blaOXA, blaSHV-, and blaTEM- extended-spectrum β-lactamases in Gram- negative strains from burn patients in Lahore, Pakistan

Introduction: Gram-negative bacterial pathogens are associated with complications of post-burn infections that showed significant association with different genotypic variants of extended-spectrum β-lactamases. In this study, we aimed to determine the distribution of ESBL gene variants among bacterial pathogens from post-burn infections. Methodology: Cephalosporin-resistant isolates were collected from Jinnah Burn and Reconstructive Surgery Centre, Lahore. Phenotypic testing: double-disk synergy test, combination disk test, multiplex PCR for blaOXA, blaTEM, and blaSHV genes were performed. Results: Of the 53.5% cephalosporin-resistant isolates, 50.7% were community-acquired and 49.3% were nosocomial pathogens. Seventy-two percent of post-burn infections were found in males (p-value = 0.92, OR = 1.04). The age of burn victims ranged from 4 to 85 years (mean = 28.9, SD = 15.6). Pseudomonas aeruginosa was most predominant at a rate of 49%. There were 83.3% multidrug-resistant isolates, which showed susceptibility to meropenem, imipenem, and amikacin in 28.7%, 25.3%, and 26% respectively. In phenotypic tests 16% ESBLs detected by the DDST and 14% confirmed by the CDT. Molecular detection proved effective for the detection of 79.7% blaTEM, 37.7% blaOXA, and 18.8% blaSHV isolates. blaTEM genes were confirmed in 18.1% CDT-positive isolates, with 62.6% diagnostic accuracy (95% CI = 54.7-70) and 88% specificity (95% CI = 80.4-93.4). Conclusions: The antimicrobial resistance associated with the ESBL-producing Pseudomonas spp. and Enterobacteriaceae is becoming a challenge for the treatment and survival of burn patients. The high rate blaTEM, blaOXA, and blaSHV genes confirm the need to improve the management of burn patients in order to prevent post-burn infections.


Introduction
Burn incidents are frequently reported from low and middle income countries [1]. According to WHO report of 2012, worldwide 195,000 deaths happen due to burns [2]. WHO also reported 7.1 million fire incidents in 2004 and the incidence rate was 110/10,000 cases worldwide. Southeast Asia and Middle-East region was found more affected with an incidence rate of 243/10,000 and 187/10,000 respectively, as compared to lowest incidence of 19/10,000 in the United States of America [3]. Post-burn infections pose a global threat as a major public health problem [4]. Nosocomial infections are predominant in burn patients and 75% of deaths occur within a few days of burn exposure due to sepsis and severity of infection [5].
Multidrug resistant Gram-negative bacterial strains are rapidly emerging as etiological agents in 50% of post-burn infections [6]. Sepsis is the ultimate consequence of infections caused by bacterial invasion of traumatized skin [7]. Both the Gram-negative and positive bacterial strains are reported to be associated with the post burn infections including Pseudomonas spp., Acinetobacter spp., Enterobacteriaceae, Staphylococcus spp., and Streptococcus pyogenes [8]. Pseudomonas aeruginosa is the predominant bacterial pathogen among clinical isolates of burn patients [9].
To date, 193 variants of bla SHV and 223 variants of blaTEM have been reported worldwide [10]. SHV enzymes are commonly found in Enterobacteriaceae including Klebsiella spp., and E. coli but other species also exit including P. aeruginosa and A. baumannii [11]. blaSHV5 and blaSHV12 from Korea and Japan, blaTEM12 and blaTEM52 from United Kingdom, blaOXA10 and blaOXA13 have been reported from Iran and France respectively [12]. Recent study from Pakistan reveals 40% of ESBLs producing bacteria are detected in burn patients [13]. Horizontal transfer by plasmids and transposons during conjugation is a principal genetic factor for worldwide dissemination of ESBLs encoding genes [14]. Self-medication is a contributing factor behind rapidly developing antimicrobial resistance [15]. Rapid and accurate diagnosis of infectious agents is necessary for appropriate antibiotic prescription [16]. The main objective of this study was to determine the frequency distribution of MDR bacterial pathogens implicated in post-burn infections. Secondly, we aimed to determine the frequency of the most prevalent types of genetic variants of blaOXA, blaSHV, and blaTEM ESBLs encoding genes that might be associated with the dissemination of antimicrobial resistance in the community acquired and nosocomial pathogens. The molecular detection of ESBLs by multiplex PCR was employed in order to test the validity of phenotypic tests to be involved as routine diagnostic procedure in order to reduce the cost and duration of the treatment.

Study design and clinical setting
A cross-sectional study was conducted at Jinnah Burn and Reconstructive Surgery Centre (JB and RSC), Lahore, Pakistan and the Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore. The burn unit at JB and RSC consists of 75 beds, where sampling was performed on inpatients and outpatients. Ethical approval was obtained from Jinnah Hospital Lahore at the 40 th meeting of the Ethical Review Board on 12 th August 2017. Bacteriological profiling, antimicrobial susceptibility testing (AST), and genotypic screening were performed between 15 th August 2017 and 15 th August 2018. Both pediatric and adult patients were included for the analysis if they had clinical signs and symptoms such as pain, swelling of burn wounds and infection. The multidrug-resistant Gram-negative isolates were susceptible to at least one antibiotic in three or more antimicrobial classes [6]. The cephalosporin-and carbapenem-resistant Gramnegative isolates were further processed for phenotypic identification and genetic profiling of ESBLs by blaOXA, blaSHV, and blaTEM multiplex PCR. Burn patients suffering from previous infections, those receiving antibiotic therapy, and cephalosporin-sensitive Gramnegative isolates were excluded.

Bacteriological profiling and data collection
A total of 358 non-repetitive samples, including wound swabs, blood and tissue biopsy specimens, were collected from the patients undergoing treatment in the outpatient department (OPD), general ward, intensive care unit (ICU), and plastic surgery ward.
Antimicrobial susceptibility testing was performed after identification of bacterial strains.

Statistical analysis
Statistical Package for Social Sciences (SPSS) version 23 was used for all statistical analyses. A pvalue < 0.05 was considered as significant for the frequency distribution of infections among males and females and the validity testing of ESBL gene detection.

Distribution of clinical isolates
From August 2017 to August 2018, 358 specimens were collected from burn patients admitted to Jinnah Burn and Reconstructive Surgery Centre (JB and RSC), Lahore. Among 280 (78.2%) positive cultures, 150 (53.5%) cephalosporin-resistant isolates were further processed for the detection of ESBLs by phenotypic tests and multiplex PCR. The patients' age ranged from a minimum of 4 years to a maximum of 85 years (Mean = 28.9, SD = 15.6).

Discussion
Multidrug resistance in Gram-negative isolates is found to be associated with the acquisition of βlactamase gene variants [22]. B-lactamases are either encoded in the plasmids or chromosomal DNA [23]. ESBLs are known for hydrolyzing the penicillin, third and fourth generation cephalosporins and monobactams [10].     Ambler classification involves four classes of βlactamases including class A, B, C, and D [24]. Class A ESBLs such as sulfhydryl variant (SHV) and Temorina Escherichia coli mutant (TEM) are inactivated by βlactamase inhibitors including clavulanic acid, sulbactam, tazobactam [25]. Particular types of ESBLs are also capable of inactivating the aminoglycosides and sulphonamides [26]. Class D oxacillinases such as OXA type ESBLs and carbapenemases including blaOXA-10 and blaOXA-48 respectively, are capable of degrading the cephalosporins and carbapenems respectively [27]. Cephamycins and carbapenems are resistant to degradation by ESBLs [24].

6%) S n (%) I n (%) R n (%) S n (%) I n (%) R n (%) S n (%) I n (%) R n (%)
This study includes assessment of cephalosporins and carbapenems resistance in burn patients' clinical isolates. The frequencies of ESBLs producing bacteria also have been determined in order to find the association with antimicrobial resistance patterns. There was no significant difference observed between the community-acquired (50.7%) and nosocomial infections (49.3%). Community acquired infections were less prevalent about a decade ago where only 16.9% previously infected burn patients were hospitalized [28]. These findings indicate that the MDR strains are currently proliferating in the environment and the community. Self-prescription and the easy access to the commercially available antibiotics and inappropriate prescriptions by physicians may be the contributing factors in the emergence of MDR strains [12,24]. Individuals with the young age work in different factories and industries. Most of the burn victims belonged to the young age of 20-30 years as previously the similar age groups of burn patients have been observed in Iran [29].
Pseudomonas spp., is the leading causative agent of burn wound infections and causes sepsis mediated mortality in 40-50% cases [30]. All of these pathogens especially Pseudomonas spp., and Klebsiella spp., are capable of adhering with and forming biofilms on inanimate objects such as catheters and surgical instruments [31]. Here, the single bacterial strains were processed instead of multiple isolates for the antimicrobial susceptibility testing in order to determine the frequency of MDR Gram-negative pathogens. Previously the multiple bacterial strains have been isolated from the burn patients with Pseudomonas spp., and Acinetobacter spp. co-infection [32].
There were 83.3% (125/150) MDR isolates showing resistance against three and more classes of antimicrobial agents. These isolates were observed with more than 70% resistance against meropenem and 69% resistance against imipenem. ESBLs positive P. aeruginosa isolates have been observed in burn patients in Pakistan with 61% resistance against imipenem [13]. E. coli strains with 47% and K. pneumoniae with 44% resistance against meropenem have been detected in burn patients in Nigeria [18]. Early investigations on burn patients differ where more than 80% isolates were resistant to imipenem and meropenem. Burn patients in Sofia, Bulgaria have been reported with 82-85% resistance against imipenem and meropenem in A. baumannii isolates [32,33]. The cephradine resistance was observed in 98% of the isolates while the remaining 2% were found to be resistant against cefoperazone and cefotaxime. The phenotypic detection of all the isolates was not helpful as DDST and CDT negative but cephalosporins resistant isolates need to be tested by molecular techniques. Molecular detection by multiplex PCR is the gold standard and more sensitive as 46% of cephalosporins resistant isolates were positive for ESBLs genes as compared to 24% ESBLs confirmed by phenotypic testing. The remaining 54% resistant isolates may harbor metallo-β-lactamases (MBLs) encoding genes and other non-enzymatic resistance mechanisms. Several phenotypically negative isolates were identified by multiplex PCR as ESBLs producers. Low specificity and lack of constant sensitivity of the phenotypic tests justifies the need to use advance molecular techniques for the rapid, specific and accurate diagnosis of ESBLs producers [34].
bla TEM was predominant in cephalosporins resistant isolates followed by blaOXA and blaSHV. These findings are in agreement with Bajpai et al., form New Delhi, India where blaTEM was detected in 48.7% isolates followed by blaSHV [35]. Shakibaie et al., reported 6.6% blaSHV and of 2.5% blaTEM from burn patients in Iran [30]. The differences in the occurrence of ESBLs genes are based on their geographical distribution as blaTEM is predominant in China and blaSHV is the leading ESBL in North America [10]. Co-existence of ESBLs genes was confirmed in our findings where blaSHV + blaTEM were detected in 17.39% and blaOXA + blaTEM in 14.5 PCR positive isolates. These findings differ from Parajuli et al., where blaSHV and blaTEM co-existed in 10% isolates [36]. blaOXA, blaSHV, and blaTEM co-existence was detected in 5.8% isolates which indicates the parallel emergence of different ESBLs in the same isolates from burn patients and their surroundings.
The clinical presentation of patients suffering from post-burn infections is very difficult to interpret which marks inappropriate prescription of antibiotics. Multidrug resistance in burn isolates develops due to slow wounds healing associated hospitalizations poor diagnosis and treatment. There is an urgent need to assess the incidence of post-burn infections by multidrug resistant bacterial pathogens. Bacteriological and genetic profiling of ESBLs and other resistance factors provides guidance for the empiric therapy and ensures proper diagnosis. The use of multiplex PCR is very reliable and promising that ensures cost-effectiveness and speedy output as compared to traditional culturing and phenotypic tests. Multiplex PCR can be used in infection control programs and to ensure higher throughput in case of outbreaks.

Conclusions
Multidrug-resistant Gram-negative bacterial pathogens are responsible for the spread of antimicrobial resistance in our community and healthcare settings. The emergence of Pseudomonas spp. as a predominant pathogen, followed by Enterobacteriaceae, is becoming problematic in the treatment of burn patients. Phenotypic testing of ESBLs is less effective due to the lower sensitivity of DDST and CDT. Molecular detection of ESBL-encoding genes by PCR is the more accurate, reliable, and specific diagnostic method. bla TEM, followed by blaOXA, ESBLs are frequently associated with the inefficacy of third-generation cephalosporins and carbapenems. blaSHV ESBLs were less in number, but all of these isolates were resistant to piperacillin. It is necessary to overcome the emergence of multidrug-resistant bacterial pathogens by understanding their antimicrobial resistance mechanisms other than the enzymatic degradation of antibiotics. The spread of multidrug-resistant strains in clinical burn settings is alarming; therefore, treatment strategy and infectioncontrol management should be improved immediately.