Bacteriological profile of wound infections and antimicrobial resistance in selected gram-negative bacteria

Background Managing wound infections is a challenging task. Understanding their resistance pattern is an essential step at reducing its burden in hospital settings. Objective To determine the bacteriological diversity of wound infections and the antimicrobial resistance exhibited by a selected Gram-negative bacterium in the Aljouf region of Saudi Arabia. Methods The study retrospectively analysed the antibiograms of wound infections from hospitalized patients for the year 2019. The European Centre for Disease Control guidelines were adopted for the classification of resistant bacteria. Multidrug-, extensive drug-, and carbapenem-resistant isolates are presented as frequencies and percentages. Results A total of 295 non-duplicate wound swab antibiograms were retrieved, 64.4% (190) and 35.6% (105) isolates were Gram-negative and Gram-positive bacterial infections respectively. Predominant pathogens included Staphylococcus species 21.0% (62), E. coli 16.3% (48) and K. pneumoniae 13.5% (40). 148 (77.9%), 42 (22.1%) and 43 (22.6%) of the Gram-negative isolates were multidrug-, extensively drug- and carbapenem-resistant. The antibiotic resistance exhibited by gram-negative bacteria was 43.4% (234/539), 59.1% (224/379) and 53.7% (101/188) towards carbapenems, 3rd - and 4th – generation cephalosporins. Conclusions The majority of wound infections are caused by multidrug-, extensively drug- and carbapenem-resistant Gram-negative bacteria. Further studies should focus on the molecular basis of this resistance.


Introduction
Infectious diseases are a common cause of morbidity and mortality 1 . Impairment of the first line of defence especially damage to the skin and mucous membranes facilitate the entry of microorganisms into the human body resulting in infections 2 . Wound infections increase the chances of wound dehiscence and delay healing 3 . Traumatic injuries are the most common etiological factor in the genesis of wounds in hospitalized patients. Traumatic injuries are categorized according to the mode of occurrence into accidental and intentionally induced wounds. Hospital-acquired wounds such as surgical incisions or intravenous medical devices are categorized as intentionally induced wounds whereas non-intentionally induced wounds include wounds such as decubitus ulcers 4 . The major cause of acquired wound infections in the hospital is surgical interventions. Surgical site infections (SSI) can be allocated into three groups namely superficial incisional SSI, deep incisional SSI, and organ-specific SSI 5 . A high rate of postoperative wound infections has been observed in developing countries [6][7][8] . Post-operative wound infection is exerting huge stress on healthcare because of its morbid and financial implications and has become a major concern in the healthcare settings asking for cost management systems to be adopted 9 . There is an urgent need to formulate surveillance programs for detecting and diagnosing surgical site infections along with the antibiotic susceptibility pattern of infecting or-ganisms in order to reduce the associated complications and morbidity 10 . Wound infections in hospitalized patients are frequently caused by Staphylococcus aureus, Escherichia coli, coagulase-negative Staphylococcus (CoNS), Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter aerogenes and Klebsiella pneumonia 11 . S. aureus has been the most dominant bacterial isolate reported in most of the studies 4,[12][13][14] . With regards to antimicrobial resistance of the bacterial isolates to multiple antibiotics is concerned, the high prevalence was observed among the gram-negative bacteria 15 . Some of the studies from Saudi Arabia have documented the gram-negative bacteria to be the predominant isolates from wound infections. A study conducted in a teaching hospital in Riyadh have found E. coli as the predominant bacteria and observed the highest antibiotic resistance in Pseudomonas 16 . An extensive review of PubMed and Google scholar did not reveal any study on the bacteriological profile and antimicrobial resistance patterns of gram-negative bacteria causing wound infections from the Aljouf region of Saudi Arabia. The current study will help us in evaluating the gram-negative spectrum and their resistance patterns in wound infections that will guide infection control measures and anti-microbial stewardship programs.

Setting and design
The present study was conducted in a specialist hospital in Sakaka, the capital city of the Aljouf region the Kingdom of Saudi Arabia. There are two specialist hospitals in Sakaka city which serve as referral hospitals for the Aljouf region. Aljouf region comprises of three governorates of Sakaka, Qurayyat, and Dumat Al-Jandal with a total population of five million and twenty thousand.. In this cross-sectional study, all antibiograms from January 1, 2019, to December 31, 2019, of hospitalized patients were included. Culture and sensitivity reports of all non-duplicate wound swabs of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii, were specifically analysed for antimicrobial resistance.

Bacterial identification and antimicrobial resistance classification
For the purpose of bacterial identification and antimicrobial sensitivity testing, an automated BD Phoenix system (BD Biosciences, Franklin Lakes, NJ, USA) was used.
Clinical and Laboratory Standard Institute recommendations were employed for the antimicrobial susceptibility testing (AST) 17 . We classified resistant microorganisms based on the European Centre for Disease Control; guidelines into multidrug-resistant (MDR), extensive drug-resistant (XDR), and pan drug-resistant (PDR) 18 . Intermediate-resistant strains were merged with the resistant strains for simplification of the results. The phenotypic characterization of carbapenem, potential carbapenem, and ESBL producers as provided by the Phoenix system was also recorded. Carbapenem and potential carbapenem producers were categorized as carbapenem producers. All the details regarding the demographic data and hospitalization data were extracted from the hospital records. STROBE-AMS guidelines were adopted to report antimicrobial resistance.

Consent and research ethics
The research protocol got approved from the Local Committee of Bio-Ethics at Jouf University (vide no: 03/04/41 dated January 6, 2020). Informed consent was not required for this study; however, it should be noted that before a sample is taken, a verbal consent is ensured by the concerned medical personnel in the presence of the patient's relative as a standing operating procedure. Patient's guardian approval is taken and recorded in the medical files for patients admitted in the intensive care units.

Statistical analysis
The data were analysed using SPSS version 20.0 for Windows (SPSS, Inc., Chicago, IL, USA). MDR, XDR, PDR, two researchers checked the completeness of data at entry. ESBl-and carbapenem producers' frequencies were calculated. The results are presented as frequencies and percentages.

Results
Of the 295 non-duplicate wound swab antibiograms, 190 (64.4%) and 105 (35.6%) were of Gram-negative and Gram-positive bacterial infections. The majority of the samples (57.3%) and (54.6%) were received from male patients and male and female surgical wards. Isolated microorganisms include forty-eight (16.3%) E. coli, forty (13.5%) K. pneumoniae, twenty-six (8.9%) P. aeruginosa and twenty-four (8.1%) as A. baumannii. Among the gram-positive bacteria, seventy-six (25.8%) isolates of Staphylococcus species were the most frequent microorganism followed by thirteen (4.4%) isolates of Strepto-coccus species causing wound infections ( Table 1). The Gram-negative profile of wound infections is shown in figure 1. Analysis of the distribution of Gram-negative wound infections revealed that the majority occurred among men (56.3%) and during the first quarter (34.8%) of the year 2019. The male and female surgical, burn and orthopaedic wards contributed to 51.6% of these isolations followed by male and female intensive care units (42.7%).

Fig 1. Gram-negative bacterial profile of wound infections
Overall, the multi-drug (54.7%) and carbapenem-resistant (60.5%) strains were isolated from male patients and the majority of these strains infected patients aged 40years and above. The multidrug-and extensively drug-resistant strains were observed in male and female intensive care unit's unit with a frequency of 45.3% and 66.7% (Table  3).   The overall antibiotic resistance rate for Gram-negative bacteria was 43.4%, 59.1% and 53.7% towards carbapenems, 3rd -and 4th -generation cephalosporins. Among aminoglycosides, amikacin continues to remain effective against E. coli, P. aeruginosa and K. pneumoniae with a sensitivity rate of > 98%, > 92% and > 75%. All four organisms under study showed a resistance rate of > 75% for 1st -generation cephalosporins. E. coli, K. pneumoniae and A. baumannii show a resistance rate of > 45%, >55% and >98% resistance against the 3rd-generation cephalosporins. A. baumannii isolates were resistant (>95%) to carbapenems. Colistin was highly effective (>90%) against all the tested isolates of gram-negative bacteria under study. Few isolates of A. baumannii and P. aeruginosa that were tested for tigecycline presented 100% resistance (Table 5).

Discussion
The information regarding the bacteriological profile of wound infections will be of immense value in the institution of proper antimicrobial therapy and in guiding the infection control measures 19 16 . The high numbers of gram-negative isolates in our study may be attributed to the inclusion of hospitalized patients only as it is wellknown that hospitalization and the procedures undertaken after hospitalization increase the risk of acquiring gram-negative infections. The other causes may include the regional variations in geographic locations and socioeconomic status of the studied population 20 . The predominance of male patients (57.3%) was observed in our study as has been noted in the majority of the other studies 3,8,12,14 . The majority of the wound samples (54.6%) were received from male and female surgical, Burn and Orthopaedic wards (  (Table 2). This increased frequency of infections may be attributed to the increased turnover of patients in these wards compared to intensive care units. Increased frequency of infections in males admitted to non-medical wards, has been observed by other researchers 21, 22 . Males are generally considered more prone to infections than females because of differences in the immune responses as well as disparity of sex-chromosome-linked genes 23 . More than 55% of these infections occurred among patients aged 40 years and above. Similar observations were made by Chang et al and Mulu et al 3,11 . Among the gram-negative bacteria, E. coli (25.3%) and K. pneumoniae (21.0%) were the frequent Gram-negative isolates (  26 . A very high degree of multidrug resistance in the range of 88.5 to 97.4% for gram-negative isolates has been observed in some of the studies 6,11,25 . The reason for the relatively high degree of resistance is the inclusion of two or more antibiotics for calculating the multidrug resistance whereas we used the three or more antibiotics for calculating the multidrug resistance. In our study, 22.1% of the cases showed extensive drug resistance which seems to be in line with other similar studies as done by Mulu et al and Muluye et al who found XDR in 22.7% and 20.6% of their cases respectively 11 , 12 . Some of the researchers have found a high degree of extensive drug resistance (>70% of cases) 6,14 . This has been ascribed to the rampant use of antibiotics in these areas. The other reasoned fact that they have employed five or more antibiotics only for calculating extensive drug resistance. Carbapenem resistance was found in 22.6% of the cases in our study whereas it was 12.5% in the study done by Enwuru et al 26 and <8% in a study done by Kader et al. 27 . A high degree of carbapenem resistance in our study may be ascribed to the increased numbers of Hajj and Umrah pilgrims visiting Saudi Arabia, unrestrained use of antibiotics and prevalence of community-acquired infections 28 .
Overall, the multidrug-(54.7%) and carbapenem-resistant (60.5%) strains were isolated from male patients especially in the age group of 40-59 years. The multidrug-and extensively drug-resistant strains were mainly observed in intensive care units ( respectively 11 . A relatively lower MDR, especially for K. pneumoniae and E. coli in our study may be because we used the criteria of three or more antibiotics for MDR calculation whereas most of the other studies have used two or more than two antibiotic criteria. Extensive drug resistance was mainly shown by A. baumannii (87.5%) followed by K. pneumoniae (20.0%), while 40% of K. pneumoniae isolates were carbapenem-resistant. We did not observe the isolation of A. baumannii in most of the studies which we used for comparison except Mohammad et al who found 100% XDR for Acinetobacter spp. and 64.7% of K. pneumoniae 14 27 . The overall antibiotic resistance rate for Gram-negative bacteria was 43.4%, 59.1% and 53.7% towards carbapenems, 3rd -and 4th -generation cephalosporin's respectively (Table 5). Among aminoglycosides, amikacin continues to remain effective against E coli, P. aeruginosa and K. pneumoniae with a sensitivity rate of > 98%, > 92% and > 75% respectively. Gamal et al found 4%, 28.8% and 25% resistance against E. coli, P. aeruginosa and K. pneumoniae 33 . The effectiveness of amikacin has been proved in other studies also 34,35 . All of the four organisms under study showed a resistance rate of > 75% for 1st -generation cephalosporins ( Table  5). A high degree of resistance of E. coli and K. pneumoniae has also been observed by Mama et al. 25 . Biadglegne et al observed 50 -70% resistance of E. coli, P. aeruginosa and K. pneumoniae for cephalothin 6 . E. coli, K. pneumoniae and A. baumannii showed a resistance rate of > 45%, >55% and >98% resistance against the 3 rd -generation cephalosporins (  36 . Colistin was highly effective (>90%) against all the tested isolates of gram-negative bacteria under study (Table 5). Tan et al in their study on the in vitro activity of colistin in gram-negative bacteria observed >90% effectiveness of colistin for E. coli and K. pneumoniae as seen in our study but found 33% resistance for P. aeruginosa which is in contrast to our study 37 . Somily et al observed that 100 % and 93.9% sensitivity of colistin against A. baumannii and P. aeruginosa respectively 38 . Few isolates of A. baumannii and P. aeruginosa that were tested for tigecycline presented 100% resistance ( Table  5). Gupta et al found 100% resistance of tigecycline to P. aeruginosa as in our study but found only 6.3% resistance for A. baumannii which is in contrast to our study 39 . Tigecycline has been generally found to be effective against A. baumannii with >90% susceptibility whereas it has limited efficacy against P. aeruginosa 40 . Al-Agamy et al in their study at a hospital in Riyadh, Saudi Arabia, observed 56% resistance of A. baumannii against tigecycline which signifies the presence of resistant strains of A. baumannii in Saudi Arabia 41 . This is the first study on wound infections from the northern region of Saudi Arabia that will add to the world literature on antimicrobial resistance. Furthermore, the study focused on selected gram-negative microorganisms of global importance. The limitation of this study rests in the lack of molecular characterization of resistance and a single centre study.

Conclusion
Wound infections are dominated by Gram-negative organisms with a higher frequency of MDR and carbapenem-resistant isolates that will challenge wound management in the light of limited treatment options. Intensive care patients are at a higher risk of acquiring resistant Gram-negative wound infections necessitating strict infection control activities. The frequent empirical antimicrobial therapy for intensive care patients should be based on the local evidence on the bacteriological profile and their resistance pattern. The study recommends strengthening surveillance activities that will guide the control of wound infection in hospitals. Furthermore, effective implementation of antimicrobial prescription guidelines coupled with patient counselling to adherence of antimicrobial consumption in primary health centres is needed.