Assessment role of phagocytic neutrophil cells among different Wagner’s grades of diabetic foot ulcers infections

Background: Foot ulcers complications in diabetes mellitus (DM) patients are one of the significant medical problems and an economic burden. The aim of this study is to assessment role of phagocytic neutrophil cells and its relationship with the incidence of diabetic foot ulcers (DFU) infection of diabetic patients. A total of 60 venipuncture blood samples collected from diabetes mellitus, diabetic foot ulcer patients and healthy persons as control group. 20 swabs from the DFU patients were collected and processed for culture and susceptibility test after the ulcers classified according to Wagner’s grades system. Phagocytic cells activity test was performed to determine the efficiency of phagocytic neutrophil cells in diabetic patients. Results: Gram positive bacteria were the most prevalent in the DFU patients 57.1% with statistical significant relationship between the type of bacteria and grades of Wagner’s classification followed by Gram negative bacteria in high grades of ulcers. Wagner’s ulcers grade 1 and 2 were the most prevalence in DFU patients 30%. There was a weak negative correlation between the efficiency of phagocytic neutrophil cells activity and grade ulcers classified (r = -0.323). Amikacin and ciprofloxacin were the most effective antibiotics ulcers with high resistance of antibiotics. In contrast, the grade of ulcer increased, the efficiency of phagocytic neutrophil cells decreased.


Bacterial species of DFU associated with Wagner's grades system
As shown in table (2), the superficial ulcer grades infections were caused by Gram positive bacteria, while Gram-negative bacteria showed predominant in deep ulcer grades infections with statistically significant association (P-value 0.057). The distribution of different bacterial species isolated from DFU cases according to Wagner's grades classification given in figure (1).

Antibiotics resistance patterns of bacterial isolates
In this study, Staphylococci species were resistant for ampicillin 100% and high sensitive to ciprofloxacin, amikacin, trimethoprim and cefaclor 100%. MRSA showed high resistant to cefixime, ampicillin, cefadroxil and trimethoprim antibiotics 100% and high sensitive for vancomycin, amikacin, ciprofloxacin, amoxyclav and cefepime 100%. Streptococci species were resistant for cefepime, cefixime, ampicillin, cefadroxil and cefaclor 100%, and sensitive for ciprofloxacin and amikacin 100%.

Antibiotics resistance patterns associated with Wagner's ulcers grades
There was an increased resistance of bacterial isolates with increased grades of Wagner's classification as shown in figure (2). According to Pearson correlation test there was statistically significant correlation (P-value = 0.05 ; r = 0.369).

Phagocytic neutrophil cells activity test
According to phagocytic cells activity test, DM and DFU patients had no high efficient phagocytic neutrophil activity, 1 and 0 respectively as given in table (4). The mean of phagocytosis was completely different between the three groups studied (control, DM and DFU patients) using LSD with high significant difference statistics as shown in table (5). 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 as given in table (6)

Discussion
Identifying the risk factors of DFU might help to develop better prevention strategies in diabetic patients. In this study we attempted to find if there is a relationship between the phagocytic neutrophil cells activity and the Wagner's grades of the diabetic ulcers. Wagner's grade 1 and 2 ulcers classification were the most common prevalence 30% in ulcers of diabetic patients, and these results were compatible with other studies conducted in different countries [13][14][15], but disagree with a previous study reported that Wagner's grade 2 and 3 ulcers were the most common prevalence [16]. Grade 4 was the lowest prevalence in this study, and this contrast with other studies revealed that the grade 4 was the most common prevalence in DFU [17,18]. Other studies showed the grade 4 was commonly infected with mixed variety of bacteria including extended spectrum beta-lactamase (ESBL) Klebsiella species and MRSA [17], also, highly multiple bacterial infection were more incidence in DFU grade 4 [19]. Our study results found that multiple bacterial infections was associated with grade 2 and 4 of Wagner's ulcers classification, while other study revealed that non-significant difference of the mean number of isolates and diabetic deep tissue infection of different grades [20].
Additionally, significant delay in wound healing cause of P. aeruginosa biofilms inhibit neutrophil movement [41]. 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 [42].
The prevalence of Klebsiella species and Esch. coli was 4.8% in this study, this agreed with other study results 4% [14], and disagreed with other study recorded a high rates of Klebsiella species 12.6% and Esch. coli 17.9% isolated from DFU [36]. Also, C. freundii and Serratia species isolates accounted 4.8% in this study, and is comparable with other results 5% [22], 3.85% [5], 4.3% [43] for C. freundii and 2.3% for Serratia species [44]. Different geographical area could have contributed to different types of bacterial isolated [35].
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 [32].
In our study, the diabetic patients have superficial wounds of Wagner's grades classification I and II were mostly infected with Gram positive bacteria. Staphylococci and Streptococci species were the predominant pathogens, whereas Gram negative bacteria were more frequently isolated from Wagner's wounds classification grades III and V. These findings are in agreement with various studies concluded that an increasing of Wagner's grades, the proportion of bacterial infections were increased [2,24,35,44,45]. However, the percentage of S. aureus decreased with increased Wagner's grades similar findings reported in other study [24]. 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 stay, increased cost and mortality [17,46]. In this study, MRSA were associated with Wagner's grade 3 ulcer classification, and other study showed that Wagner's ulcers grade 3 were commonly infected with MRSA [17].
This study showed high level resistance of bacterial isolates to ampicillin 95%, this ratio showed in other studies reported 92.4% [44], 94.9% [47,48], and 50% [35]. This increased of resistance may be attributed to the fact that ampicillin has been widely abused and frequently implicated in selfmedication [47]. In contrast, other studies showed high resistance amoxyclav 79% [44], 81.8% [54]. Cefexime showed low sensitivity 9.5% for the isolated bacteria in our study. Similar low rate reported in other study 3.2% [59]. While the sensitivity for Gram negative bacteria to cefexime was 70.7% [5], and for Gram positive bacteria was 77.3% [33]. The sensitivity of cefaclor was 47.6% of bacterial isolates in this study. This was compatible with the results of other study showed the second generation of cephalosporin was 50% [44]. Other studies showed that cefaclor was active 58.4% against Gram positive bacteria [54], and 92.8% against Gram negative bacteria [35].
Our study revealed that a low significant positive correlation between the antibiotics resistant and increasing of Wagner's grades classification. Such relation have previously reported that an increasing of Wagner's grades, the resistance rates to some antibiotics increased [60].
This study indicated a significant reduction in phagocytic activity in DM and DFU patients compared with control group. In addition, our baseline results confirm and extend previous reports concerning the impairment of phagocytosis that occurs in neutrophil cells from diabetic patients than non-diabetic patients [61]. Other studies showed a significant reduction in phagocytic cells activity in Type-1 DM and Type-2 DM [62,63]. This difference may be due to that hyperglycemia impairs granulocyte functions including adherence, chemotaxis, phagocytosis and bactericidal activity [64]. So, hyperglycemia reduced response of neutrophil function and disorders of humoral immunity as one of the long-term effects of elevated mean blood glucose (MBG) or HbA1c, also the impaired micro-vascular circulation in patients with diabetic foot limits the access of phagocytes favouring development of infection [3,26,65].
One of the strengths of our study results find the relationship between the manner of immune response involving phagocytic cells activity and the degree of wound. 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). However, in our knowledge, there have been no reports in the literature regarding the correlation between phagocytic cells activity and the degree of wound in diabetic foot patients to support our result or to know the reasons of this relation we need further deep studies to be clarified.

Conclusion
When the grade of ulcer increased, the bacterial 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. Further immunological and bacteriological investigations needed to control the increasing of antibiotics resistance in diabetic foot ulcers patients. We recommended when the grade of ulcer classified is high, a bacterial culture and sensitivity testing should be done to avoid complications of the increasing bacterial resistance to antibiotics. Then the swabs kept at room temperature and 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 [7][8][9][10] with minor modifications. In briefly, normal saline suspension of pathogenic strain Staphylococcus aureus (S. aureus) colonies was prepared and the visible turbidity was adjusted to 0.5 McFarland turbidity standard yielding an approximately 1.5×10 8 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 water 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 phagocytic cells activity.

Isolation and identification of bacterial pathogens
All swab samples of ulcers were inoculated on the blood agar and MacConkey agar media (Oxoid/England). The culture plates were incubated under aerobic conditions at 37°C for 24 hours.
After incubation, the growing of bacterial isolates were recognizable by colonies features.
Identification of bacterial species was made based on reaction of Gram stain, morphology and biochemical characteristics using different available tests, catalase; coagulase; DNase; mannitol fermentation; urea; citrate; Kligler iron agar, sulphide indole motility and oxidase [11].

Antibiotic susceptibility testing
Antibiotic susceptibility testing was done using Kirby-Bauer disc diffusion method on Mueller Hinton agar (Oxoid/England) according to the standard guidelines of the Clinical Laboratory Standards Institute (CLSI) for testing ampicillin, cefepime, cefadroxil, ciprofloxacin, cefixime, cefaclor, trimethoprim, amoxyclav, methicillin, amikacin and vancomycin [12].

Statistical analysis
Data analyzed using the software of Statistical Package for Social Sciences (SPSS) version 25. The graphs presented using the software program (Excel for Windows Microsoft) version 10.
Descriptive statistics (frequencies and percentages) for study variables were obtained and compared using least significant difference test (LSD) and one away ANOVA test. The association between different groups of the explanatory variables was measured and compared using Pearson Chi-square (χ2) test. The relationship between the variables examined by the Pearson correlation (r) test. The level of significance was set at P-value less than 0.05. Great thanks expressed to department of biology, faculty of science, Hadhramout university/Yemen for their efforts in developing scientific research.

Authors' contributions
All authors conceived, designed the experiments and wrote the manuscript. Maryam Hamed Baras analyzed the experimental results, performed the statistical analysis and interpreted all of the data.
Eidha Ali Bin-Hameed reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding
Non funding

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Ethics approval and consent to participate
Ethical approval for this study was obtained from Hadhramout University before commencing the study. The information were taken from the participants after they agreed to it verbally according to the informed consent with confidentiality of each participant as well as the results.

Consent for publication
Not applicable.