanthamoeba isolates would be necessary to determine whether protozoan hosts contribute to hvKp persistence or virulence modulation in aquatic systems.

Conclusion

This study provides critical evidence into the co-occurrence of Acanthamoeba and K. pneumoniae in freshwater systems and their potential interaction, suggesting that Acanthamoeba may stimulate K. pneumoniae in natural environments. The widespread detection of K. pneumoniae in urban river systems, along with the presence of Acanthamoeba, indicates that natural aquatic environments could serve as reservoirs for hvKp. This interaction likely enhances the bacterium's survival and virulence, raising significant public health concerns about the potential for waterborne transmission of these highly pathogenic strains. These findings emphasize the need for continuous monitoring and control strategies to mitigate the growing threat of hvKp in environmental settings.

Data availability

Data is provided within the supplementary information files.

Abbreviations

cKp:

Classical K. pneumoniae

hvKp:

Hypervirulent K. pneumoniae

LPS:

Lipopolysaccharide

ESPs:

Excretory-secretory proteins

PCR:

Polymerase chain reaction

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Acknowledgements

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Funding

This study was supported by grants from the China Medical University, Taiwan (CMU113-N-17, YJW).

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Author notes

  1. Hsin-Yu Hsieh and Yu-Xiang Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Nursing, Feng Yuan Hospital, Ministry of Health and Welfare, Taichung, Taiwan

    Hsin-Yu Hsieh

  2. Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan

    Yu-Xiang Zhang

  3. School of Medicine, China Medical University, Taichung, Taiwan

    Yen-Zhu Zhuang

  4. Department of Parasitology, School of Medicine, China Medical University, Taichung, Taiwan

    Yu-Jen Wang

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  1. Hsin-Yu Hsieh
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  3. Yen-Zhu Zhuang
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Contributions

YJW, HYH, and YXZ initiated the idea and designed the experiments; HYH and YXZ performed the experiments; YZZ performed the bioinformatic analysis; HYH and YXZ wrote the manuscript; YJW performed the English editing; YJW, HYH, and YXZ revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yu-Jen Wang.

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Hsieh, HY., Zhang, YX., Zhuang, YZ. et al. Detection and co-occurrence of Acanthamoeba and Klebsiella pneumoniae in freshwater river systems of Taichung, Taiwan. BMC Microbiol 25, 143 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03867-y

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Keywords

BMC Microbiology

ISSN: 1471-2180

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