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India accounts for more than half of the global leprosy burden.1 Plantar ulcers are one of the most common complications of leprosy and occur due to sensory loss and consequent chronic wound formation.2 Plantar ulcers lead to disability, which is associated with stigma and discrimination and further impacts the quality of life of the people affected by leprosy.3 The majority of the chronic wounds in leprosy begin as minor traumatic, penetrating injuries on the feet, which would normally heal like acute wounds within a few days or weeks. Most of these wounds however, tend to recur as non-healing wounds leading to ulceration, due to factors such as hypoxia, malnutrition and bacterial colonisation. Chronic wounds result from the disruption of the normal wound healing sequence of hemostasis, proliferation of immune cells to release cytokines, inflammation and tissue remodelling.4 Wound hypoxia leads to tissue necrosis and decreased proliferation of immune cells, while malnutrition reduces the inflammatory process and thus disrupts normal wound healing.
Colonisation of wounds, with obligate bacterial pathogens such as S. aureus and Pseudomonas spp., is another important causative factor for the chronicity of open wounds.5 Bacterial colonisation delays healing not only due to direct damage to the host tissue but also due to the triggering of the inflammatory cascade and inhibition of wound closure.6 Moreover, bacteria in chronic wounds may produce biofilms, which consist of an extra-cellular matrix that embeds the bacterial cells, protecting them from host immune responses.7 Secondary bacterial infection of plantar ulcers thus forms one of the possible causative factors for chronicity and if unattended, may progress to cellulitis and osteomyelitis, warranting surgical interventions such as amputation of the infected limb to avoid further spread of infection.8
Understanding secondary microbial infections therefore forms an important basis for effectively managing leprosy plantar ulcers and preventing leprosy related disability. There are reports from various leprosy endemic countries on the profile of bacterial infections in leprosy plantar ulcers and antimicrobial resistance of the bacterial isolates.9–13 Secondary infection with antimicrobial resistant strains such as MRSA from plantar ulcers has been reported.14
Biofilm formation is known to be one of the key features of pathogenic bacteria, enabling them to become established on wounds and to develop resistance to antimicrobials.15 Normal skin healing involves a short inflammatory phase of 2–3 days, which then switches to tissue regeneration.16 However, the presence of biofilm alters the healing process by prolonging the inflammatory phase.17 It is therefore important to understand the bacterial colonisation and the effect of biofilms on chronic wounds in order to formulate effective management strategies.
Bacterial biofilms have been studied in other chronic wound conditions, such as diabetic foot ulcers.18 However, there is limited evidence on bacterial biofilm in infected plantar ulcers in leprosy and its correlation with antimicrobial resistance, the subject of this study. To our knowledge this is first such study to be reported. The study has been approved by the Institutional Ethics Committee (IEC) of LEPRA Society, Blue Peter Public Health and Research Center (BPHRC) (Ref no. LEPRA/BPHRC/IEC/2016-17/02).
Material and methods
Subject
Leprosy patients with plantar ulcers who were registered at LEPRA Society, BPHRC, Hyderabad, India during May 2017–March 2019 (n = 64) were enrolled, after obtaining informed consent.
Clinical examination
Leprosy patients were examined for leprosy and associated clinical manifestations. The demographic and clinical data including age, sex, type of leprosy, duration of ulcer, site were documented.
Sample collection
A total of 64 swabs (one from each ulcer) were collected from the base of the plantar ulcer (Figure 1) with sterile pure viscose swab (Hi-media, Mumbai) and immediately transported to the microbiology laboratory for further processing.
Figure 1.
A typical plantar ulcer in leprosy.
Isolation and identification of bacteria
Each swab was inoculated on Blood and Mac Conkey’s agar plates and incubated overnight at 37 °C for aerobic bacterial culture. Overnight cultures were subjected to standard bacterial identification methods such as colony morphology, gram stain, oxidase, catalyse, motility and biochemical tests.7 All the isolates were subjected to antimicrobial susceptibility testing and in-vitro biofilm formation.
Antimicrobial susceptibility testing
The antimicrobial susceptibility testing (AST) was determined by the Kirby-Bauer disc diffusion method as previously described,8 against nine commonly used antibiotics in eight classes, namely aminoglycosides (amikacin: 30 μg, gentamicin: 10 μg), cephalosporins (cefixime 30 μg), fluoroquinolones (ciprofloxacin 5 μg), macrolides (erythromycin 15 μg), penicillin (amoxicillin 25 μg), penicillin lactamase inhibitors (amoxy-clav 30 μg), sulphonamides (co-trimoxazole 25 μg), and tetracyclines (doxycycline hydrochloride 30 μg). The antibiotics were selected from those commonly used to treat bacterial infections in the study area. The disk diffusion test was performed using Mueller–Hinton agar (HiMedia, India) plates, which were inoculated with bacteria equivalent to 0.5 McFarland standards and incubated for 18–24 h at 37 °C aerobically, as recommended by Clinical and Laboratory Standards Institute (CLSI). Isolates were classified as susceptible, intermediate and resistant as per standard guidelines.9 Bacterial isolates which was found resistant to multiple antimicrobial agents (at least three classes of antibiotics) was considered as multidrug-resistant (MDR).10
Biofilm screening by Congo Red Agar (CRA)
Biofilm formation was detected by the Congo Red method (CRA) as described by Kumar et al.11 A special medium composed of Brain Heart Infusion (BHI) broth (37 gm/L), sucrose (50 gm/L), agar no.1 (10 gm/L) and Congo Red stain (0.8 gm/L) was used. Congo Red was prepared as a concentrated aqueous solution and autoclaved at 121 °C for 15 min, separately from other medium constituents and was added when the agar had cooled to 55 °C. Plates were inoculated and incubated aerobically for 24 h at 37 °C. Colonies were observed for biofilm production. Colonies of biofilm producing bacteria were black in colour, while non-producers were red coloured (Figure 2).
Figure 2.
The Congo Red method: colonies of biofilm producing bacteria were black in colour, while non-producers were red coloured.
Microtitre plate method (MTP)
The Microtitre plate method (MTP) was performed as described by Christensen et al.,12 for quantitative measurement of biofilm production in clinical isolates of bacteria. A single colony from each agar plate was inoculated in a 15 mL falcon tube containing five ml of Tryptic Soy Broth containing 1% glucose. The tubes were incubated overnight at 37 °C under aerobic conditions. Two hundred micro litres containing a 1:100 dilution of overnight broth culture of the test organism was transferred in the wells of a flat-bottomed micro-well plastic plate and incubated overnight at 37 °C. After incubation, planktonic cells were washed thrice with Phosphate buffered saline (PBS) and air dried, then stained with crystal violet solution; glacial acetic acid was added to the wells and optical density was read at 590 nm. An optical density (OD) greater than 0.240 indicated a strong biofilm producer, an OD between 0.120–0.240 a moderate biofilm producer and an OD of less than 0.120, a non-biofilm producer (Figure 3). S. aureus ATCC 25923 was used as the control organism for biofilm production.
Figure 3.
The Microtitre plate method: high optical density in biofilm producers.
Statistical analysis
All data were recorded in Excel (Microsoft-2007) and analyzed with SPSS software (SPSS-16.0). Chi-square test was done to evaluate the relationship between bacterial biofilm and antibiotic resistance. A p-value < 0.05 was considered as significant.
Definitions
Acute ulcer: Persistence of an ulcer for less than six weeks and presenting with signs of acute inflammation.
Chronic ulcer or non-healing ulcer: Ulcer failing to show signs of healing after six weeks of treatment with complete rest and sterile dressings.
Infected ulcers: Wounds with clinical signs of erythema, edema, cellulitis, increased exudates.
Non-infected ulcers: Wounds without clinical signs of erythema, edema, cellulitis, exudates.
Release from treatment (RFT): Leprosy patients who have successfully completed treatment with multi drug therapy (MDT) and who are regarded as cured.
Newly registered: Patients who are newly registered for leprosy and who are on treatment with MDT.
Mono-bacterial: Occurrence of a single bacterial species was termed mono-bacterial.
Poly-bacterial: Occurrence of more than one bacterial species was termed poly-bacterial.
Susceptible: A bacterial strain is said to be susceptible to a given antibiotic when it is inhibited in vitro by a concentration of this drug that is associated with a high likelihood of therapeutic success.
Intermediate: The sensitivity of a bacterial strain to a given antibiotic is said to be intermediate when it is inhibited in vitro by a concentration of this drug that is associated with an uncertain therapeutic effect.
Resistant: A bacterial strain is said to be resistant to a given antibiotic when it is inhibited in vitro by a concentration of this drug that is associated with a high likelihood of therapeutic failure.
Multidrug-resistant (MDR): Bacterial isolates which were non-susceptible to at least one agents in three or more antimicrobial categories.
Biofilm: A biofilm is a sessile community of microorganisms growing in a self-produced matrix of extracellular polymeric substances (EPS).
Results
A total of 64 leprosy patients with non-healing ulcers (Figure 1) were enrolled. Of these, 57 (89%) were treated for leprosy in the past and the remaining 7 were newly registered for leprosy treatment at the time of enrolment. Twenty-one (33%) were female. All patients had a single ulcer with mean diameter 3.85 cm (SD 4.44) (Figure 1). Forty-eight (75%) were clinically infected, based on clinical signs. Fifty-seven (89%) were chronic ulcers and seven were acute wounds. Forty-one (64%) occurred on the metatarsal area, 10 (16%) on the calcaneus/heel, 7 (11%) on the greater toe, and 6 (9%) on the mid-foot.
Among the 64 swabs tested, 61 (95%) were found to be culture positive, of which 55 (90%) were mono-bacterial. Of the 67 bacterial isolates, 73% (49/67) were gram-positive and 27% (18/67) were gram-negative organisms. The most commonly isolated organism was S. aureus in 36/67 (54%), followed by P. aeruginosa in 8/67 (12%), Streptococcus spp (8/67), P. mirabilis (4/67), E. coli (3/67), K. pneumonia (2/67), M. morganii (2/67) and A. buamannii (1/67). All forty-eight subjects with clinical signs of infection were culture positive for pathogenic bacteria and 13/16 (81%) with no clinical signs of infection also yielded pathogenic bacteria. All the clinical isolates tested demonstrated resistance to at least one or more antimicrobials (Data not shown).
Of the 67 bacterial isolates screened for biofilms by the CRA method, 29.8% (n = 20) were biofilm producers (BP) and 70.1% (n = 47) were biofilm non-producers (NBP). Using the MTP method, 28/67 (41.7%) of isolates were BP, (of which, seventeen were strong and eleven were moderate BP), while 39/67 (58.2%) were NBP. The frequencies of resistance to various antimicrobials in BP and NBP cases are shown in the Table 1. Resistance to amikacin (85.7%) was found to be highest among BP isolates followed by resistance to amoxicillin and doxycycline, whereas resistance to gentamicin (56.4%) was highest among NBP isolates (Table 1). In total, 27 (55%) of the 49 MDR isolates were BP.
Table 1
The frequencies of resistance to various antimicrobials amongst biofilm producers and non-producers
| Characteristics | Biofilm producers % (n = 28) | Biofilm non producers % (n = 39) | p value |
| Multi drug resistance | *0.0003 | ||
| Yes | 96.4 (27) | 56.4 (22) | |
| No | 03.6 (01) | 43.6 (17) | |
| Antibiotic resistance | |||
| Gentamicin 10 μg | 67.9 (19) | 59.0 (23) | 0.46 |
| Erythomycin 15 μg | 57.1 (16) | 56.4 (22) | 0.95 |
| Cefexime 30 μg | 57.1 (16) | 41.0 (16) | 0.19 |
| Amikacin 30 μg | 85.7 (24) | 38.5 (15) | *0.0001 |
| Amoxicillin 25 μg | 78.6 (22) | 28.2 (11) | * <0.0001 |
| Doxycycline 5 μg | 78.6 (22) | 25.6 (10) | * <0.0001 |
| Ciprofloxacin 5 μg | 57.1 (16) | 23.1 (09) | *0.004 |
| Cotrimoxazole 25 μg | 57.1 (16) | 23.1 (09) | *0.004 |
| Amoxyclave 30 μg | 42.9 (12) | 23.1 (09) | 0.085 |
Discussion
Plantar ulceration is one of the most common complications of leprosy, often leading to disfigurement and disability.19 Plantar ulcers, which result from loss of sensation of feet and subsequent proneness for injuries, tend to occur even after completion of leprosy treatment.20 People affected by leprosy make multiple visits to the health care facility in order to seek treatment for chronic ulcers with cost implications in terms of loss of work hours and health expenditure.21 It is hence important that plantar ulcers are appropriately managed so as to prevent their recurrence and minimise patient visits to the health care facility and thus reduce the cost associated with ulcer care.
In the present study, the majority of the subjects had ulcers years after completing multi-drug therapy (MDT), indicating the need for incorporating appropriate interventions for identifying and treating plantar ulcers even after MDT is completed. Health education for self-care practices for prevention of ulcers, periodic screening for early detection and treatment may be undertaken at all levels of the leprosy control programme. It is alarming to find plantar ulcers at the time of leprosy diagnosis, which clearly indicates a delay in diagnosis, which suggests the need for better case detection strategies. We observed a male preponderance among the study subjects; it is possible that males are at higher risk for foot injuries by virtue of their mobility and mostly outdoor work. The metatarsal region of foot seems to be the commonest anatomical location for the occurrence of plantar ulcers, followed by the calcaneus region. The metatarsal region is the main weight-bearing region, undergoing shearing pressures while walking and standing, leading to ulceration.22
The study found that more than three quarters of ulcers were secondarily infected with pathogenic bacteria. It is however interesting to note that even ulcers without any clinical indication of infection also yielded pathogenic bacteria. We presume that chronic wounds could form a niche for bacterial colonisation. However, the role of the bacterial colonies on apparently non-infected ulcers is yet to be understood and beyond the scope of the current study. This finding also suggests that mere presence of microbes may not lead to clinical complications of infection, and hence it may not be necessary to treat all chronic ulcers with antibiotics. Further studies involving larger samples and advanced microbiological tools could throw more light on this.
There have been reports from within India and elsewhere during the past decades demonstrating the nature of secondary infections in leprosy plantar ulcers, causative bacterial pathogens and their antibiotic susceptibility pattern.13–16 There has been a certain level of diversity observed as far as the bacterial profile that is reported by various studies. Most of these reports indicated the presence of antimicrobial resistance among the wound isolates. While we found S. aureus as the commonest organism followed by P. aeruginosa, streptococcus spp and proteus spp., Ashwini et al.,13 reported S. aurues, P. aeruginosa, Proteus spp, Corynebacterium spp., E. coli and other species. Majunmdhar et al.,10 reported E. coil, Proteus spp. and Pseudomonas spp. Ramos et al.,9 reported Proteus spp, followed by E. coli, S. aureus and P. aeruginosa. It is known that the bacterial profile of chronic wounds varies across the clinical setting and over time, even in the same setting.
While Ramos et al.,9 reported less than 30% resistance to gentamicin and ciprofloxacin, making them the drugs of choice for empirical treatment, we found a higher percentage of resistance for these two drugs, probably due to the current clinical use of these two drugs as presumptive antimicrobials for treating plantar ulcers. Considerably higher frequency of AMR isolates as reported in this study suggests that most of the antimicrobials that are routinely used are no more effective for treating these infections. Hence, it is very important to identify the profile of bacterial pathogens and their antimicrobial susceptibility patterns, before treating the plantar ulcers with antibiotics. Future research in this area should focus on therapeutic approaches alternative to antimicrobials. Interventions which either prevent the microbial colonisation or promote the wound healing could be more effective than presumptive antibiotic usage.
The study reports for the first time the occurrence of bacterial biofilms on plantar ulcers in leprosy, with about half of the clinical isolates screened testing positive for biofilm production, which is an interesting finding. MTP is supposed to be a more sensitive method than CRA for screening of biofilm production, as indicated by the additional eight isolates that produced biofilm on MTP. We also found that biofilm formation was significantly associated with antimicrobial resistance (see Table), as also reported by Dumaruet et al.23 The association of biofilm formation with multi-drug resistance has been already reported on chronic diabetic foot ulcers.24 Swarna et al.,25 reported that about three-quarters of the MDR organisms isolated from chronic diabetic foot ulcers were biofilm formers. In the present study, almost half of the MDR isolates were biofilm producers indicating the significance of bacterial biofilms as an emerging clinical problem. This can be explained by the fact that biofilms consist of intercommunicating microbial cells leading to horizontal gene transfer, which potentially alters the microbial phenotype towards antimicrobial resistance.26 We presume that similar mechanisms could potentially occur in plantar ulcers, which have more or less similar pathophysiology to that of diabetic ulcers. It may be deduced that biofilm producing pathogenic strains pose a higher threat of antimicrobial resistance in plantar ulcers in leprosy, indicating the need for formulating appropriate interventions. Our study findings highlight the importance of bacterial biofilm in understanding the antimicrobial resistance in chronic plantar ulcers in leprosy. It is important for wound care strategies to adopt suitable interventions to address bacterial biofilm.
The present study, which reported the presence of biofilm for the first time in leprosy ulcers, is limited by a small sample size. Further studies on larger samples across various settings are recommended, in order to further validate the role of biofilm in plantar ulcers in leprosy. Further studies are also needed to implicate biofilm in the clinical outcome of ulcers, which could pave the way for better strategies for ulcer care in leprosy patients. Addressing plantar ulcer treatment forms one of the most important components in our efforts towards the global leprosy control strategy of ‘Zero disability’ due to leprosy.
Acknowledgements
The study is funded by the Department of Science and Technology, Science and Engineering Research Board, Govt. of India (National Post Doctoral Fellowship). BPHRC is core supported by Lepra, Colchester, UK. Authors acknowledge the study subjects for consenting to participate and support of staff of BPHRC clinic.
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