Bacteriological Profile and Antibiotic Resistance Patterns of Pus/Wound Samples in Humans with Infected Wounds in North Central Algeria

The present study was carried out between 2016 and 2020 in


INTRODUCTION
Antibiotic resistance is a critical cosmopolitan public health issue causing increasing serious public concerns.2][3] This bacterial resistance is the main cause of a multitude of difficult-to-treat wound infections. 4Several epidemiological studies showed important fluctuations in antibiotic resistance according to the nature of wounds, surgical procedures, and regions. 5Different patterns of bacteria such as staphylococci, Enterobacteriaceae, and Pseudomonas, cause of pus infections.
Depending on the causative bacteria, active infections are either purulent or serosanguinous. 6These pyogenic infections interfere with the wound healing process, leading to a dramatic increase of treatment costs, morbidity, and mortality. 7,8Bacteria identification, to the level of species and strain, and monitoring of the organisms' response to the therapeutic protocol require microbiological research implementation in both surgery and pharmacology 6 .Knowing the local epidemiology and the antibiotic resistance pattern will therefore help decreasing treatment failure risks. 7The intensive uncontrolled use of antibiotics increases microbial resistance rate and speed.This resistance induces the development of chronic wounds and in extreme cases stops the healing. 9n the world, several research were conducted to estimate the frequency of pyogenic infections, causative bacteria, and their antibiotic susceptibility.Different wound infection rates were reported in several countries.It was reported to be low in France (2.2%) 10 but very high in developing countries such as Sudan (25.23%). 11n Algeria, the incidence of methicillinresistant S. aureus (MRSA) isolated from surgical site infections between 2008 and 2010 was estimated to 4.2%. 12In the north west of Algeria, Bouharkat et al reported that 18.3% of the 60 S. aureus isolates were MRSA. 13However, there is lack of information regarding bacterial resistance evolution in Algeria.
The present study aimed to investigate antimicrobial resistance patterns, including multidrug resistance, among bacteria isolated from various types of pyogenic infected wounds in humans in Makour Hamou Hospital in Ain Defla, Algeria.

Samples' collection
The present study was a prospective investigation.It was carried out during five years, from the 1st of January 2016 to the 31st of December 2020 in Makour Hamou public hospital, Ain Defla district, North Centre of Algeria.A total of 620 patients consisting of 262 males and 358 females (sex ratio M:F = 0.73) presenting wound purulent infections and at least one of the wound infection symptoms were including in the sampling. 14The patients' ages ranged from one month to 90 years (mean age: 37.7±20.69years).
Pus/wound samples were collected aseptically using disposable sterile swabs or syringes.The samples were identified and immediately transported to the laboratory of medical analyses.To prevent duplicate samples, each patient was included in the study only once during their entire hospitalization period.

Bacterial isolation and identification
The samples were primarily cultivated using standard media: blood agar, Mannitol salt agar, and Hekteon agar (Tmmedia Labs, Rajasthan, India) and incubated at 37°C for 24-48 h.Blood agar plates were incubated in an atmosphere rich in CO 2 .In the case of a polymicrobial culture, distinct colonies were randomly selected and streaked into new plates until pure cultures were obtained.The identification was made according to standard bacteriological diagnostic procedures: macroscopic examination of colonies, unstained wet-mount test, Gram staining, and biochemical tests.Control strains as E. coli (ATCC 25922), S. aureus (ATCC 25923), and P. aeruginosa (ATCC 27853), were included in the study.

Antimicrobial susceptibility testing
Antibiotic susceptibility test was performed using Muller-Hinton agar (Tmmedia Labs, Rajasthan, India) plates by disk diffusion method according to Guidelines of Clinical and Laboratory Standards Institute. 15A total number of 26 antibiotic discs (Cypress Diagnostic, Langdrop, Belgium) were used to test antibio-susceptibility of the strains (Table 1).
Methicillin-resistant S. aureus was detected by disc diffusion test using cefoxitin (30 µg) on Muller-Hinton Agar.Bacteria producing Extended-Spectrum b-lactamases (ESBLs) were screened using disc diffusion on Muller-Hinton Agar and confirmed by double disc method (Amoxicillin-Clavulanate (20/10 µg)/Cefotaxime (30 µg)). 15 recommended by the international expert for standard, multidrug resistance (MDR) was defined as acquired non-susceptibility to at least one antibiotic from at least three antimicrobial groups. 16

Data analysis
All data were analysed in a Microsoft Excel® 2019 spreadsheet.Percentages were compared using chi-square test at 5% threshold with Epi info 2000 software. 17
Penicillins antibiotics had the highest rate of resistance followed by cephalosporins among Gram-negative bacteria, as indicated by dark squares (Figure 3).Moreover, the lowest antibiotic resistance was due to aminoglycoside antibiotics.

DISCUSSION
The antibiotic resistance limits the choice of antibiotics to treat wound infections.The frequency of these infections varied according to time and region.
The present survey was performed in an Algerian hospital located in Ain Defla district; it allowed the collection of 620 pus samples from infected wounds during five years.9][20] In the United States, there were 26.5% more infection cases in August than in January 20.This discrepancy could be related to the scheduling of surgeries outside the summer season in Algeria where the temperature exceeds 45°C during the summer season.Vickers et al. did not found any statistically significant relation between the climate and frequency of infections in two regions (tropical and subtropical Australian hospitals). 21he bacteriological profile of pus/wound swabs revealed that more than half of pus samples showed positive cultures (407/620; 65.65%).The rates obtained here are similar to those reported by Rai et al. 22 who worked on children in Nepal (265/450; 59%) and lower than those reported by Tchakal-Mesbahi et al. 23 in Algeria (79.12%; 1585 samples), Shimekaw et al. 24 in Ethiopia (72.6%; 201 samples), and Kassam et al. 25 in Tanzania (91.4%; 93 samples) using standard bacteriological diagnostic procedures.Despite the presence of pus and signs of wound infections, the rate of negative cultures was 34.35% (213/620).This could be due to presurgical antibio-prevention or early antibiotic prescription during the initial stages of infection.In addition -in these cases-bacteriological identification and antibiotic susceptibility testing are performed after therapeutic failure.
The frequency of Gram-positive (66.12%) was higher compared to Gram-negative bacteria (33.38%).This is consistent with the study of Duwadi et al. in Nepalese Tertiary Care Cancer Patients, in which the rate of Gram-positive isolates (63.9%) outnumbered Gram-negative isolates (36.1%). 26Similar results were reported also in Nepal by Upreti et al. in 182 samples from patients with clinical features of wound infection. 27 study performed in Algeria on patients with burn wound infection revealed that 68.95% of swabs contained Gram-negative bacteria (68.95%).23 The lower isolation frequency of Gram-negative bacteria can be attributed to their limited ability to survive in the dry conditions of normal skin, allowing the domination of Gram-positive bacteria.28 Escherichia coli (30.34%),K. pneumoniae (25.52%), and P. aeruginosa (23.45%) were the major bacteria among Gram-negative isolates.The same trend was reported in several regions in the world.In India, Rai et al. found that P. aeruginosa was the most common isolate (45/102; 44%) followed by K. pneumoniae (28/102; 27%) and E. coli (13/102; 13%) in wound infections among child. 22n a study performed in Tanzania by Manyahi et al., P. aeruginosa was the most frequent Gram-negative bacteria (24/147; 16.3%).29 Causative bacterial species varies according to geographic area and the wounds' nature.In Algeria, Tchakal-Mesbahi et al. reported that P. aeruginosa was the most frequent species (33.91%) followed by K. pneumoniae (25.14%) and Acinetobacter baumanii (16.37%).23 S. aureus represented the second most frequent (27.21%)Gram-positive bacteria after CoNS. Thsame trend was reported in 8,569 French patients (35% and 23% for CoNS and S. aureus, respectively).10 Staphylococci are the most common colonizers bacteria of the skin and the primary causes of nosocomial infections and skin infections in the community.30 Among S. aureus isolates, 31.17%(24/77) were MRSA.Zerouki et al. found that S. aureus was isolated in 39.5% (30/76) of the Department of Orthopaedic Surgery and Traumatology, University Military Hospital of Constantine, Algeria, among them, 63.3% was MRSA.12 Also, in Algeria, Bouharkat et al. revealed that S. aureus is the most isolated species (25.97%) in diabetic patients suffering of foot infections where MRSA represented 18.3%.13 In Morocco, S. aureus rate in burn wound infections was 33.85% (44/86), MRSA represented 86.36% of them.31 These discrepancy between studies can be explained mainly by the microbial ecology of the hospitals.12 The high prevalence of S. aureus is expected since this species belongs to the normal flora of healthy persons' skins.32 In the present study, the rate of penicillin resistance was 70.13% for S. aureus.In Morocco, the highest rate was noted for S. aureus (86.36%).31 According to Que et al., penicillinase production is the most common mechanism liable for the emergence of penicillin resistance where the prevalence is close to 80% between hospitals and the community.33 A high level of amoxicillin resistance was found in E. coli (86.36%),K. pneumoniae (97.30%), and other Enterobacteriaceae species.In addition, the combination amoxicillin-clavulanate has very important resistance (68.18 and 83.78% for E. coli and K. pneumoniae, respectively).Furthermore, El Hamzaoui et al. found that more than 80% of the different strains were resistant to this antibiotics association. 31 The most common reason for resistance to b-lactam antibiotics among Enterobacteriaceae is the expression of b-lactamase enzymes that hydrolyse the b-lactam ring, resulting in antibiotic inactivation.34,35 In the present study, we get favourable clinical responses to treatments with vancomycin against Gram-positive infection.In Ethiopia, Mama et al. reported that all the 145 isolates were sensitive to vancomycin.36 This is a glycopeptide antibiotic used as last-resort treatment against resistant infections.Several generations of vancomycin were developed to prevent conceivable resistance, 37 they are only used in hospitals and is administered intravenously.38 Amikacin and imipenem were the effective against Gram-negative bacteria.Both of them are expensive, they have only clinical use.Amikacin is characterized by a high level of resistance to bacterial enzyme modifications; as a result, several Gram-negative bacteria are susceptible to this antibiotic. 39The high rate of antibiotic resistance to penicillins is due to the misuse of these antibiotics.Also, the important level of resistance to the cephalosporins antibiotic can be related to their frequent used in surgery.40 To limit the emergence and spread of resistance to aminoglycosides, it is imperative to minimize selective pressures that arise within hospital settings and areas with unregulated antibiotic usage.Continuous monitoring of resistance genes in humans, animals, and food sources is vital to delay the dissemination of aminoglycoside resistance.41 In the present study, 31.54% of isolated strains showed a MDR.This could delay the wound healing process and even alter the patient's general condition causing for example septicaemia.Furthermore, this resistance reduces the therapeutic choice and increases duration of hospitalizations.42 In the present study, the MDR in Gram-negative isolates represented 42.76% while 25.79% of Gram-positive bacteria were MDR.These resistance levels were lower than the findings of Duwadi et al. (60 and 40% for Gram-positive and Gram-negative bacteria, respectively).26 In addition, the MDR varied significantly with bacterial species.23,42 There was a significant correlation between age and MDR rate.This could be explained by the build-up with age, the irrational use of antibiotics, especially self-medication.

CONCLUSION
In this study, CoNS and S. aureus are the predominant bacteria isolated from pus/ wound infections from patients at Ain Defla district, North Centre Algeria.Very high resistance rate has been recorded to commonly used antibiotics like amoxicillin (more than 80% for the different bacterial species).Amikacin and vancomycin were the most effective against Gram-negative and positive bacteria, respectively.Almost one-third of the isolates were multidrug resistant.This resistance was higher among Gram-positive bacteria.To slowdown the bacterial resistance emerging, an antibiotic susceptibility test before any antibiotic therapy is mandatory.Also, the respect of hygienic practices is necessary to prevent infections, particularly nosocomial infections.Furthermore, increasing public awareness of the risks associated with self-medications.Additional epidemiological studies are needed to perform risk analysis of antibiotic resistance in Algerian bacterial strains.Similarly, genetic background investigation is needed to understand the mechanisms of these antibiotic resistance.

Figure 2 .Figure 3 .
Figure 2. Frequency of bacteria isolated in the present study from pus/wound samples