Amoxyclav Resistance Pattern and Aerobic Bacterial Profile in Diabetic Foot Infection Patients in Bangladesh

Introduction: The goal of this study was to determine the frequency of bacterial isolates cultured from diabetic foot infections and to assess their amoxyclav resistance and susceptibility. Methods: A total of 378 diabetic foot lesions were included in this prospective analysis. The antibiotic susceptibility pattern of bacteria isolated from foot lesions was assessed using the Kirby-Bauer disk diffusion method. Results: The most commonly isolated Gram-positive bacteria were Staphylococcus aureus , followed by Enterococcus spp. and CoNS. The most commonly isolated Gram-negative bacteria were Klebsiella spp., Pseudomonas spp., Proteus spp., Escherichia coli, Enterobacter spp., Citrobacter spp., Citrobacter freundii , and Proteus vulgaris . Amoxyclav was found to be 100.00% resistant against Pseudomonas aeruginosa and followed by Enterococcus spp. (89.50%), Proteus spp. (87.50%), Staphylococcus aureus (84.30%), Escherichia coli (81.50%), Klebsiella spp. (70.50%) and Enterobacter spp. (69.20%). Conclusion: The present study confirmed the prevalence of amoxyclav drug resistant pathogens (84.90%) in diabetic foot ulcers. The diverse bacteria infecting the wound must be evaluated, as well as the antibiotic susceptibility patterns of the isolates from the infected lesion. This information is critical for selecting the right medications, eliminating resistance trends, and lowering healthcare costs.

all contribute to this high prevalence [12].
Infection in a diabetic foot is a limb threatening condition because the consequences of deep infection in a diabetic foot are more disastrous than elsewhere mainly because of certain anatomical peculiarities. The foot has several compartments, which are inter-communicating and the infection can spread from one into another, and lack of pain allows the patient to continue ambulation further facilitating the spread. The foot also has soft tissues, which cannot resist infection, like plantar aponeurosis, tendons, muscle sheaths, and fascia. A combination of neuropathy, ischemia, and hyperglycemia worsens the situation by reducing the defense mechanism [6].
The correct antibiotic selection based on the antibiograms of isolates from diabetic foot infections is critical for the proper management of these infections. As a result, the current study sought to assess the bacteriology of diabetic foot ulcers at Hospital Geral de Palmas in Tocantins, Brazil, in order to determine the relative frequencies of bacterial isolates cultured from foot infections and to assess the isolated bacteria's in vitro antibiotic resistance and susceptibility to amoxyclav antibiotic.

Materials and Methods Materials Study Design
This cross-sectional study was designed to assess the bacterial profile cultured from the wounds of diabetic foot patients, and to assess the functional pattern of the amoxicillin antibiotic on the cultured microorganisms.

Data Collection
Laboratory data from May 2020 to May 2021 were routinely collected from the microbiology department from Bangladesh Institute of health and sciences hospital (BIHS) Dhaka, Bangladesh. The total sample volumes were 378.

Swab sampling
Wound beds were prepared before specimen collection, where the wound immediate surface exudates and contaminants were cleansed off with moistened sterile gauze and sterile normal saline solution. Dressed wounds were cleansed with non bacteriostatic sterile normal saline after removing the dressing. Aseptically the end of a sterile cotton-tipped applicator was rotated over 1 cm 2 area for 5 seconds with sufficient pressure to express fluid and bacteria to surface from within the wound tissue [13]. The specimens were placed into sterile transport containers and sent to the microbiology laboratory for aerobic culturing within 20 minutes. Anaerobic culturing was not performed in this study [14,15].

Bacterial Culture
Cultures were processed following the standard procedure [14,15]. Samples were inoculated on MacConkey agar (Oxoid), Chocolate agar (Oxoid) and Blood agar (Oxoid) media plate under class-II laminar airflow (NUVO SanajiMalzemelzeni, ImalatVcTicaret A.S, Turkey). The inoculum on the plate was streaked out for discrete colonies with a sterile wire loop sterilized by auto loop sterilizer (Germany) following standard procedures. The culture plates were incubated at 37°C by an incubator (Germany) for 48 hours and observed for the growth of bacteria through formation of colonies. All the bacteria were isolated and identified using morphological, microscopy (Japan) and biochemical tests like TSI (HiMedia), MIU (HiMedia) and Simmons Citrate (HiMedia) agar following standard procedures.

Antibiotic Susceptibility Assessment
The disc diffusion technique was used for antibacterial susceptibility testing of the isolates [16][17][18] using commercial antibiotics containing discs. We used the commercial antibiotic disc. Bacterial susceptibility was determined by the Kirby-Bauer disc diffusion method using antibiotic containing discs from Oxoid Ltd, UK. Amoxyclav antibiotic discs were used in this study. Interpretation of results was analysed using zone sizes. Zones of inhibition ≥21mm will be considered sensitive, 16-20mm intermediate and <15 mm resistant. Isolates were classified as either sensitive or resistant based on the definition of the Clinical and Laboratory Standard Institute [17].

Statistical Analysis
Data were assessed by using the free software GNU PSPP stable release 1.4.1/ September 5, 2020. It has a graphical user interface and conventional command-line interface. It is written in C and uses GNU Scientific Library for its mathematical routines. The name has "no official acronymic expansion". Statistical frequencies were analyzed here.

Results
A diabetic treatment center provided us with 378 participants for this research. They had all been diagnosed with diabetes and had diabetic feet. Males made up 247 (60.0%) of the subjects, with 165 females (40.0%) (Figure-1).

Figure-4: Sex distribution of gram positive (+ ve) and gram negative (-ve) microorganisms among diabetic
foot patients. Figure-4 described sex distribution regarding specific microorganism. i) In respect to Acinetobacter spp. pattern was 3.24% for male and 2.42% for female, ii) 0.01% male was found for Citrobacter feundii iii) 2.02% of males and 3.03% of females were found infected with Citrobacter spp. iv) 2.43% male was found for CoNS and female was 3.64%. v) 7.29% male was found for Enterobacter spp. and female was found 5.45%. vi) 6.07% of males were infected with Enterococcus spp. and 9.09% female. vii) Escherichia coli found in 7.29% male, female percentage was 7.27. viii) Male found 11.74% and 13.94% female in respect to Klebsiella spp. ix) Proteus spp. was found in 10.93% male and in 9.09% female. x) Proteus vulgaris was found in 0.81% of males and in 0.61% of females. xi) 1.62% of males were found with Pseudomonas aureginosa infection. 2.42% females were found with Pseudomonas aureginosa infection. xii. No female was found with Serratia marcescens and 100.00% were males. xiii. Staphylococcus aureus was found as a big name in our experiment. 34.41% of males were infected by this organism and the female percentage was 33.94.

Figure-5: Amoxyclav's functional profile.
As a whole, we got a scenario that amoxyclav was sensitive for 15.10% of diabetic foot patients and 84.90% of patients found resistant (Figure-5 Figure-2 described resistant and sensitivity pattern against amoxyclav. i) In respect to Acinetobacter spp. pattern was 0.00-100.00%, that means 0.00% was for sensitivity and 100.00% was for resistant ii) 10.00% sensitivity was found for Citrobacter feundii and 90.00% for resistant iii) 100.00% sensitive applied for Citrobacter spp. iv) 100.00% sensitivity was found for CoNS spp. v) 30.80% sensitivity and 69.20% were found for Enterobacter spp. vi) 10.50% resistant Enterococcus spp. was found rest 89.50% was sensitive. vii) Escherichia coli showed 18.50% resistance against amoxyclav, sensitivity percentage looked high (89.50%). viii) amoxyclav found 29.50% resistant and 70.50% sensitive against Klebsiella spp. ix) Proteus spp. was found 12.50% resistant and 87.50% sensitive. x) Proteus vulgaris infection, 33.30% diabetic foot infection with this organism showed resistance against amoxyclav and 66.70% were fine with amoxyclav. xi) 100.00% diabetic foot infection with this organism showed resistance against amoxyclav. xii) Serratia marcescens showed 100.00% resistant. xiii) Staphylococcus aureus was found as a big name in our experiment. 15.70% of this organism showed resistance against target antibiotic and 84.30% showed sensitive.

Discussion
Foot infections in patients with diabetes are a common, complex, and costly problem [19]. In the present study, we found that elderly patients (˃60 years of age) constituted the majority of patients with foot infections. This may be explained by the fact that foot lesions occur commonly among patients with diabetes, particularly the elderly and those with sensory neuropathy [20]. Previous studies have shown that the susceptibility to foot infections is greater in male patients than in female patients [1,5]. However, in our study, we did not find differences between male and female patients, which may be because of the limited number of patients.
Diabetic foot ulcers are colonized by pathogenic bacteria that may predispose a susceptible patient to a lower extremity infection, defined as the invasion and multiplication of microorganisms in body tissues associated with tissue destruction or host inflammatory responses [21]. In the present study, we found that the majority of lesions were located on the right toe and plantar region, and varied in duration from 1 day to more than 90 days. Additionally, recent lesions (1-30 days) were the most common. Our findings are in accordance with the results of Donoso et al. (2013) [22]. This study is limited by the fact that cultures for anaerobic bacteria could not be performed. In diabetic foot infections, the role of anaerobic bacteria is particularly unclear; some studies have reported that anaerobic bacteria play a minor role [23,24], while other studies found a high incidence of anaerobic bacteria [5,25].
The most common pathogens isolated were Gram-positive cocci, such as Staphylococcus aureus and Staphylococcus saprophyticus (CONS) and Gram-negative rods, such as Proteus spp. and Enterobacter spp. Although the findings of our study are consistent with the results of previous studies showing that Gram bacteria were predominant in diabetic foot infections [25], other studies have reported that Gram-negative bacteria were predominant in particular regions [26,27]. These results suggest, in part, differences in the type and severity of infections [5,28]. Aerobic Gram-negative bacteria (mainly Enterobacteriaceae and sometimes Pseudomonas aeruginosa or other Gram-negative species) are usually isolated in conjunction with Gram-positive cocci in patients with chronic or previously treated infections [26], which is consistent with our findings. Polymicrobial infections accounted for 70% of all infections. Although polymicrobial etiology has been implicated in diabetic foot infections [28], a previous study reported the predominance of monomicrobial infections [28]. These discrepancies suggest differences in diabetic foot infections, with severe infections usually having polymicrobial isolates and mild infections usually having monomicrobial isolates [28,29].

Conclusions
The present study report has some limitations because cultures for anaerobic bacteria could not be performed and sample size was small. However, it confirmed the high prevalence of multidrug-resistant pathogens in diabetic foot ulcers. Diabetic foot infections were predominantly due to Gram-positive bacteria, such as Staphylococcus aureus, or were polymicrobial infections. Many studies on the bacteriology of diabetic foot infections have reported results that vary and are often contradictory [25,26,28]. In such cases, application of molecular techniques may lead to more accurate microbial characterizations and targeted antibiotic therapy. Therefore, it is necessary to evaluate the different microorganisms infecting the wound on a routine basis and to know the antibiotic susceptibility patterns of the isolates from the infected wound in patients with diabetic foot lesions. This knowledge is crucial for planning the treatment of these patients with the appropriate antibiotics, reducing resistance patterns, and minimizing healthcare costs. We hope the data presented on this article can assist the clinicians in determining the multidrug-resistant pathogens in diabetic foot ulcers. In this study amoxyclav was found sensitive for 15.10% of diabetic foot patients and 84.90% of patients were found resistant.