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Year : 2011  |  Volume : 54  |  Issue : 1  |  Page : 90-95
Isolation of bacteriophages to multi-drug resistant Enterococci obtained from diabetic foot: A novel antimicrobial agent waiting in the shelf?

1 Department of Microbiology and Surgery, S. S. Institute of Medical Sciences and Research Centre, Davangere, Karnataka, India
2 Department of Microbiology, St. Johns Medical College, Bangalore, Karnataka, India
3 Department of Microbiology, Madras Medical College, Chennai, Tamil Nadu, India

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Date of Web Publication7-Mar-2011


Introduction: While foot infections in persons with diabetes are initially treated empirically, therapy directed at known causative organisms may improve the outcome. Many studies have reported on the bacteriology of diabetic foot infections (DFIs), but the results have varied and have often been contradictory. The purpose of the research work is to call attention to a frightening twist in the antibiotic-resistant Enterococci problem in diabetic foot that has not received adequate attention from the medical fraternity and also the pharmaceutical pipeline for new antibiotics is drying up. Materials and Methods: Adult diabetic patients admitted for lower extremity infections from July 2008 to December 2009 in the medical wards and intensive care unit of medical teaching hospitals were included in the study. The extent of the lower extremity infection on admission was assessed based on Wagner's classification from grades I to V. Specimens were collected from the lesions upon admission prior to the initiation of antibiotic therapy or within the first 48 h of admission. Results: During the 18-month prospective study, 32 strains of Enterococcus spp. (26 Enterococcus faecalis and 06 E. faecium) were recovered. Antibiotic sensitivity testing was done by Kirby-Bauer's disk diffusion method. Isolates were screened for high-level aminoglycoside resistance (HLAR). A total of 65.6% of Enterococcus species showed HLAR. Multidrug resistance and concomitant resistance of HLAR strains to other antibiotics were quite high. None of the Enterococcus species was resistant to vancomycin. Conclusion: Multidrug-resistant Enterococci are a real problem and continuous surveillance is necessary. Today, resistance has rendered most of the original antibiotics obsolete for many infections, mandating the development of alternative anti-infection modalities. One of such alternatives stemming up from an old idea is the bacteriophage therapy. In the present study, we could able to demonstrate the viable phages against MDR E. faecalis.

Keywords: Bacteriophage, diabetic foot, Enterococcus faecalis, high-level aminoglycoside resistance

How to cite this article:
Vinodkumar C S, Srinivasa H, Basavarajappa K G, Geethalakshmi S, Bandekar N. Isolation of bacteriophages to multi-drug resistant Enterococci obtained from diabetic foot: A novel antimicrobial agent waiting in the shelf?. Indian J Pathol Microbiol 2011;54:90-5

How to cite this URL:
Vinodkumar C S, Srinivasa H, Basavarajappa K G, Geethalakshmi S, Bandekar N. Isolation of bacteriophages to multi-drug resistant Enterococci obtained from diabetic foot: A novel antimicrobial agent waiting in the shelf?. Indian J Pathol Microbiol [serial online] 2011 [cited 2022 May 20];54:90-5. Available from: https://www.ijpmonline.org/text.asp?2011/54/1/90/77333

   Introduction Top

Enterococci are Gram-positive facultative anaerobic bacteria which are found in soil, water, food, humans, and animal bodies. [1] They are regarded as commensals of the gastrointestinal tract but can also be the cause of dangerous infections, such as bacteraemia, endocarditis, urinary tract infections, and diabetic foot infection. [1],[2] Among Enterococci, the most prevalent is Enterococcus faecalis, causing 80-90% of such infections. [1]

Foot ulcers are among the leading causes of morbidity in diabetics and are the most common indication for admission in this population. [3],[4] Devitalized tissue is the site where the bacteria responsible for the nonhealing ulcers inflict damage. [4],[5] Most diabetic foot lesions have a polymicrobial aetiology; however, the bacterial yield varies with the method of sample collection. Foot infections in the diabetics constitute a tremendous clinical and financial burden to the patients involved, the clinicians caring for these patients, and the community as a whole. Adverse consequences of polyetiology and multidrug resistance have led to approximately 20% admitted to the hospital for their foot problems, and 50-70% of all nontraumatic amputations are performed every year on these diabetic patients. [6] Infection caused by multidrug-resistant Enterococci leads to adverse outcomes and is a real challenge and it threatens to undo the dramatic advances in human health that were ushered in with the discovery of these drugs in the mid-1900s. [6],[7],[8] Today, resistance has rendered most of the original antibiotics obsolete for many infections. The emergence of multidrug-resistant Enterococci to most, if not all, currently available antimicrobial agents has become a critical problem in modern medicine, particularly because of the concomitant increase in immuno-suppressed patients. The concern that humankind is re-entering the "preantibiotics" era has become very real and the development of alternative anti-infection modalities has become one of the highest priorities of modern medicine. One of such substitutes stem up from an old idea is the bacteriophage therapy. Bacteriophage therapy is a method of antibacterial treatment that harnesses the bacteria-killing properties by harmless viruses. [9],[10],[11]

The purpose of research work is to call wakefulness to an alarming plait in the antibiotic-resistant Enterococci problem in diabetic foot and to check the practicability of the bacteriophage in the treatment of multidrug-resistant Enterococci isolated from diabetic foot.

   Materials and Methods Top

Adult diabetic patients admitted for lower extremity infections from July 2008 to December 2009 to the medical wards, surgical wards, and intensive care unit of medical teaching hospitals at S. S. Institute of Medical Sciences and Research Centre, Davangere, and Chigateri Government General Hospital, Davangere, were included in the study. The extent of the lower extremity infection on admission was assessed based on Wagner's classification [3],[4],[5] from grades I to V. Specimens were collected from the lesions upon admission prior to the initiation of antibiotic therapy or within the first 48 h from admission and processed as per the standard microbiological techniques. [11] The identification of Enterococci up to the species level was done by using the batteries of biochemical tests as suggested by Facklam and Collins. [11],[12] Antimicrobial susceptibility testing of the Enterococci was performed by the disk diffusion method and high-level aminoglycoside resistance (HLAR) was evaluated by the agar dilution method as recommended by the Clinical and Laboratory Standard Institute (CLSI). [13] E. faecalis ATCC 29212 was used as control.

The interpretive category of microbial susceptibility was determined according to CLSI interpretive criteria. The CLSI-accepted gentamicin HLAR breakpoint is ≤1000 μg/ml as sensitive, and >1000 μg/ml as resistant, and for streptomycin, the HLAR breakpoint is ≤2000 μg/ml as sensitive, and >2000 μg/mL as resistant. [13]

Isolation of the Enterococci Phage

The phages were isolated from raw sewage at a municipal sewage treatment plant, Davangere, by the method of Smith and Huggins. [14] The sewage water (50 ml) was collected in a sterile conical flask and treated with a few drops of chloroform. [14] To this, an equal volume of the lactic phage broth and 1 ml of the 24-h old broth culture of E. faecalis CSV-35 was added. The sample inoculated with bacterial pathogens was incubated at 37°C for 12-24 h in a shaker water bath. After 1224 h, the lysate was shaken with a few drops of chloroform for about 10 min, centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through 0.22-μm pore size Acrodisc membrane filters (PALL, German Laboratory) to remove the bacteria and subjected to the plaque-forming unit (PFU) assay using the double-layer agar method described by Smith and Huggins. For the recovery of the phage, a single, well-isolated plaque was picked and replated twice to ensure the isolation of a single phage type. The phage isolated was designated as E. faecalis phage Ř SH-56.

In vitro Confirmation of the Bacteriophage Activity on E. faecalis CSV-35

The bacterial lawn was prepared on nutrient agar plates employing 1.0 ml of the 24-h E. faecalis CSV-35 culture by flooding and draining out the excess. Wells were dug into the agar by employing a sterile cork borer and the 20 μl E. faecalis phage Ř SH-56 suspension (3 × 10 9 PFU/ml) was loaded into each of the well. Sterile distilled water served as the control. The plates were incubated at 37°C for 24 h. Thereafter the zone of inhibition, if any, was recorded. [15],[16]

Host Range

The susceptibility of E. faecalis phage Ø SH-56 was also investigated with other bacteria isolated from diabetic foot infection like Streptococcus pyogenes, Staphylococcus aureus, Coagulase-negative Staphylococci, E. faecium, and Pseudomonas aeruginosa to evaluate genus and species specificity. [17],[18]

Propagation of the Phage

The method of Duff and Wyss (1961) was used for the propagation of the phage in the lactic phage broth at 37°C. Complete clearing of the broth cultures usually occurred in 3-4 h. After the removal of bacteria by passage through a membrane filter, the lysate was stored in 10-ml amounts in sterile vials at 4-6C. Phage concentrations of 10 9 plaque-forming particles (PFP)/ml were obtained. [14],[17]

Morphology Study by a Transmission Electron Microscope

The E. faecalis phage Ø SH-56 solution was filtrated with the Acrodisc® filter to remove soluble biological macromolecule fragments of host bacteria. After washing three times with the 0.1 M ammonium acetate solution (pH 7.0), the retained phage solution was used directly for negative staining. [19],[20] Photographs were taken with a transmission electron microscope.

   Result Top

Demographic Data and Clinical Characteristics

A total of 32 diabetic patients, 20 males (62.5%) and 12 females (37.5%), aged between 30 and 85 years, duration of diabetes 4-28 years, were investigated. The mean age of all the patients was 49 years (SD 12.5). Out 32 known diabetic patients prior to admission, 24 (75%) were maintained on oral hypoglycemic agents and 8 (25%) were maintained on insulin. The mean duration of illness was 15 years (SD 8.9). A total of 21.9% of the subjects had leukocytosis during admission [Table 1].
Table 1: Demographic, clinical, and microbiologic characteristics of patients with diabetic foot infections

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The prevalence rate of enterococcal infection was 9.8%. Majority of enterococci were isolated from Wagner's II and IV.

Antimicrobial Susceptibility Pattern

Out of 32 Enterococcus spp. isolated, 26 were E. faecalis and 06 were E. faecium. A total of 21 (65.6%) isolates showed high-level resistance to gentamicin and/or streptomycin by both high-content disc diffusion and agar dilution methods.

Antibiotic sensitivity testing revealed that 50% of these isolates had high-level gentamicin resistance (HLGR) and 15.6% had high-level streptomycin resistance (HLSR) [Table 2]. All the isolates with HLSR also had HLGR. Combined resistance to both the aminoglycosides was much higher in E. faecium (33.3%) as compared to E. faecalis (11.8%). There was a difference in the resistance of E. faecalis to gentamicin (53.8%) and streptomycin (11.8%), and in E. faecium it was higher to gentamicin (66.7% alone and 33.3% overall) than to streptomycin (33.3% alone, 33.3% overall). None of the 32 enterococcal isolates were vancomycin resistant.
Table 2: Distribution of two high-level aminoglycoside resistant Enterococcal species with respect to the resistance to aminoglycoside combination

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Both E. faecalis and E. faecium showed multidrug resistance, the former to as many as seven and latter to as many as six drugs. It was observed that the resistance of high-level gentamicin-resistance and high-level streptomycin-resistance isolates to various antibiotics was more in E. faecalis than E. faecium.

Concomitant resistance of HLGR and HLSR strains to β-lactam antibiotics was quite high in both the species. In the case of HLGR and HLSR, overall it was 100% with penicillin for both the enterococci species. In the case of HLGR, 91.7% of E. faecalis was resistant to ampicillin and it was 75.0% for E. faecium. In the case of HLSR, both E. faecalis and E. faecium were 100% resistant to ampicillin [Table 3].
Table 3: Resistance of HLGR and HLSR E. faecalis and E. faecium to other antibiotics

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Phage Strain Antibacterial Activity

E. faecalis phage Ø SH-56 was found to form plaques on 79% [Figure 1] of the MDR E. faecalis clinical isolates and inhibited bacterial growth of an additional 8% of the strains, thus exhibiting an antibacterial effect against 87% of the strains in our collection. The inhibition of bacterial growth in strains that the phage could not form plaques is most likely due to partial expression of the phage genome, sufficient for killing but not enough for phage production to a level necessary for plaque formation.
Figure 1: Enterococci faecalis phage Ø SH-56.

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Electron Microscopy

E. faecalis phage Ø SH-56 had an icosahedral head, about 65 nm in diameter, and a 100-nm-long tail, thus being morphologically similar to phages belonging to Siphoviridae family [Figure 2].
Figure 2: Electron micrograph of  Enterococcus faecalis Scientific Name Search  SH-56 particles. Phage is negatively stained with potassium phospotungstate

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Host Range

None of the bacteria tested was found susceptible to E. faecalis phage Ø SH-56. Phage SH-56 did not even form plaques on E. faecium. The results indicated that phage Ø SH-56 had a narrow host range, and it is highly genus and species specific.

   Discussion Top

Recent years have witnessed an increased interest in Enterococci not only because of their ability to cause serious deep foot infections but also because of their increasing resistance to many antimicrobial agents. [2],[7] In the present study, only two species E. faecalis (81.3%) and E. faecium (18.7%) were recovered in contrast to additionally more by others from India under different clinical conditions. [21],[22] Our isolation rate corroborates with that from Nagpur [23] (E. faecalis 86% and E. faecium 14%). However, the report of higher isolation of E. faecium (80.7%) over E. faecalis (19.2%) has been there from Mumbai. [24]

Antibiotic resistance in Enterococci is either intrinsic or acquired. Intrinsic traits expressed by Enterococci include resistance to semisynthetic penicillinase-resistant penicillins, cephalosporins, a low level of aminoglycosides, and a low level of clindamycin whereas acquired resistance includes resistance to chloramphenicol, erythromycin, a high level of clindamycin, tetracycline, a high level of aminoglycosides, penicillin, fluroquinolones, and vancomycin. [1] HLAR is due to the release of various aminoglycoside-modifying enzymes.

In the present study, 65.6% of Enterococci showed HLAR. The overall prevalence of HLGR and HLSR was higher among E. faecium (33.3%) compared to E. faecalis (11.8%). Our finding correlates with Gordon et al.'s [25] whereas other investigators have found a high incidence of HLGR and HLSR among E. faecalis.[26],[27],[28],[29]

Resistance to aminoglycosides in Enterococci is often associated with multidrug resistance. [1] In our study, E. faecalis showed resistance to as many as seven drugs and E. faecium to as many as six drugs. Concomitant resistance of HLGR and HLSR strains to the two β-lactam antibiotics (penicillin and ampicillin) was quite high in both the species and it was higher to penicillin than ampicillin in E. faecalis. Associated HLAR, high-level penicillin resistance, and resistance to vancomycin have been reported in 16% isolates by Aggarwal et al.[23] from Nagpur.

HLGR has also been linked to β-lactamase production, and resistance to ciprofloxacin [27] and chloramphenicol. [1],[19] In fact Schouten et al.[27] reported that as the prevalence of HLGR increases, β-lactamase production in Enterococci may also increase. Though none of our isolates was a β-lactamase producer, as also reported by Jessudason et al., [2] both E. faecium and E. faecalis showed concomitant resistance to ciprofloxacin and chloramphenicol.

Enterococci are resistant to many commonly used antimicrobial agents (aminoglycosides, aztreonam, cephalosporins, clindamycin, the semisynthetic penicillins, nafcillin, and oxacillin, and trimethoprim-sulfamethoxazole). [24] In the present study, 100% of the isolates were resistant to one of the third-generation cephalosporins. Exposure to cephalosporins is a particularly important risk factor for colonization and infection with Enterococci. Thus, the era in which safe and effective cephalosporins became widely available has also been an era of enterococcal ascendance.

Diabetic foot is a leading cause of morbidity and mortality in developing countries. [3],[4] But, the indiscriminate overuse of antibiotics for the treatment of diabetic foot has triggered an increase in the development of multidrug-resistant "super bugs" and has been posing serious problems. [28]

Therefore, in the present investigation an attempt is made to develop an alternative to conventional drugs. One possible option is to use bacteriophages as antimicrobial agents. The bacteriophages, which are highly specific for the target bacteria without affecting the normal micro-flora, are effective against multiple-drug-resistant bacteria. Although phages were discovered nearly a century ago, [29] Western medicine's interest in them as therapeutic agents was relatively short-lived, in part because of the eventual discovery and instant success of antibiotics and in part because of the highly empirical and counterproductive approach that had been used by phage practitioners in the early era. In the modern era (1980s and 1990s), some rigorously controlled animal experiments have been conducted by Smith Soothill, [14],[30] but the clinical reports in this same era have been subjective in nature rather than unfolding controlled studies. [31] The experiments in the present study represent elucidations to many of the problems that hindered the prior applications of phage therapy. For example, the relatively narrow host range of most phages which caused many of the early attempts of phage therapy to succeed can be trounced by isolating phages that have a broad host range within the species being targeted. For example, phage Ø SH-56 forms plaques on more than 79% of the MDR Enterococci isolates tested and inhibits growth of an additional 7%, thus providing an antibacterial effect against more than 86% of approximately 32 clinical isolates of MDR Enterococci isolated from diabetic foot cases.

The potential of phage therapy has been the subject of several recent reviews [18],[30],[32],[33] and the present study strengthens the view that phage therapy is worth exploring. We resolute our efforts on MDR Enterococci because we predicted that positive results would reveal the prospective of this form of therapy in situations where few substitutes are available. It is enticing to advocate research investigations into enterococcal infections for which animal models for infection are available and for which phages may be isolated.

This study revealed the prevalence of multidrug-resistant HLAR strains of E. faecalis and E. faecium in diabetic foot infection. The problem of vancomycin-resistant Enterococci (VRE) may not be very high in India [21],[23],[24] as also seen in our hospital at present. But monitoring of VRE is the need of hour, since it appears to be an emerging pathogen in India. If the incidence of VRE increases, we may be left with no other alternative to treat VRE; as a result it would seem timely to begin to look anew at the viability of using phages for treatment, and a scientific methodology can make phage therapy as a stand-alone therapy for infections that are fully resistant to antibiotics.

   Acknowledgments Top

Authors would like to thank Department of Pathology, NIMHANS, Bangalore; St. Johns Medical College, Bangalore; and S. S. Institute of Medical Sciences and Research Centre, Davangere, for the facilities.

   References Top

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Correspondence Address:
C S Vinodkumar
Department of Microbiology, S.S. Institute of Medical Sciences and Research Centre, Davangere - 577 005, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.77333

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