Assessment of hypermucoviscosity as a virulence factor for experimental Klebsiella pneumoniaeinfections: comparative virulence analysis with hypermucoviscosity-negative strain
- Yi-Chun Lin†1,
- Min-Chi Lu†2, 3,
- Hui-Ling Tang4, 5,
- Hsu-Chung Liu1, 6,
- Ching-Hsien Chen1,
- Keh-Sen Liu7,
- Chingju Lin8,
- Chien-Shun Chiou9,
- Ming-Ko Chiang10,
- Chuan-Mu Chen1Email author and
- Yi-Chyi Lai2, 3Email author
© Lin et al; licensee BioMed Central Ltd. 2011
Received: 24 October 2010
Accepted: 8 March 2011
Published: 8 March 2011
Klebsiella pneumoniae displaying the hypermucoviscosity (HV) phenotype are considered more virulent than HV-negative strains. Nevertheless, the emergence of tissue-abscesses-associated HV-negative isolates motivated us to re-evaluate the role of HV-phenotype.
Instead of genetically manipulating the HV-phenotype of K. pneumoniae, we selected two clinically isolated K1 strains, 1112 (HV-positive) and 1084 (HV-negative), to avoid possible interference from defects in the capsule. These well-encapsulated strains with similar genetic backgrounds were used for comparative analysis of bacterial virulence in a pneumoniae or a liver abscess model generated in either naïve or diabetic mice. In the pneumonia model, the HV-positive strain 1112 proliferated to higher loads in the lungs and blood of naïve mice, but was less prone to disseminate into the blood of diabetic mice compared to the HV-negative strain 1084. In the liver abscess model, 1084 was as potent as 1112 in inducing liver abscesses in both the naïve and diabetic mice. The 1084-infected diabetic mice were more inclined to develop bacteremia and had a higher mortality rate than those infected by 1112. A mini-Tn5 mutant of 1112, isolated due to its loss of HV-phenotype, was avirulent to mice.
These results indicate that the HV-phenotype is required for the virulence of the clinically isolated HV-positive strain 1112. The superior ability of the HV-negative stain 1084 over 1112 to cause bacteremia in diabetic mice suggests that factors other than the HV phenotype were required for the systemic dissemination of K. pneumoniae in an immunocompromised setting.
As a common pathogen responsible for a wide range of clinical illnesses, K. pneumoniae has long been the principal cause of pneumonia , emerging as the major pathogen associated with pyogenic liver abscesses over the past decade . K. pneumoniae has been implicated in 7-12% of hospital-acquired pneumoniae in ICUs in the United States [3, 4], accounting for 15, 32, and 34% of community-acquired pneumoniae in Singapore , Africa , and Taiwan , respectively. In the 1990 s, K. pneumoniae surpassed E. coli as the number one isolate from patients with pyogenic liver abscesses in Taiwan , where more than 1,000 cases have been reported . Liver abscesses caused by K. pneumoniae (KLA) have become a health problem in Taiwan and continue to be reported in other countries. Metastatic lesions, such as meningitis and endophthalmitis, develop in 10-12% of KLA patients and, worsening the prognosis of this disease . Cases of KLA in Taiwan typically occur in diabetic patients with a prevalence rate from 45% to 75% [9, 10].
Diabetes mellitus (DM), the most common endocrine disease, is a predisposing factor for infections of K. pneumoniae . Type 1 diabetes (IDDM) is a form of DM resulting from autoimmune triggered destruction of insulin-producing β cells of the pancreas. Type 2 diabetes (NIDDM) is characterized by high blood glucose within the context of insulin resistance and relative insulin deficiency. In 2000, approximately 171 million people in the United States were affected by diabetes, and this number is expected to grow to 366-440 million by 2030 . Diabetes can lead to a variety of sequelae, including retinopathy, nephropathy, neuropathy, and numerous cardiovascular complications, and patients with diabetes are more prone to infection. Several factors predispose diabetic patients to infection, including genetic susceptibility, altered cellular and humoral immune defense mechanisms, poor blood supply, nerve damage, and alterations in metabolism .
Clinical K. pneumoniae isolates produce significant quantities of capsular polysaccharides (CPS). Several CPS-associated characteristics have been identified in correlation with the occurrence of KLA, including serotype K1 or K2  and a mucopolysaccharide web outside the capsule, also known as the hypermucoviscosity (HV) phenotype . We collected 473 non-repetitive isolates from the foci of K. pneumoniae-related infections. Interestingly, the incidence of strains displaying the HV phenotype in the K. pneumoniae abscess isolates was 51% (48/94), which was significantly lower than that reported by Yu et al. (29/34, 85%)  and Fang et al. (50/53, 98%) . A decline in the HV-positive rate suggests the emergence of etiological HV-negative strains and urges a re-evaluation of whether the HV phenotype acts as a virulence determinant for clinical K. pneumoniae isolates. Due to the significant susceptibility of diabetic patients, this study established two infection models recapitulating pneumonia and KLA in diabetic and naïve C57BL/6J mice. The role of the HV phenotype in the pathogenesis of K. pneumoniae was determined in these mouse models by comparatively analyzing bacterial virulence for two clinically isolated K1 strains, 1112 and 1084, which were well-encapsulated with similar genetic backgrounds; however, only 1112 exhibited the HV-phenotype.
Emergence of HV-negative K. pneumoniaerelated to tissue abscesses
Analysis of comparative virulence for HV-positive and-negative K. pneumonia
Requirement of HV-phenotype for K. pneumoniae1112 virulence
Virulence characteristics of K. pneumoniae 1112, KPG6, and 1084.
Serum killing a
Oral LD50 b
6.9 × 106
9 × 106
Intracellular survival (%) in Raw264.7 cell c
60.29 ± 5.04
46.77 ± 1.61
57.82 ± 2.42
A capsule-associated mucopolysaccharide web, also known as the hypermucoviscosity (HV) phenotype, was previously considered a characteristic associated with pyogenic K. pneumoniae infections [14, 15]. Nevertheless, the prevalence of K. pneumoniae negative for HV-phenotype in our pyogenic cases (49%; 46/94) suggests that HV-negative strains have emerged as etiologic in the formation of tissue abscesses. HV-negative-associated infections were related to diabetic conditions, as diabetic patients suffering from pyogenic infections were more frequently associated with HV-negative strains than with HV-positive strains (70% vs. 56%). Therefore, in this study, we aimed to assess how essential the HV-phenotype is for K. pneumoniae pathogenesis by comparing the virulence of clinically isolated strains that were naturally HV-positive or -negative. Because K1 is the predominant serotype in KLA cases, we selected two K1 strains, 1112 and 1084, which have relatively high genetic similarity among our clinical isolates. Not surprisingly, the HV-positive strain 1112 demonstrated greater virulence than the HV-negative strain 1084 in either a pneumonia or KLA infection model in naïve mice. However, in the KLA model of diabetic mice, 1084 was as potent as 1112 in inducing liver abscesses, exhibiting an ability superior to that of 1112 in causing bacteremia and mortality in mice. The advantageous tissue-invasive ability of 1084 indicates that the HV-phenotype per se is not a determinant for K. pneumoniae virulence in a diabetic host.
Genetic loci, including magA , the cps gene cluster , the wb gene cluster , and rmpA , have been associated with the HV-phenotype. Mutations of these genes have resulted in the loss of the HV-phenotype in conjunction with defects in capsular integrity, confirming the findings of Fang et al. , who reported that capsule-related properties, including serum resistance, anti-phagocytosis, and virulence to mice, were drastically attenuated in the magA mutants. Ideally, the capsule and HV-phenotype should be investigated independently. However, all of the HV-phenotype-associated genes identified thus far are involved in the regulation or the biosynthesis of capsular polysaccharides. Given that significant quantities of clinically isolated K. pneumoniae are well-encapsulated but negative for HV-phenotype, these naturally- selected HV-negative strains could be used as an ideal control for HV-positive strains to minimize the influence of defects on the capsule. Consistent with previous thoughts, the HV-positive strain 1112 was more likely to cause pneumonia or KLA in naïve mice than 1084. Although the idea that the HV-phenotype is a determinant for K. pneumoniae virulence was suggested by the fact that the isogenic HV-negative mutant of 1112, KPG6, notably lost its virulence to mice, we could not exclude the possibility that the mutation of pgi influenced the integrity of the capsule and disrupted the synthesis of exopolysaccharides as the anti-phagocytic ability of KPG6 in Raw264.7 macrophages was attenuated. Unlike KPG6, naturally-selected HV-negative strain 1084 exhibited the wild-type level capsule-related characteristics, including serum-resistance, anti-phagocytosis, and virulence to mice. The findings suggest that HV-phenotype-related properties are not necessarily the same as the properties related to capsules. Further studies are required to differentiate the roles of the HV-phenotype and capsule in K. pneumoniae pathogenesis.
Diabetes is a risk factor for K. pneumoniae infections [2, 22]. To clarify the role of HV-phenotype in diabetic individuals, we produced diabetes in mice using a STZ-induction method . The STZ-treated diabetic mice were raised to the age of thirty weeks to avoid immunomodifying effects of STZ occurring after administration of the drug , to ensure the physiological properties of clinical diabetes occurring in mice, and to mimic middle-aged diabetic persons, the population most susceptible to K. pneumoniae infections [2, 24]. In pneumonia or the KLA model generated in the diabetic mice, bacteremia was more likely to develop following an intratracheal- or oral-infection with the HV-negative strain 1084 compared to that of 1112. The pathological advantages of HV-negative K. pneumoniae in diabetic mice implies that diabetes might provide a specialized environment permitting these strains to disseminate from local tissues, such as the lungs and intestines into the blood. Although previous studies have indicated that the hyperglycemic state of diabetes provokes a functional decline of neutrophils [25, 26], phagocytosis by neutrophils from diabetic patients of K. pneumoniae 1112 was comparable to that of 1084 (data not shown). Moreover, pulmonary infections caused by K. pneumoniae 1112 and 1084 caused similar apoptosis levels of the alveolar macrophages in both diabetic and naïve mice (data not shown). Given that capsules play a pivotal role in the protection of K. pneumoniae from phagocytosis , it is not surprising that the well-encapsulated K. pneumoniae 1084 interacted with phagocytes in the same manner as 1112. This implied that the HV phenotype was not essential for the antiphagocytosis of K. pneumoniae. Thus, a mutant library of 1084 generated using a signature-tagged mutagenesis technique is currently under in vivo screening in diabetic mice. Identification of the genetic requirement of 1084 with regard to virulence will provide insights into the means by which 1084 gains an advantage in dissemination and proliferation in the blood of diabetic mice. To our knowledge, this is the first study using naturally-selected strains to evaluate the requirements of HV-phenotype for K. pneumoniae virulence in diabetic mice. Our findings suggest that the HV-negative strain 1084 is more virulent than the HV-positive strain 1112 under diabetic conditions, the naturally-selected strain 1084 may serve as an ideal model for identifying virulence factors, rather than relying on the HV phenotype that contributes significantly to the pathogenesis of K. pneumoniae.
HV-phenotype is a virulent determinant for clinically isolated HV-positive K. pneumoniae. However, factors other than the HV-phenotype contribute significantly to the virulence of K. pneumoniae isolates displaying no HV-phenotype, particularly for systemic dissemination under diabetic conditions.
During a fifteen-month period from April 2002, a total of 473 non-repetitive K. pneumoniae were isolated from the infection foci of patients who had K. pneumoniae-related infections treated at a referral medical center in central Taiwan. The clinical isolates, which were confirmed as K. pneumoniae using the API 20E system (BioMerieux), were collected from various infection foci: 11.6% were from blood; 4%, from liver aspirates; 0.4%, from eye aspirates; 0.8%, from cerebrospinal fluid; 26.2%, from non-hepatic abscesses; 22.8%, from sputum; 8.5%, from wound pus; and 25.6%, from other body fluids. Due to the difficulty in determining whether K. pneumoniae is the primary pathogen in a urinary tract infection, urine isolates were excluded. If cultures were concomitantly positive in more than one site, only that culture which was isolated from the primary infection focus was included. One isolate per patient was analyzed, and each isolate represented a single case. Isolates were cultured in Luria-Bertani (LB) broth and stored at -80°C until use. Medical records were reviewed and information related to clinical manifestations and underlying diseases was collected. Clinical research was conducted according to the human experimentation guidelines of Chung-Shan Medical University. Ethical approval was not needed for the present study.
Determination of the hypermucoviscosity (HV) phenotype and detection of HV-related genes
The HV phenotype display was examined with a string-formation test as described by Fang et al . Bacterial strains to be tested were inoculated onto 5% sheep blood plates and incubated at 37°C for 16 h. Positive of hypermucoviscosity phenotype was defined as the formation of viscous strings > 5 mm in length when a standard inoculation loop was used to stretch the colony on blood agar plates. K. pneumoniae isolates, capable of displaying the HV-phenotype from three independent tests were described as HV-positive and those that were unqualified in string forming were HV-negative.
Induction of diabetes in mice
Six-week-old male C57BL/6J mice were purchased from the National Laboratory Animal Center (NLAC, Taiwan) and allowed to acclimatize in the animal house for one week before experiments. Mice (25-30 g body weight) were randomly divided into two groups. One group received intraperitoneal injection of the pancreatic β-cell toxin streptozotocin (STZ; Sigma) for five days (55 mg/kg per day in 0.05 M citrate buffer, pH 4.5) . The other group received injections of citrate buffer as the control. The serum glucose concentrations and body weights of the mice were determined at indicative time points after the multi-injection of STZ.
Pneumonia or KLA infection models
To recapitulate a pneumonia infection, thirty-week-old mice were anesthetized with isoflurane and intratracheally inoculated with 104 CFU of K. pneumoniae by intubation with a blunt-ended needle . At 20 h post-inoculation, lungs and blood were retrieved, homogenized, and plated onto M9 agar for enumerating bacterial counts. Based on the KLA infection model established in our previous study , groups of two to four thirty-week-old diabetic or naïve mice were orally inoculated with 105 or 108 CFU of K. pneumoniae, respectively. Twenty microliter of blood was retrieved from the retroorbital sinus of infected mice at 24, 48, and 72 h post-inoculation for enumeration of bacterial counts. Survival of the infected mice was monitored daily for seven days. For histological examination, livers retrieved from mice were fixed in 4% paraformaldehyde, paraffin embedded, and stained with haematoxylin and eosin. All the animal experiments were performed according to NLAC guidance and the Institutional Animal Care and Use Committee approved protocols.
Results are expressed as means ± SD. The two-sample t test was used to test for differences between the groups indicated. Statistical significance was determined based on a P value ≤ 0.05. All experiments were repeated a minimum of three times to ensure reproducibility.
Acknowledgements and Funding
This work was supported by the National Science Council and China medical University of Taiwan R.O.C. (NSC98-2320-B-040-013- to Yi-Chyi Lai, NSC92-2314-B-039-008- to Min-Chi Lu and CMU-95-109 to Chingju Lin).
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