Assessment the Role of Phagocytic Neutrophil Cells among Different Wagners Grades of Diabetic Foot Ulcers Infection

Introduction

Diabetes mellitus (DM) is one of the main global health prob- lems. It's defined as a metabolic disorder resulting from a de- fect in insulin secretion, insulin action, or both, which leads to chronic hyperglycemia [1]. Several polymorphonuclear leukocyte cells (PMNLs) affected by hyperglycemia, neutrophil's functions impair occur in diabetic subjects including impaired migration, phagocytosis, intracellular killing and chemotaxis, which may be due to decreased PMNLs membrane fluidity [2]. There is clinical evidence pointing to the higher prevalence of infectious diseases among individuals with DM [3]. Foot ulcers remain one of the most distressing complications of a diabetic patient and it's one of the most significant and devastating complications of diabetes [4]. Wagner’s ulcer grades classification is one of the most widely used and universally accepted grading systems for diabetic foot ulcers (DFU), consisting of six simplistic wound grades used to assess ulcer depth as following: grade-0; high risk foot and no ulceration, grade-1; superficial ulceration, grade-2; deep ulcer (cellulitis), grade-3; osteomyelitis with ulceration or abscess, grade-4; gangre- nous patches and grade-5; gangrene of entire foot [5].

Phagocytic cells activity is affected by blood sugar levels, and in- fection of lesions grades of DFU also affected by phagocytic neu- trophil cells activity. This study aimed to assessment the phago- cytic neutrophil cells and its role to fight the bacterial infection of diabetic foot ulcers classified according to Wagner’s grades sys- tem, as well as to determine the antibiogram patterns of bacterial species isolated from DFU.

Materials and Methods

Study Design

A case-control analytical study was conducted on diabetes mellitus and diabetic foot ulcer patients.

Research Setting

Some hospitals in Mukalla city, Hadhramout, Yemen were selected to achieve the research.

Participants

A total of 60 volunteers were selected randomly and classified into 20 DFU patients, 20 DM patients and 20 healthy control group.

Preparation of Samples

About 10ml of venipuncture blood samples were collected from all participants in ethylene diamine tetraacetic acid (EDTA) for phagocytic neutrophil activity test.

A total of 20 wound swabs were collected from DFU after cleaned with sterile saline to remove accumulated drainage and transient skin flora, then the swabs delivered to the microbiology laboratory for culture [6].

Classification of Diabetic Ulcers

The ulcers of diabetic foot patients were classified according to Wagner’s grades classification system as an assessment of five grades at the time of study period.

Phagocytic Neutrophil Cells Activity

Test Phagocytic cells activity test was performed according to the methods described by Shodja et al. [7] with minor modifications. In briefly, normal saline suspension of pathogenic strain Staph- ylococcus aureus (S. aureus) colonies was prepared and the vis- ible turbidity was adjusted to 0.5 McFarland turbidity standard yielding an approximately 1.5×108 CFU/ml. About 1ml of EDTA whole blood mixed with 1ml of saline bacterial suspension, then incubated for one hour at 37°C. One drop of incubated mixture was placed on a slide of microscope to make a thin smear. The smear was lifted to dry for 3 minutes, and fixed with ethanol 96%, then placed in hematoxylene stain for 10 minutes. After water washing, the smear placed in eosin stain for 30 seconds, then washed by wa- ter and examined under light microscope with 100x oil immersion. The total number of S. aureus ingested within 100 neutrophil cells were counted and divided by 100 to give the percentage of phago- cytic cells activity. The results were interpreted as the following: less than 40% (low phagocytosis activity), 40%-70% (intermedi- ate phagocytosis activity) and more than 70% (high phagocytosis activity).

Isolation and Identification of Pathogenic Bacteria

Samples of ulcers swab were inoculated into blood agar and Mac- Conkey agar media (Oxoid/England). The culture plates were incubated under aerobic conditions at 37°C for 24 hours. Iden- tification of bacterial species was made based on bacteriological standard methods [8].

Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was done using Kirby-Bauer disc diffusion method on Mueller Hinton agar (Oxoid/England) for testing ampicillin, cefepime, cefadroxil, ciprofloxacin, cefixime, cefaclor, trimethoprim, amoxicillin/clavulanic acid, amikacin and vancomycin. Resistant to methicillin was determined by the disc diffusion method to identify methicillin resistance S. aureus (MRSA) isolates. All susceptibility testing methods done accord- ing to the standard guidelines of the Clinical Laboratory Standards Institute (CLSI) [9].

Statistical Analysis

Data analyzed using the software of Statistical Package for Social Sciences (SPSS) version 25 (IBM Corp., Chicago Illinois, USA). Descriptive statistics for study variables were obtained and com- pared using least significant difference test (LSD) and one away ANOVA test. The association between different groups of the ex- planatory variables was measured and compared using Pearson Chi-square (χ2) test. The relationship between the variables exam- ined by the Pearson correlation (r) test. The level of significance was set at P-value less than 0.05.

Results

Diabetic Foot Ulcers Classification

As an assessment of DFU grades by Wagner's classification sys- tem, the results showed that grade I and II accounts 6(30%), grade III 5(25%), grade IV 1(5%) and grade V 2(10%).

Distribution of Bacterial Species Isolated from DFU

Twenty-one bacterial species were isolated from DFU according to the conventional methods for identification of bacteria as pre- sented in Table (1). Gram-positive bacteria showed an increased prevalence 57.1% in diabetic ulcers. The most common bacterial isolates recovered from diabetic ulcers were Staphylococci spe- cies 40.8%, with high rate of S. aureus and S. epidermidis 14.0% for each, S. saprophyticus 10.0% and MRSA 5.0%. Streptococci species were the second most commonly isolated pathogens in our study 14.0%. P. mirabilis and M. morganii accounted 9.0% of pre- dominant Gram-negative bacteria in DFU in this study. The prev- alence of P. aeruginosa, Klebsiella species, Esch. Coli, C. freundii and Serratia species isolates accounted 5.0% for each.

Table 1: Bacterial Species Isolated from Diabetic Ulcers Samples

Bacterial Species of DFU Associated with Wagner’s Grades System

As shown in table (2), the diabetic patients have superficial wounds of Wagner’s grades classification I and II were mostly infect- ed with Gram-positive bacteria. Staphylococci and Streptococci species were the predominant pathogens, whereas Gram-negative bacteria were more frequently isolated from deep wounds (grades III and above) with Statistically significant of P-value 0.057.

Table 2: Distribution of Bacterial Isolated according to Wagner’s Grades of Ulcers

Antibiotics Resistance Patterns of Bacterial Isolates

The results of the antibiotics sensitivity assay showed the pres- ence of resistant patterns of the isolated bacteria to antibiotics. The highest percentage of bacteria resistance was to ampicillin 95.2, followed by cefixime 90.5, cefadroxil 52.4%, cefepime and cefa- clor 47.6% as given in Table (3).

Staphylococci species were resistant for ampicillin 100% and highly sensitive to ciprofloxacin, amikacin, trimethoprim and ce- faclor. MRSA showed high resistant to cefixime, ampicillin, ce- fadroxil and trimethoprim 100% and highly sensitive for vanco- mycin, amikacin, ciprofloxacin, amoxicillin/clavulanic acid and cefepime 100%. Streptococci species were resistant for cefepime, cefixime, ampicillin, cefadroxil and cefaclor 100%, and sensitive for ciprofloxacin and amikacin 100%. Gram-negative bacteria showed high resistance to ampicillin and cefixime 88.8% followed by cefadroxil, cefaclor, cefepime 77.8% and trimethoprim 55.6%. Most of the isolates were susceptible to amikacin 77.8% and cip- rofloxacin 55.6%.

Table 3: Antibiotics Susceptibility Patterns of Bacterial Isolates

Antibiotics Resistance Patterns Associated with Wag- ner's Ulcers Grades

Our study revealed that a low significant positive correlation be- tween the antibiotics resistant and increasing of Wagner’s grades classification. When the resistance of bacterial isolates increased the grades of Wagner's classification increased too according to Pearson correlation test (P-value = 0.05; r = 0.369).

Phagocytic Neutrophil Cells Activity Test

According to phagocytic cells activity test, DM and DFU patients had no highly efficient phagocytic neutrophil cells activity, 1 and 0 respectively as given in table (4).

Table 4: Phagocytic Neutrophil Cells Activity among Studied Groups

As shown in table (5), the mean of phagocytosis was completely different between the three groups studied (control, DM and DFU pa- tients) using LSD test with high significant difference statistics.

Table 5: The Mean of Phagocytic Neutrophil Cells Activity among Studied Groups

In this study we correlated the degree of wounds with phagocytic efficiency to understand the development of the ulcer grade and role of neutrophil phagocytic cells. Statistically, there was low insignificant correlation between these two variables. Also, the study results showed a weak inverse correlation between the ratio of phagocytic cells activity and the degree of wound in Wagner’s classification (P-value 0.164; r = -0.323), as given in table (6).

Table 6: Correlation between Phagocytic Neutrophil Cells Ac- tivity and Wagner's Grades of Ulcer

Discussion

In the present study, the results of the assessment DFU grades by Wagner's classification system showed high prevalence of grades I and II followed by grade III. These results were compatible with other studies conducted in different countries [10-12], but disagree with a previous study reported that Wagner’s grade II and III ulcers were the most common prevalence [13]. Grade IV was the lowest prevalence in this study, and this contrast with other studies re- vealed that the grade IV was the most common prevalence in DFU [14-15]. Identifying the risk factors of DFU might help to develop better prevention strategies in diabetic patients.

In this study, Gram-positive bacteria showed an increased prev- alence 57.1% in diabetic ulcers with similar results of different studies 77% [16], 35% [17] and 51.1% [5]. The most common bac- terial isolates from diabetic ulcers in this study were Staphylococci species 40.8% compatible to other findings showed that Staphy- lococci species accounted 43.37% [18], whereas Gram-negative bacteria were the most frequently pathogenic agents reported in other studies 51% [19], 56.1% [20], 61% [21], 67% [22] and 51.2% [23].

Interestingly, our study revealed high rate of S. aureus 14.3%, S. epidermidis 14.3%, S. saprophyticus 9.5% and MRSA 4.8%. These results were compatible with the findings of other studies showed that S. aureus 19.4%, S. epidermidis 18.4% and other Staphylococci species 3.6% [24]. Other study showed the most commonly isolated S. aureus 30%, followed by S. saprophyticus 19% and S. epidermidis 10% [25]. Another study showed that S. aureus 25%, S. saprophyticus 15% and S. epidermidis 7% were the most isolated from DFU [11].

Streptococci species were the second most commonly isolated pathogens in our study 14.3%, this is nearly with other studies iso- lated Streptococci species 17% [26], 16% [27] and 15.6% [28]. Poor control of blood glucose was associated with a high relative abundant colonization by Staphylococci species and Streptococci species [29]. So, the most cases of DFU who have mixed infection of Staphylococci species and Streptococci species were poor gly- cemic control patients in our study.

P. mirabilis and M. morganii accounted the predominant Gram-neg- ative bacterial isolated from DFU in this study. High prevalence of Proteus species was reported in different studies [19,27,30], while M. morganii showed low prevalence isolated 3.16% [31]. P. aeru- ginosa accounted 4.8% with resistance to the most antibiotics used in this study. Similar results found that Pseudomonas species were the most common isolated from DFU [28-30,32]. P. aurginosa may cause an invasive form, sometimes in diabetics which fails to re- spond to topical antibiotic therapy [33]. Additionally, significant de- lay in wound healing cause of P. aeruginosa biofilms inhibit neutro- phil movement [34]. So, the coordinated control of the production of virulence and antibiotic resistance factors and the ability to adapt to various environmental changes is a likely and important reason that P. aeruginosa is a successful and common pathogen [35].

In this study, the prevalence of Klebsiella species and Esch. coli agreed with other study results 4% [11], and disagreed with other study recorded a high rate of Klebsiella species 12.6% and Esch. coli 17.9% isolated from DFU [31]. Also, the prevalence of C. freundii and Serratia species isolates is comparable with other re- sults 5% [17], 3.85% [5], 4.3% [36] for C. freundii and 2.3% for Serratia species [37]. Different geographical area could have con- tributed to different types of bacterial isolated [38]. The results of numerous studies conducted on the bacterial profiles of DFU showed a varied and often contradictory findings [39]. The reason for this difference in findings could be attributed to the difference in the causative agents, geographic variations or the severity of the infections [27].

In the current study, the diabetic patients have superficial wounds of Wagner’s grades classification I and II were mostly infect- ed with Gram-positive bacteria. Staphylococci and Streptococci species were the predominant pathogens, whereas Gram-negative bacteria were more frequently isolated from deep wounds (grades III and above). These findings are in agreement with various stud- ies concluded that an increasing of Wagner’s grades, the propor- tion of bacterial infections were increased [2, 21, 30, 40]. Howev- er, the percentage of S. aureus decreased with increased Wagner’s grades similar findings reported in other study [19]. MRSA is an increasing problem in industrialized and developing countries. It is commonly believed to be an important cause of poor outcome, increased duration of hospital stays, increased cost and mortality [14].

In this study, MRSA were associated with Wagner’s grade III ulcer classification, and other study showed that Wagner’s ulcers grade III was commonly infected with MRSA [14]. This study showed the presence of antibiotics resistant patterns of bacterial isolated. The highest percentage of bacteria resistance was to ampicillin, cefixime, cefadroxil, cefepime and cefaclor, and these finding were similar to another previous studies reported high resistant patterns in a rate of 92.4% [37], 94.9% [41-42] and 50% [38]. The increased resistance may be attributed to the fact that ampicillin has been widely abused and frequently implicat- ed in self-medication[40]. Cefepime was active against 47.6% of isolates, and this result agreed with other reports 41% [36], 61.9% [41], 42.4% [27] and 46.7% [42].

Our results revealed that amikacin was the most effective antibiot- ic against 90.5% of the bacterial isolates. This result was similar to other results showed 75.4% [43], 96.7% [37], 82.9% [44] and 90% [21], while its less activity showed in other studies 54.7% [43], 54.5% [45], 62.7% [46]. Also, ciprofloxacin showed greater sus- ceptibility to the isolates 81%, and this result was approved with several studies reported sensitivity of ciprofloxacin was 63.7% [47], 61.8% [42], 78.8% [28] and 77.3% [48], while other stud- ies showed high resistant bacteria to ciprofloxacin 51.2% [49] and 71.8% [45].

In this study, vancomycin showed 83.3% sensitivity of Gram-pos- itive bacterial isolates. This similar to other studies that showed 90.1% [47], 95% [31] and 98.2% [19], while other study revealed low sensitivity of vancomycin 44.5% [37]. However, other studies showed 100% of Gram-positive bacteria susceptible to vancomy- cin [5, 24, 29, 30, 44, 50].

In our study, amoxicillin/clavulanic acid was active against bac- terial isolates 61.9%, and this result was in accordance with the studies showed amoxicillin/clavulanic acid sensitivity were 67.4% [22], 49% [47] and 57.2% [49]. In contrast, other studies showed high resistance amoxicillin/clavulanic acid 79% [37] and 81.8% [45]. Cefexime showed low sensitivity 9.5% for the isolated bac- teria in this study. Similarly, low rate reported in other study 3.2% [51]. While the sensitivity for Gram-negative bacteria to cefexime was 70.7% [5], and for Gram-positive bacteria was 77.3% [28]. The sensitivity of cefaclor was 47.6% of bacterial isolates in this study, and this was compatible with the results of other study showed the second generation of cephalosporin was 50% [37]. Other studies showed that cefaclor was active 58.4% against Gram-positive bac- teria [45], and 92.8% against Gram-negative bacteria [38].

Trimethoprim had sensitivity 52.4% of bacterial isolates in this study. It's similar with the results of other studies 50% [37] and 70.3% [38], while the sensitivity to co-trimoxazole was low in oth- er studies 16% [31] and 14.3% [48]. Cefadroxil was active 52.4% against bacterial isolates. Other study indicated that first genera- tion of cephalosporin was sensitive 41.7% [36] and 16.1% [27]. Other studies showed the sensitivity of cefadroxil to Gram-nega- tive bacteria was 44.4% [42] and 11% [19].

Our study revealed that a low significant positive correlation be- tween the antibiotics resistant and increasing of Wagner’s grades classification. When the resistance of bacterial isolates increased the grades of Wagner's classification increased too according to Pearson correlation test (P-value = 0.05; r = 0.369). Such relation has previously reported that an increasing of Wagner’s grades, the resistance rates to some antibiotics increased [52].

This study indicated a significant reduction in phagocytic activity in DM and DFU patients compared with control group. In addi- tion, our baseline results confirm and extend previous reports con- cerning the impairment of phagocytosis that occurs in neutrophil cells from diabetic patients than non-diabetic patients [53]. Other studies showed a significant reduction in phagocytic cells activity in Type-1 DM and Type-2 DM [54-55]. This difference may be due to that hyperglycemia impairs granulocyte functions includ- ing adherence, chemotaxis, phagocytosis and bactericidal activity [56]. So, hyperglycemia reduced response of neutrophil function and disorders of humoral immunity as one of the long-term ef- fects of elevated mean blood glucose (MBG) or HbA1c, also the impaired micro-vascular circulation in patients with diabetic foot limits the access of phagocytes favoring development of infection [3, 57, 23].

One of the strengths our study results find the relationship between the manner of immune response involving phagocytic cells activi- ty and the degree of wound. In this study, we correlated the degree of wounds with phagocytic efficiency to understand the develop- ment of the ulcer grade and role of neutrophil phagocytic cells. Statistically, there was low insignificant correlation between these two variables. Our study results showed a weak inverse correla- tion between the ratio of phagocytic cells activity and the degree of wound in Wagner’s classification (P-value 0.164; r = -0.323). However, in our knowledge, there have been no reports in the lit- erature regarding the correlation between phagocytic cells activity and the degree of wound in diabetic foot patients to support our results to know the reason of this relation we need further deep studies to be clarified.

Conclusion

This study confirmed when the grade of ulcer increased, the bac- terial resistance to antibiotics increased, and this was emphasis the correlation with prevalent of Gram-negative bacteria in the high grade of ulcers with high resistance of antibiotics. In contrast, the grade of ulcer increased, the efficiency of neutrophil phagocytic cells decreased. This study discover the correlation between dia- betic foot ulcers and bacterial infection that can be beneficial for the surgeons to study antibiotics resistance in diabetic foot ulcer patients. The study will help the researchers to uncover the rela- tionship between the manner of immune response involving the role of neutrophil phagocytic cells activity and the development degree of foot ulcer. Further immunological and bacteriological investigations needed to control the increasing of antibiotics resis- tance in diabetic foot ulcers patients.

Abbreviations

DM: Diabetes mellitus; DFU: Diabetic foot ulcers; PMNLs: Polymorphonuclear leukocyte cells; EDTA: Ethylene diamine tetraacetic acid; CLSI: Clinical Laboratory Standards Institute; SPSS: Statistical Package for Social Sciences; LSD: Least signifi- cant difference test; MBG: Mean blood glucose; MRSA: Methicil- lin resistance Staphylococcus aureus.

Acknowledgment

The authors thanks all the participants in the research work. Great thanks expressed to the administrations of the hospitals for their cooperation.

Author’s Contribution

Maryam Baras: conceptualization the idea, methodology, data analysis and writing the first draft preparation. Eidha Bin-Hameed: reviewing the manuscript formats, paper structure, final reviewing and editing.

Funding

Not applicable.

Availability of Data and Materials

All the data are available from corresponding author if required.

Ethics Approval and Consent to Participate

The study was approved by the board of Faculty of Science, Hadh- ramout University, Yemen. Ethical approval was also obtained from the hospitals in Mukalla City, Hadhramout.

Consent for Publication

Not applicable.

Conflict of Interest

It is declared that they have not conflict of interest.

References

1. Aynalem, S. B., & Zeleke, A. J. (2018). Prevalence of diabetes mellitus and its risk factors among individuals aged 15 years and above in Mizan-Aman town, Southwest Ethiopia, 2016: a cross sectional study. International journal of endocrinology, 2018.
2. Vasanthan, K., Vengadakrishnan, K., & Surendran, P. (2018). Clinical Profile of Diabetic Foot Infections. INTERNATION- AL JOURNAL OF SCIENTIFIC STUDY, 6(1), 99-103.
3. Casqueiro, J., Casqueiro, J., & Alves, C. (2012). Infections in patients with diabetes mellitus: A review of pathogenesis. Indian journal of endocrinology and metabolism, 16(Suppl1), S27.
4. Rosyid, F. N. (2017). Etiology, pathophysiology, diagnosis and management of diabetics’ foot ulcer. International Journal of Research in Medical Sciences, 5(10), 4206-4213.
5. Malepati, S., Vakamudi, P., Kandati, J., & Satish, S. (2017). Bacteriological study of diabetic foot ulcer according to Wag- ner’s classification: a one-year study. International Surgery Journal, 5(1), 98-104.
6. Karmaker, M., Sanyal, S. K., Sultana, M., & Hossain, M. A. (2016). Association of bacteria in diabetic and non-diabetic foot infection–An investigation in patients from Bangladesh. Journal of Infection and Public Health, 9(3), 267-277.
7. Shodja, M. M., Knutsen, R., Cao, J., Oda, K., Beeson, L. E., Fraser, G. E., & Knutsen, S. (2017). Effects of glycosylated hemoglobin levels on neutrophilic phagocytic functions. Ja- cobs journal of diabetes and endocrinology, 8(2), 9.
8. Tille, P. M., & Bailey, S. (2014). Diagnostic microbiology. Misouri: Elsevier. p. 202-927.
9. Clinical and Laboratory Standards Institute (CLSI), (2019). Performance standards for antimicrobial susceptibility test- ing. CLSI supplement M100. Wayne, PA. CLSI.
10. Konar, J., & Das, S. (2013). Bacteriological profile of dia- betic foot ulcers, with a special reference to antibiogram in a tertiary care hospital in eastern India. Journal of Evolution of Medical and Dental Sciences, 2(48), 9323-9329.
11. Sugandhi, P., & Prasanth, D. A. (2014). Bacteriological profile of diabetic foot infections. Int J Innov Res Sci Eng Technol, 3, 14688-14692.
12. Madmoli, M., Karami, M., & Madmoli, Y. (2019). Some ef- fective risk factors on diabetic foot ulcer: study on 2643 cases of patients with diabetes. Journal of Research in Medical and Dental Science, 7(3), 139-143.
13. Edo A, Edo G, & Ezeani I. (2013). Risk factors, ulcer grade and management outcome of diabetic foot ulcers in a Tropical Tertiary Care Hospital. Niger Med. J, 54(1), 59-63.
14. Pal B, & Gupta S. (2016). Study on the relation of the severity of diabetic foot ulcers with the type of bacterial flora isolated from the wounds. Int. Surg. J, 3(1), 189-194.
15. ÃÂ?°çer M, & Durgun H. (2017). Factors Affecting Amputations in Patients with Diabetic Foot Ulcer Referring to the Emer- gency Units. Dicle TÃÂ?±p Dergisi/Dicle Med. J, 44(1), 91–97.
16. Yates, C., May, K., Hale, T., Allard, B., Rowlings, N., Free- man, A., ... & Wraight, P. (2009). Wound chronicity, inpatient care, and chronic kidney disease predispose to MRSA infec- tion in diabetic foot ulcers. Diabetes Care, 32(10), 1907-1909.
17. Bello, O. O., Oyekanmi, E. O., Kelly, B. A., Mebude, O. O., & Bello, T. K. (2018). Antibiotic susceptibility profiles of bacte- ria from diabetic foot infections in selected Teaching Hospi- tals in Southwestern Nigeria.
18. Ahmad, W., Khan, I. A., Ghaffar, S., Al-Swailmi, F. K., & Khan, I. (2013). Risk factors for diabetic foot ulcer. Journal of Ayub Medical College Abbottabad, 25(1-2), 16-18.
19. Wu, M., Pan, H., Leng, W., Lei, X., Chen, L., & Liang, Z. (2018). Distribution of microbes and drug susceptibility in pa- tients with diabetic foot infections in Southwest China. Jour- nal of Diabetes Research, 2018.
20. Saltoglu, N., Ergonul, O., Tulek, N., Yemisen, M., Kadanali, A., Karagoz, G., ... & SargÃÂ?±n, F. (2018). Influence of multidrug resistant organisms on the outcome of diabetic foot infection. International Journal of Infectious Diseases, 70, 10-14.
21. Jain, S. K., & Barman, R. (2017). Bacteriological profile of diabetic foot ulcer with special reference to drug-resistant strains in a tertiary care center in North-East India. Indian journal of endocrinology and metabolism, 21(5), 688.
22. Abd Al Hamead, H., Al Metwally, R., Khaled, M., Mahmoud, M., Ayman, F., Ramah, E., ... & SatarLotfi, A. (2013). Bacteri- ological study of diabetic foot infection in Egypt.
23. Al Benwan, K., Al Mulla, A., & Rotimi, V. O. (2012). A study of the microbiology of diabetic foot infections in a teaching hospital in Kuwait. Journal of infection and public health, 5(1), 1-8.
24. Dezfulian, A., Salehian, M. T., Amini, V., Dabiri, H., Rad, M.A.,Aslani, M. M., ... & Zali, M. R. (2011). Bacteriological study of , Aslani, M. M., ... & Zali, M. R. (2011). Bacteriological study of diabetic foot infections in an Iranian hospital. Iranian Red Crescent Medical Journal, 13(8), 590.
25. Perim, M. C., Borges, J. D. C., Celeste, S. R. C., Orsolin, E.D. F., Mendes, R. R., Mendes, G. O., ... & Pranchevicius, M.C. D. S. (2015). Aerobic bacterial profile and antibiotic re- sistance in patients with diabetic foot infections. Revista da Sociedade Brasileira de Medicina Tropical, 48, 546-554.
26. Anand, A., Biswal, I., Soni, R. K., Sinha, A., Rynga, D., & Deb, M. (2016). A clinico-microbiological study of diabetic foot ulcer patients to identify risk factors and their correlation with prognosis in tertiary care hospital in India. International Surgery Journal, 3(2), 669-673.
27. Priyadarshini Shanmugam, J. M. (2013). The bacteriology of diabetic foot ulcers, with a special reference to multidrug resistant strains. Journal of clinical and diagnostic research: JCDR, 7(3), 441.
28. Anyim, O., Okafor, C., Young, E., Obumneme-Anyim, I., & Nwatu, C. (2019). Pattern and microbiological characteristics of diabetic foot ulcers in a Nigerian tertiary hospital. African Health Sciences, 19(1), 1617-1627.
29. Misic, A. M., Gardner, S. E., & Grice, E. A. (2014). The wound microbiome: modern approaches to examining the role of microorganisms in impaired chronic wound healing. Advances in wound care, 3(7), 502-510.
30. Aamir, A. H., Nasir, A., Jadoon, M. Z., Mehmood, K., & Ali,S. S. (2011). Diabetic foot infections and their management in a tertiary care hospital. Journal of Ayub Medical College Abbottabad, 23(1), 58-62.
31. Nageen, A. (2016). The most prevalent organism in diabetic foot ulcers and its drug sensitivity and resistance to different standard antibiotics. J Coll Physicians Surg Pak, 26(4), 293-6.
32. Pappu, A. K., Sinha, A., & Johnson, A. (2011). Microbiolog- ical profile of diabetic foot ulcer. Calicut Med Journal, 9(3), 1-4.
33. Gillespie, S., & Hawkey, P. M. (Eds.). (2006). Principles andpractice of clinical bacteriology. John Wiley & Sons.
34. Mendoza, R. A., Hsieh, J., & Galiano, R. D. (2019). The im- pact of biofilm formation on wound healing. Wound heal- ing-current perspectives, 10.
35. Yun, E. H., Kang, Y. H., Lim, M. K., Oh, J. K., & Son, J.M. (2010). The role of social support and social networks in smoking behavior among middle and older aged people in ru- ral areas of South Korea: a cross-sectional study. BMC Public Health, 10(1), 1-8.
36. Samant, S., Victor, S., & Rai, S. (2018). Aerobic bacterial Pro- file of Diabetic Foot Ulcers and their Antibiotic Sensitivity Pattern. Int J Curr Microbiol App Sci, 7(1), 1412-1418.
37. Ahmed M, Said M, Naira E, Fatania A, Emad E, & Soma A.(2013). Infection in diabetic foot. Menoufia Med. J, 26, 49–53.
38. AW, N. H. H., Intan, N. S., Syafinaz, A. N., Zalinah, A., Ak- mar, L., & Devnani, A. S. (2015). Clinical presentation and microorganisms sensitivity profile for diabetic foot ulcers: a pilot study. The Medical Journal of Malaysia, 70(3), 182-187.
39. Shashanka, R., & Rajanna, B. (2016). Bacteriological Profi le of Diabetic Foot Ulcers: A Clinical Study. IJSS Journal of Surgery, 2(3), 38-41.
40. Yang, Y., Zhang, H., Zuo, J., Gong, X., Yi, F., Zhu, W., & Li, L. (2019). Advances in research on the active constituents and physiological effects of Ganoderma lucidum. Biomedical Dermatology, 3(1), 1-17.
41. Manisha, J., Mitesh, P., Nidhi, S., Dhara, M., & Vegad, M. (2012). Spectrum of microbial flora in diabetic foot ulcer and its antibiotic sensitivity pattern in tertiary care hospital in Ahmedabad, Gujarat. Nat J Med Res, 2(3), 354-357.
42. Divya G, Pavithra B, Sathish B, Naveena R, Ashwini V, & Sri Kiruthika K. (2017). Clinical and Microbiological Char- acteristics of Diabetic Foot Infections in Teaching Hospital at Kancheepuram District. Int. J. Phar. Tech, 9(1), 28488-28502.
43. Bengalorkar, G. M., & Kumar, T. N. (2011). Culture and sen- sitivity pattern of micro-organism isolated from diabetic foot infections in a tertiary care hospital. Int J Cur Biomed Phar Res., 1(2), 34-40.
44. Mathangi, T., Prabhakaran, P., Nadu, T., & Nadu, T. (2013). Prevalence of bacteria isolated from type 2 diabetic foot ul- cers and the antibiotic susceptibility pattern. International Journal of Current Microbiology and Applied Sciences, 2(10), 329-337.
45. Zubair, M., Malik, A., & Ahmad, J. (2010). Clinico-bacte- riology and risk factors for the diabetic foot infection with multidrug resistant microorganisms in north India. Biol Med, 2(4), 22-34.
46. Otta, S., Debata, N. K., & Swain, B. (2019). Bacteriological profile of diabetic foot ulcers. CHRISMED Journal of Health and Research, 6(1), 7.
47. Singh, S., Banerjee, G., Agarwal, J., Kumar, V., & Usman, K. (2018). Spectrum of microbiota in diabetic foot infections in a teaching hospital of Uttar Pradesh. International Journal of Medical Science and Public Health, 7(9), 741-748.
48. Amaefule, K. E., Dahiru, I. L., Okpe, I. O., Aliyu, S., & Aru- na, A. A. (2019). Clinico-microbial profile of diabetic foot in- fections in Zaria, North-West Nigeria. Sahel Medical Journal, 22(1), 28.
49. Kumar, A., Agrawal, A. K., Kumar, M., Sharma, A. K., & Ku- mari, P. (2017). Aerobic Bacterial Profile of Diabetic foot and its Antibiogram in RIMS, Ranchi-a Tertiary Care Hospital. Proteus, 2, 1-5.
50. Mehta, V. J., Kikani, K. M., & Mehta, S. J. (2014). Microbio- logical profile of diabetic foot ulcers and its antibiotic suscep- tibility pattern in a teaching hospital, Gujarat.
51. Maryam, U. (2015). Isolation of Pathogens Causing Sepsis, Pus and Infected Wounds from Critical Care Unit: A Retro- spective Study. Annals of clinical and laboratory research, 3(4), 50.
52. Xie, X., Bao, Y., Ni, L., Liu, D., Niu, S., Lin, H., ... & Luo, Z.(2017). Bacterial profile and antibiotic resistance in patients with diabetic foot ulcer in Guangzhou, Southern China: focus on the differences among different Wagner’s grades, IDSA/IWGDF grades, and ulcer types. International journal of en-docrinology, 2017.
53. Shnawa, I. M. (2011). The mucosal and systemic immune sta- tus for diabetic and non-diabetic Dentialvcolar infected pa- tients. Baghdad Sci. J, 8(1), 489-493.
54. A Al-Shahery, M., & G AL-Allaff, R. (2012). Estimation of phagocytic activity in diabetic patients (type 1, 2) in Mosul city. Rafidain Journal of Science, 23(8), 31-40.
55. Huang, J., Xiao, Y., Xu, A., & Zhou, Z. (2016). Neutrophils in type 1 diabetes. Journal of diabetes investigation, 7(5), 652-663.
56. Ena, J., Casa, R., Carratalá, M. J., & Leutscher, E. (2012). Ef- fect of a program to control perioperative blood glucose on the incidence of nosocomial infections in patients with diabetes: A pilot study.
57. Umadevi, S., Kumar, S., Joseph, N. M., Easow, J. M., Kand- hakumari, G., Srirangaraj, S., ... & Stephen, S. (2011). Micro- biological study of diabetic foot infections. Indian journal of medical specialities, 2(1), 12-17.