Characterization of small colony variants of Klebsiella pneumoniae: Correlation with antibiotic resistance and biofilm formation

Dania Hassan; Michael Magaogao; Ashfaque Hossain

doi:10.4103/bbrj.bbrj_154_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 3 | Page : 438-442

Characterization of small colony variants of Klebsiella pneumoniae: Correlation with antibiotic resistance and biofilm formation

Dania Hassan, Michael Magaogao, Ashfaque Hossain
Department of Medical Microbiology and Immunology, RAK College of Medical Sciences, RAK Medical and Health Sciences University, Ras Al Khaimah, United Arab Emirates

Date of Submission	20-May-2022
Date of Decision	22-Jul-2022
Date of Acceptance	25-Aug-2022
Date of Web Publication	17-Sep-2022

Correspondence Address:
Dania Hassan
Department of Medical Microbiology and Immunology, RAK College of Medical Sciences, RAK Medical and Health Sciences University, Ras Al Khaimah
United Arab Emirates

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_154_22

Abstract

Background: Small colony variants (SCVs) of bacterial pathogens are smaller, slow-growing variants which often pose a challenge to the clinical microbiologist in their identification and characterization. SCVs are receiving much attention in recent years due to their association with several types of chronic infections. In this study, we aimed to develop a suitable culture media for high frequency generation and stable maintenance of SCV of Klebsiella pneumoniae. We also intended to compare different phenotypic characteristics such as growth, antibiotic resistance pattern, and biofilm-forming potential of SCVs with the original parental strain. Methods: We used Mueller–Hinton agar containing the extract of clove (Syzygium aromaticum) for the generation of SCV. Antibiotic sensitivity was determined using disk diffusion method and minimum inhibitory concentration determinations using microdilution method. Biofilm formation was assessed using crystal violet dye binding assay. Results: Mueller–Hinton agar (MHA) containing clove (Syzygium aromaticum) extract (10% volume/volume; MHA-C10) supported generation of SCV from K. pneumoniae at high frequency. SCVs were smaller in colony size and grew slowly in comparison to the wild-type original strain. In addition, SCVs exhibited increased resistance to aminoglycoside group of antibiotics (gentamicin and kanamycin). Crystal violet dye binding spectrophotometric method showed increased biofilm formation potential by SCVs in comparison to their parental counterparts. Conclusions: The findings of this study show that MHA-C10 can be used as a bacterial culture media for the formation of SCV by K. pneumoniae. SCVs, thus, generated on MHS-C10 exhibited typical characteristics of SCVs.

Keywords: Antibiotic resistance, biofilm, Klebsiella pneumoniae, small colony variants

How to cite this article:
Hassan D, Magaogao M, Hossain A. Characterization of small colony variants of Klebsiella pneumoniae: Correlation with antibiotic resistance and biofilm formation. Biomed Biotechnol Res J 2022;6:438-42

How to cite this URL:
Hassan D, Magaogao M, Hossain A. Characterization of small colony variants of Klebsiella pneumoniae: Correlation with antibiotic resistance and biofilm formation. Biomed Biotechnol Res J [serial online] 2022 [cited 2023 Mar 26];6:438-42. Available from: https://www.bmbtrj.org/text.asp?2022/6/3/438/356148

Introduction

Klebsiella pneumoniae is a Gram-negative, nonmotile, encapsulated, rod-shaped bacterium. It is frequently found in the flora of the mouth, skin, and intestines of humans and other animals as well as in natural environments. It is an important opportunistic nosocomial pathogen involved in a variety of multidrug-resistant infections worldwide, particularly in immunocompromised patients.^[1],[2] K. pneumoniae now ranks as the second most common cause of bacteremia. The grave situation of antibiotic resistance is further complicated by the ability of the pathogen to form biofilm.^[3] Small colony variants (SCVs) are typically smaller morphotypes of pathogenic bacteria, usually slow growing with altered biochemical properties in comparison to the parental strains.^[4] SCVs are spontaneously formed in vitro and in vivo, in response to nutrient limitations and stress, as a survival strategy.^[5] In in vivo settings, SCV formation has been found to be associated with chronic infection. SCVs have now been described for a wide range of Gram-positive and Gram-negative bacteria, and because of their slow growth, different morphology, altered biochemical properties, and fastidious growth requirements, isolation and identification of SCV from infection site often offer a great challenge. As the antibiotic sensitivity pattern is often altered, it also poses a therapeutic challenge.^[6] We discovered that an K. pneumoniae American Type Culture Collection (ATCC) strain 700603 formed SCV at a high frequency when grown on MHA containing clove (Syzygium aromaticum) extract while developing a selective medium by incorporating it into Mueller Hinton agar.

Biofilm is an association of microorganisms, enclosed in a self-produced extracellular polymeric material (EPM), adhered to a biotic or abiotic surface.^[5],[7] Bacteria form biofilm, usually in response to a stress such as nutrient limitation or exposure to antibiotics. Bacteria in biofilm exhibit enhanced resistance to antibiotics due to diffusional barrier of EPM.^[5],[7] Minimum inhibitory concentration (MIC) of antibiotics for different bacteria in biofilm is in the range of 10–1000 times higher in comparison to the free floating, planktonic bacteria. Moreover, phagocytes cannot eliminate bacteria in biofilm as efficiently as free floating, planktonic bacteria, making eradication of these bacteria from infection site a challenge. According to the National Institutes of Health, about 65% of all microbial infections and 80% of all chronic infections are associated with biofilm.^[8],[9] In this study, we report the optimum condition for production of SCV and describe few properties of SCV thus generated such as antibiotic resistance and biofilm production, in comparison to the original bacterial strain from which the SCVs derived.

Methods

Bacterial strains and culture media

K. pneumoniae strain 700603 obtained from ATCC and one clinical strain (wound isolate) K. pneumoniae were used in this study. Mueller–Hinton agar (MHA) and Mueller-Hinton broth (MHB) were used to culture bacteria as needed. Modified MHA containing clove extract was used for the generation of SCVs.

Preparation of clove extract

Clove extract was prepared as described by Al-Blooshi et al.^[10] Dried and powdered clove (Al Faris Spices, Salmabad, Bahrain) was used to prepare a water extract. A 20% (weight/volume) clove powder suspension in hot distilled water was prepared and mixed using a magnetic stirrer hot plate for 30 min at 50°C. Then, the extracted material was filtered using Whatman filter paper and stored at 4°C until used. Mueller-Hinton agar (MHA) containing different concentrations of the clove extract (5%–20%, volume/volume) was prepared by adding different volumes of the clove extract and autoclaved. We followed the manufacturer's instruction in the preparation of the MHA plates and the volume of water to be added to the media to be prepared was adjusted according to the volume of the clove extract to be added for each concentration of extract. We labeled the plates MHA-C5 (MHA containing 5% extract volume/volume), MHA-C10 (MHA containing 10% extract volume/volume), and so on for other concentrations. K. pneumoniae was cultured on both MHA for regular culture and MHA containing clove extract for the generation of SVCs. Although K. pneumoniae strains grew on MHA generating regular-sized colonies following incubation at 37°C for 18–24 h; culture of MHA-C10 needed 48 h for the formation of SCV. The SCVs were much smaller, typically 1/10 the size of a colony grown on MHA [Figure 1].

Figure 1: Culture of Klebsiella pneumoniae on MHA (a) and MHA-C10 (b) Strain-dependent production of SCV. MHA: Mueller–Hinton agar, SCV: Small colony variant

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Antibiotic sensitivity test

Antibiotic sensitivity of parental stain and SCVs was determined using Kirby–Bauer disk diffusion method. Bacterial inoculum was placed on the agar surface followed by deposition of paper disk containing antimicrobial agents on agar, incubation at 37°C and measuring the zone of inhibition.

Gentamicin (10 mcg, CP), ciprofloxacin (5 mcg, CIP), aztreonam (30 mcg, AT), and meropenem (10 mcg, MRP) were used.

Minimum inhibitory concentration determinations

MIC of gentamicin and kanamycin for SCV and the parental strain was determined using microtiter plate-based antibacterial assay. Briefly, the test involved preparation of 2-fold serial dilution of the test antibiotic in microtiter plates was made, then addition of 10⁴ bacteria (adjusted from McFarland standard 0.5 = 10⁸ bacteria/ml) to each well and observing for visible growth after 24 or 48 h as needed following incubation at 37°C.

Biofilm assay

Crystal violet dye binding spectrophotometric method was used to quantitate biofilm production by the strains as described earlier.^[11] Briefly, overnight cultures of bacteria in MHB or MHB containing 10% clove extract for SCV were diluted 1:100 and added to the wells of microtiter plates in 100 microliter volumes. The cultures were allowed to grow at 37°C in a static condition for 24–48 h, as needed. Biofilms attached to the wall of the wells were washed to remove unbound bacteria and stained with 1% (w/v) crystal violet for 10 min at room temperature. After washing with water, the stained biofilms were dissolved in 100% ethanol and the absorbance at 570 nm was determined spectrophotometrically using an enzyme-linked immunoassay reader.

Results

When experimenting on antibacterial properties of clove, we observed that culture of K. pneumoniae on MHA plates containing clove extract resulted in formation of colonies which are smaller in size, presumably SCVs. We then carried out a series of experiments to determine the optimum concentration of clove extract that led to maximum formation of SCV. These experiments showed that inclusion of 10% clove extract to MHA, which we designated as MHA-C10, was the most suitable medium for SCV formation [Table 1] and [Figure 1], without interfering with growth (in terms of number of colonies) of K. pneumoniae. [Figure 1] shows culture of K. pneumoniae on MHA (A) and on MHA-C10 (B). The SCV colonies were approximately 1/10 the size of K. pneumoniae colonies grown on normal culture media, i.e., MHA [Figure 1]a and [Figure 1]b. Culture of bacteria on MHA-C15 also resulted in the formation of SCV, but the number of colonies originating from the same inoculum size was less on MHA-C15 in comparison to MHA-C10. On MHA-C20, the growth of K. pneumoniae was completely inhibited [Table 1]. Hence, we used MHA-C10 in subsequent experiments to culture and propagation SCV. Then, we tested whether the capacity of SCV formation by K. pneumoniae, as we observed for the ATCC 700603 strain, was a generalized property of this bacteria. We observed that on the same MHA-C10 plate, a clinical isolate of K. pneumoniae failed to produce SCV, whereas ATCC strain produced large number of SCVs [Figure 2].

Table 1: Growth of Klebsiella pneumoniae strain American Type Culture Collection 700603 on Mueller–Hinton agar and Mueller–Hinton agar containing different concentrations of clove extract

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Figure 2: Growth of a clinical isolate of a Klebsiella pneumoniae, KP-1 (a) and Klebsiella pneumoniae strain ATCC 700603 (b). ATCC: American Type Culture Collection

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SCV formation is reported to be accompanied by changes in antibiotic resistance property. In our study, we also observed the same phenomenon, i.e., formation of SCV was accompanied by changes in sensitivity to antibiotics [Figure 3]a and [Figure 3]b in disk diffusion assay. Out the four antibiotics tested, the dramatic change in antibiotic sensitivity pattern was noted in the case of aminoglycoside, gentamicin. As formation of biofilm is reported to be associated with SCV formation, we investigated whether this previously reported property of SCV also holds true for the SCVs derived from K. pneumoniae strain in our study. Three randomly selected SCVs were tested from biofilm formation by crystal violet dye binding assay.^[11] The results are presented in [Figure 4]. In comparison to the parental strain, all the three SCVs produced relatively higher amount of biofilm formation.

Figure 3: Disk diffusion assay for wild type Klebsiella pneumoniae strain ATCC 700603 (a) And the SCV derived from it (b) 1: Gentamicin, 2: Meropenem, 3: Aztreonam, and 4: Ciprofloxacin. Decrease in the zone of inhibition of all the antibiotics were noted with SCV in all the antibiotics tested, while it was totally resistant to gentamicin. ATCC: American Type Culture Collection, SCV: Small colony variant

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Figure 4: Biofilm production by Klebsiella pneumoniae strain ATCC 700603 and 3 SCVs derived from it. Biofilm production and assay methods are described in the materials and methods section. ATCC: American Type Culture Collection

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Discussion

Although initially SCVs are not considered clinically relevant as these were thought to be nonvirulent, the role of SCV in chronic infection is increasingly being recognized and their persistence and antibiotic resistance have been receiving considerable attention.^[4],[5],[12] Studies on SCV need stable maintenance of the phenotype during repeated passage on the laboratory. As SCVs once formed, they tend to change into different morphotypes.^[13],[14] Development of bacterial culture media which will permit formation and stable maintenance of SCV phenotype is essential. Our effort in this study is a step in that direction.

In this study, we investigated the formation of SCV by K. pneumoniae. It is established that SCVs are generated spontaneously at low frequency while growing on conventional bacteriological media. However, a dramatic increase in frequency of SCV formation occurs when the bacteria is exposed to different stress condition such as exposure to antibiotics, oxidative stress, and low pH.^{[15],[16],[17]} Here, we report exposure of K. pneumoniae to a naturally occurring material, a plant extract, water extract of clove (Syzygium aromaticum), which also induced SCV high frequency generation in K. pneumoniae.

The observation that MHA-C15 also permitted SCV formation but the number of colonies formed was reduced (in comparison to MHA-C10), and on MHA-C20, the growth of K. pneumoniae was completely inhibited [Table 1] is probably due to the fact that extract concentration may have reached toxic levels, as reported by Al-Blooshi et al.^[10] We observed that formation of SCV by K. pneumoniae was strain dependent. While K. pneumoniae strain ATCC 700603 produced SCV at high frequency on MHA-C10, a fresh clinical isolate of K. pneumoniae, KP-1 failed to do so [Figure 2]. Bacteria belonging to the same genus and species are reported to possess subspecies differences reflected in difference in size, in biochemical reaction, virulence and other phenotypic characteristics.^{[18],[19],[20],[21]} It will be interesting to delineate the molecular basis of production of SCV by one strain of K. pneumoniae, but not by the other. The presence of efflux pumps, which expels antimicrobial agents form the bacterial cells, is likely to be a reason, which may be present or more efficient the clinical isolate of K. pneumoniae in comparison to the ATCC strain. Screening of large number of K. pneumoniae strains from different clinical and environmental sources for SCV formation on MHA-C10 will provide further insights on this phenomenon.

Formation of SCV is reported to be associated with alterations in sensitivity to different antibiotic in different pathogens including K. pneumoniae. Our findings, which show that SCV formation is accompanied by changes in antibiotic resistance to the aminoglycoside group of antibiotic gentamicin, are consistent with previous findings.^[14],[22] We also observed that for other three antibiotics (meropenem, aztreonam and ciprofloxacin), there was also a change in antibiotic sensitivity [Figure 3]. The original parental strain exhibited relatively more sensitivity to all the antibiotics tested. The corresponding SCV variants exhibited less sensitivity to all the drugs tested; however, it became totally resistant to gentamicin.

Alterations in antibiotic resistance as bacteria form SCV are also linked with their capacity to form biofilm in different pathogens, including K. pneumonia.^[23],[24] A study reported that biofilm produced by Staphylococcus aureus exhibited high-level resistance to different antibiotics. This enhanced resistance is attributed to the presence of higher proportion of SCV persisters in biofilm in comparison to their corresponding planktonic (nonbiofilm) culture.^[13] As an extension of this in vitro observation to in vivo clinical setting, the finding reported in this study that SCVs produced more biofilm may be implicated in the probable scenario of frequent presence of SCVs in biofilm-associated chronic infections.^[8.9] This interesting observation connecting SCVs and biofilm formation is that SCVs exhibit enhanced potential to produce biofilm^[25] which are difficult to eradicate by antibiotic treatment.^[14] In the present study, we observed that each of the three randomly selected K. pneumoniae SCVs produced higher amounts of biofilm in comparison to the strain from which the SCVs derived [Figure 4], adding newer evidence to the previously observed phenomenon.

Conclusions

SCVs generated from K. pneumoniae strain ATCC 700603 growing on MHA containing 10% extract of clove (MHA-C10) differed in colony size, sensitivity to aminoglycoside group of antibiotics, growth characteristics, and biofilm formation potential from the that grow on normal MHA (without nay extract added). Increased resistance to aminoglycoside antibiotics such as kanamycin and gentamicin and enhanced production of biofilm may have clinical implications as these properties may help the bacteria to survive better in the host and thus make eradication difficult, leading to chronic infections. This is the first report on the development and use of a special media using a plant material for studies on SCVs of K. pneumoniae, which permits development and stable maintenance of SCV phenotype. Further investigation into the usefulness of this medium in the generation and maintenance of the SCV phenotype of a large number of K. pneumoniae strain from different sources and other pathogenic bacterial species is warranted.

Limitation of the study

One of the major limitation of the study is large number of fresh clinical isolates of K. pneumoniae were not tested, which would strengthen the findings of this study. Another limitation that can be perceived is that other antibiotics could be tested.

Ethical approval

This research was approved by the RAK Medical and Health Sciences University (RAKMHSU) Research and Ethics Committee. Approval number for this project is RAKMHSU-REC-066-2018.

Acknowledgment

The constant encouragement of the President of RAKMHSU is gratefully acknowledged. We acknowledge the excellent technical help of Mr. Michael Magaogao of RAKMHSU.

Financial support and sponsorship

The financial support for this research was provided by the RAKMHSU.

Conflicts of interest

The authors declare that none of the authors have any competing interests.

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