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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 3  |  Issue : 2  |  Page : 88-93

Antimicrobial activity of Vitamin C demonstrated on uropathogenic Escherichia coli and Klebsiella pneumoniae


1 Medical Student, Pondicherry Institute of Medical Sciences, Kalapet, Puducherry, India
2 Department of Microbiology, Believers Church Medical College, Thiruvalla, Kerala, India
3 Research Associate, Believers Church Medical College, Thiruvalla, Kerala, India

Date of Submission21-Jul-2017
Date of Acceptance23-Sep-2017
Date of Web Publication8-Jan-2018

Correspondence Address:
Dr. Stephen K Mathew
Department of Microbiology, Believers Church Medical College, Kuttapuzha P.O., Thiruvalla - 689 103, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrsm.jcrsm_35_17

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  Abstract 

Purpose: Studies have demonstrated the ability of Vitamin C (ascorbic acid) to inhibit pathogenic bacteria and inhibit biofilms. The effect of varying concentrations of ascorbic acid on bacterial growth was studied on uropathogenic Escherichia coli and Klebsiella pneumoniae. The concentration at which maximal inhibition occurred was determined.
Methods: All uropathogenic strains of E. coli and K. pneumoniae isolated from patients over a 3-month period were incubated in varying concentrations (5, 10 and 20 mg/ml) of Vitamin C-supplemented Trypticase Soy Broth. Effect on bacterial growth was quantified as a change in absorbance measured by spectrophotometry (450 nm), as compared to controls. Independent samples t-test was used to calculate P value.
Results: Bacterial growth was inhibited at all Vitamin C concentrations. Mean absorbances of E. coli and K. pneumoniae broths containing 5, 10, and 20 mg/ml Vitamin C were significantly less than absorbances of growth control broths without Vitamin C (P < 0.005). This inhibition was independent of antimicrobial resistance profiles of isolates. Differences between mean absorbance at 10 and 20 mg/ml Vitamin C for both species were not significant (P > 0.005). Thus, the inhibitory activity of Vitamin C appears to be dose-dependent, with 10 mg/ml being the optimum concentration of ascorbic acid.
Conclusions: Ascorbic acid's ability to inhibit bacterial growth may find novel clinical applications. Vitamin C may find potential use in topical antibacterial applications, or urinary bladder irrigation fluid for catheterized patients with urinary tract infections or during bladder instrumentation. There is a need to further explore the possibility of using Vitamin C safely as an effective antimicrobial agent.

Keywords: Antibacterial agents, antioxidants, ascorbic acid, multidrug resistance, urinary tract infections, Vitamins


How to cite this article:
Verghese RJ, Mathew SK, David A. Antimicrobial activity of Vitamin C demonstrated on uropathogenic Escherichia coli and Klebsiella pneumoniae. J Curr Res Sci Med 2017;3:88-93

How to cite this URL:
Verghese RJ, Mathew SK, David A. Antimicrobial activity of Vitamin C demonstrated on uropathogenic Escherichia coli and Klebsiella pneumoniae. J Curr Res Sci Med [serial online] 2017 [cited 2018 Apr 25];3:88-93. Available from: http://www.jcrsmed.org/text.asp?2017/3/2/88/222415


  Introduction Top


In today's world, bacterial resistance to antimicrobials has become a major cause for concern. Rising antimicrobial resistance is threatening to derail much of the progress made in the field of medicine over the last century. A review by O'Neill estimates that 300 million people will die as a result of drug resistance over the next three decades.[1] New multidrug-resistant (MDR) strains of bacteria are appearing frequently, and infections that were once considered as trivial now progress to fatal, untreatable septicemia.[2] Urinary tract infections (UTIs) are among the most common infections seen around the world, especially among women.[3] They are also a common complication in catheterized patients.[4] Moreover, they have become an important cause of morbidity with the rising incidence of antibiotic resistance in common causative bacteria such as Escherichia coli and Klebsiella pneumoniae.[5]

With no significant progress in the search for a new, effective antimicrobial, researchers now fear a return to the “preantibiotic era.” As governments hasten to formulate policies and protocols to regulate the sale and use of antibiotics, particularly in developing countries, there is also a need to look at alternative therapeutic modalities that can help control the pandemic of antimicrobial resistance.

Vitamin C (ascorbic acid) presents one such alternative. It is cheap, easily available and has few or no adverse effects. Frequently prescribed as a nutritional supplement, it has established antioxidant effects and has even been used as an adjuvant in cancer chemotherapy. Ever since Szent-Gyorgi recognised Vitamin C in citrus as the cure for scurvy in 1932, numerous studies have been carried out to elucidate its various biological properties.[6] Currently, ascorbic acid is the most widely used vitamin supplement in the world.[7] Increased Vitamin C levels are said to reduce the risk of arthritis, asthma, cataracts, periodontal disease, and stroke.[8]

All the known biological functions of Vitamin C originate from its chemical property as a reducing agent. It plays an important role in the maintenance of collagen, thus playing a critical role in wound repair and healing. Ascorbic acid is a cofactor in the conversion of dopamine to norepinephrine; is involved in the synthesis of catecholamines; catalyses enzymatic reactions necessary for the activity of certain hormones.[7] Its role in mitigating oxidative injury, restoring microcirculatory flow and boosting antibacterial defense seems to promote recovery in patients with sepsis, burns, and multiorgan dysfunction.[9]

Although the history of Vitamin C is not free from controversy [10],[11] it has been the subject of many studies that demonstrated and assessed its antibacterial action. It has been shown to have an inhibitory effect on the growth of Staphylococcus aureus, Enterococcus faecalis,[12]Helicobacter pylori, Campylobacter jejuni,[13]Mycobacterium tuberculosis[14] and even on fungi such as Aspergillus.[15] Furthermore, in vitro studies have shown that Vitamin C can enhance the inhibitory effect of antibiotics such as levofloxacin [16] and azithromycin.[17] El-Gebaly et al. also demonstrated the effectiveness of Vitamin C in catheter-associated UTIs through its inhibitory effect on biofilm development.[16]

Despite evidence in its support, Vitamin C is still not considered in routine clinical practice for its antibacterial action, for want of more substantiated data. In this study, we demonstrated the effect of increasing concentrations of Vitamin C on bacteria commonly isolated from urine samples of patients with suspected UTIs.


  Materials and Methods Top


Study design

This was an experimental study carried out in the Department of Microbiology. All uropathogenic strains of E. coli and K. pneumoniae isolated from urine samples of patients with suspected UTIs, between July and September 2015 were included in the study. A waiver of consent was obtained from the Institutional Ethics Committee since the study did not require patient information or participation.

Methods

Urinary bacterial isolates were cultured and identified as per standard bacterial identification protocols.[18] Susceptibility patterns of the bacterial isolates to recommended antibiotics, as per CLSI guidelines, were recorded.[19] Strains resistant to at least one agent in three or more antimicrobial categories were considered as MDR, while those resistant to fewer than three antimicrobial categories were considered sensitive.[20] Freshly prepared broths of E. coli and K. pneumoniae in peptone water, incubated for 2 h were used for the tests. Three sets of Trypticase Soy Broth (TSB) (Himedia, India) were supplemented with L-Ascorbic Acid (Himedia, India) such that the final ascorbic acid concentrations were 5, 10, and 20 mg/ml, respectively. These concentrations were used as per the study by Isela et al.[12] Test tubes containing 1000 μL of this solution were inoculated with 250 μL of bacterial broth (E. coli and K. pneumoniae) and incubated at 37°C overnight, under aerobic conditions. Bacterial broths in TSB without Vitamin C, uninoculated TSB without Vitamin C, and uninoculated TSB containing 20 mg/ml Vitamin C served as positive, negative, and Vitamin C controls, respectively. The absorbance of the inoculated broths and controls were then measured by spectrophotometry (BIO-RAD Model 680 microplate reader) at 450 nm.[21]

Statistical analysis

The results were tabulated and statistical analysis was carried out using the independent samples t-test (SPSS 20.0, BM Corp., Armonk, NY, USA) and P < 0.005 was considered statistically significant.


  Results Top


During the study, 50 strains of E. coli and 20 strains of K. pneumoniae were isolated from urine samples sent to the laboratory for culture. Of the 50 E. coli strains, 42 (84%) were MDR, and 8 (16%) were sensitive; among the 20 K. pneumoniae strains, 16 (80%) were MDR and 4 (20%) were sensitive.

The mean absorbance values for E. coli and K. pneumoniae revealed a decreasing trend as the concentration of Vitamin C increased. The fall in absorbance at each Vitamin C concentration was significant (P = 0.001) when compared to the positive controls [Table 1]. Frequency distributions of the absorbance values of E. coli and K. pneumoniae at the various Vitamin C concentrations are plotted [Figure 1] and [Figure 2].
Table 1: Comparison of the absorbance values of the bacterial solutions

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Figure 1: Frequency distribution of absorbance values of Escherichia coli broths at various Vitamin C concentrations

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Figure 2: Frequency distribution of absorbance values of Klebsiella pneumoniae broths at various Vitamin C concentrations

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The absorbance values at Vitamin C concentrations of 5, 10, and 20 mg/ml were compared to 0, 5, and 10 mg/ml Vitamin C concentrations, respectively [Table 2]. These results show that while maximum inhibition was seen at 20 mg/ml Vitamin C, the difference between the absorbance values of 10 and 20 mg/ml Vitamin C for both, E. coli (P = 0.315) and K. pneumoniae (P = 0.259) was not significant. The differences in the absorbance between 0 and 5 mg/ml, and between 5 and 10 mg/ml Vitamin C for E. coli and K. pneumoniae were significant (P = 0.001). This suggests that there is no significant enhancement of the inhibitory effect of Vitamin C in our study beyond a concentration of 10 mg/ml.
Table 2: Relative difference in absorbance with increasing Vitamin C concentrations

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The difference between the absorbance values for sensitive and MDR strains of E. coli and K. pneumoniae at varying concentrations of Vitamin C was statistically not significant (P > 0.005). Growth inhibition of the bacterial strains by the various concentrations of Vitamin C could be said to be independent of the antibiotic susceptibility of the strains [Figure 3] and [Figure 4].
Figure 3: Comparison of mean absorbance values of sensitive and resistant Escherichia coli strains at various Vitamin C concentrations (P < 0.05; significant)

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Figure 4: Comparison of mean absorbance values of sensitive and resistant Klebsiella pneumoniae strains at various Vitamin C concentrations (P < 0.05; significant)

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  Discussion Top


The results show that there was a significant reduction (P = 0.001) in the absorbance of E. coli and K. pneumoniae broths containing Vitamin C, compared to the respective positive controls [Table 1]. This fall in absorbance implies that bacterial dry weight decreased with the addition of Vitamin C, showing that Vitamin C inhibits the growth of E. coli and K. pneumoniae.

Various studies have demonstrated a similar inhibitory effect of Vitamin C on bacterial species such as S. aureus,[12]H. pylori and C. jejuni,[13]Bacillus cereus,[17] and M. tuberculosis,[14] and on fungi such as Candida albicans,[12]Aspergillus niger, and A. flavus.[15] The inhibitory effect of Vitamin C has been seen on biofilm formation with bacteria such as Streptococcus mutans, Porphyromonas gingivalis, E. faecalis, and S. aureus[12] and on the enzymatic activity of Streptococcus pneumoniae “spreading factor,” hyaluronate lyase.[22] The concentrations used vary widely, from 128 μg/ml to 20 mg/ml.[12],[13] In contrast, some studies failed to observe an inhibitory effect on E. coli, Pseudomonas aeruginosa, S. epidermidis, or Candida albicans.[13],[23] This could be due to a difference in the experimental methods, as well as in the concentrations of Vitamin C used. In fact, a report from 1938 found that the growth of anaerobes was stimulated by ascorbic acid.[24]

The inhibitory effect was demonstrated in vivo by a decrease in H. pylori gastric colonization in Mongolian gerbils that were administered Vitamin C orally for 7 days. Moreover, epidemiological evidence seems to suggest that the decline in the incidence of gastric carcinoma, and H. pylori seropositivity prevalence might be linked to the consumption of Vitamin C supplements by 40% of the U.S. population.[13]

El-Gebaly et al. showed that Vitamin C had an inhibitory effect on the growth of E. coli, Klebsiella sp., Citrobacter sp., Enterobacter sp. Proteus sp., and Pseudomonas sp., and that this inhibitory effect was consistent irrespective of the susceptibility pattern on the tested isolates.[16] We too observed that the inhibitory effect of Vitamin C was independent of the antibiotic susceptibility pattern of the organisms. The difference in the mean absorbance between the susceptible and MDR strains of both, E. coli and K. pneumoniae at the various Vitamin C concentrations was not statistically significant (P > 0.005) [Figure 3] and [Figure 4]. While the authors observed inhibition at concentrations of 80-100 mg/ml,[16] we observed it at much lower concentrations. Vilchèze et al. observed that Vitamin C sterilized cultures of drug-susceptible as well as drug-resistant M. tuberculosis.[14]

Various explanations for the inhibitory effect of Vitamin C have been offered. A structural change in bacteria, such as irregularly constricted cells observed by phase contrast microscopy,[13] or elongated cells with disorganized membranes seen under the scanning electron microscope.[16] The inhibitory effect of Vitamin C on bacterial biofilms may be due to its anti-quorum sensing activity.[25] Other explanations include the presence of antioxidants, flavonoids, and phenolics in Vitamin C,[17] or the ability to lower the pH,[16] as observed when Vitamin C intake consistently produced acidic urine in subjects.[23] The lowered pH might account for the enhanced activity of antibiotics such as levofloxacin [16] and azithromycin.[17] Inhibition of H. pylori was found to be increased markedly, along with a lowering of the minimum inhibitory concentration (MIC) when the pH was adjusted from 7.4–5.5.[13] A higher rate of degradation of Vitamin C also occurs at ph 7.0 than at pH 6.0.[26]

We observed a progressive fall in the absorbance for E. coli and K. pneumoniae broths as the Vitamin C concentrations were raised incrementally from 5 through 20 mg/ml [Figure 1] and [Figure 2]. This suggests a concentration-dependent inhibition of bacterial growth. Our observation is similar to that of Isela et al.[12] who calculated a MIC of ascorbic acid required to inhibit microbial growth to be 10 mg/ml. They also observed a reduction in the number of bacterial cells by as much as 90% at 20 mg/ml Vitamin C, as compared to controls.

We compared the absorbance at different Vitamin C concentrations using the independent samples t-test. While the difference between absorbance at concentrations of 5 and 10 mg/ml was found to be significant for both species (E. coli, P = 0.001; K. pneumoniae, P = 0.001), the difference in absorbance between 10 and 20 mg/ml Vitamin C was not statistically significant (E. coli, P = 0.315; K. pneumoniae, P = 0.259) [Table 2]. This suggests a maximal inhibition at 10 mg/ml Vitamin C in our study, and that a further increase in concentration might not significantly increase the inhibitory effect. To prove this threshold effect however, studies with larger sample sizes studying the effects of Vitamin C over a wider range of concentrations are needed.

Adverse reactions to Vitamin C are said to be rare. Creagan et al. observed that Vitamin C in doses of up to 10 g/day orally can be given in most patients with no toxicity,[27] and a prospective study by Curhan et al. could not find any association between a high daily intake of Vitamin C and the risk of renal stone formation.[28] However, a few case reports suggest that unusually high intake of Vitamin C may be associated with secondary oxalosis and acute renal failure [29],[30] and with acute hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency.[31],[32] Stein et al. observed that Vitamin C at a dose of 8 g/day orally precipitated uricosuria in only one of the volunteers.[33] However, adverse reactions to Vitamin C are said to be rare at dosages <4 g/day.[34] Therefore, further studies are required to determine the dose required to achieve and sustain inhibitory concentrations safely in vivo and effectiveness trials.

The ability of Vitamin C to inhibit bacterial growth has been demonstrated in a few clinical settings. Habash et al. demonstrated in volunteers that Vitamin C supplementation reduced numbers of E. coli and E. faecalis in urine samples.[23] Nakanishi showed that scattering of Vitamin C, along with the application of 1% sulfadiazine cream on a bed sore helped to eradicate organisms (methicillin-resistant S. aureus, P. aeruginosa) that were previously resistant to sulfadiazine alone.[35] In a study conducted on women with bacterial vaginosis, the cure rate was higher in women who were prescribed silicon-coated Vitamin C vaginal tablets for 6 days as compared to the placebo group.[36]

Vitamin C can perhaps be used in the near future as a topical antibacterial agent, or for urinary bladder irrigation in cases of catheter-associated UTIs. Further studies are needed to determine the range of bacteria that may be inhibited by Vitamin C, the MIC and dosage, and the spectrum of infections where Vitamin C can be an effective antimicrobial agent.


  Conclusions Top


We have shown that Vitamin C exerts a concentration-dependent inhibitory effect on E. coli and K. pneumoniae that, notably, is independent of the antimicrobial susceptibility profile. Through well-designed studies, the possibility of using Vitamin C as a safe and effective antimicrobial agent, aiding the battle against rising antimicrobial resistance needs to be further explored.

Acknowledgment

RJV contributed to study design, laboratory work, statistical analysis and writing. SKM contributed to study design, statistical analysis and writing. AD contributed to statistical analysis, interpretation and writing. The authors would like to express their warm gratitude to the technical staff and faculty of the Department of Microbiology, Pondicherry Institute of Medical Sciences, and in particular, Dr. Reba Kanungo, Professor and Head, for their constant support and guidance toward this work.

Financial support and sponsorship

This study was awarded a grant from the Indian Council of Medical Research (F. No. 21/1/2015-BMS-STS).

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Vilchèze C, Hartman T, Weinrick B, Jacobs WR Jr. Mycobacterium tuberculosis is extraordinarily sensitive to killing by a Vitamin C-induced Fenton reaction. Nat Commun 2013;4:1881.  Back to cited text no. 14
    
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El-Gebaly E, Essam T, Hashem S, El-Baky R. Effect of levofloxacin and Vitamin C on bacterial adherence and preformed biofilm on urethral catheter surfaces. J Microb Biochem Technol 2012;4:131-6.  Back to cited text no. 16
    
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