Distribution of bl aOXA, bla SHV, and bla TEM type extended-spectrum β-lactamase genes in multi-drug resistant Gram-negative isolates from burn patients of Lahore, Pakistan

Background Multidrug resistant Gram-negative bacterial pathogens are becoming a lethal source of infections and associated complications of sepsis and multiple organ failure following burn injuries. Genotypic variants of bla OXA , bla SHV and bla TEM type extended-spectrum-β-lactamases (ESBLs) have been detected in Pseudomonas aeruginosa , Enterobacteriaceae and other type of bacteria. These hydrolytic enzymes are responsible for the degradation of broad-spectrum antimicrobials including third generation cephalosporins. We aimed to determine the distribution of ESBLs gene variants among MDR pathogens from post burn infections. Methods A total of 358 specimens were collected during the period from 15 th August 2017 to 15 th August 2018 from burnt patients at Jinnah Burn and Reconstructive Surgery Centre (JB&RSC). Results 53.57% cephalosporins resistant isolates were found to be associated with a slightly higher frequency of 50.7% community-acquired and 49.30% nosocomial infections. 72% of these infections was found to be associated with males (p-value = 0.919, OR = 1.038). The age of burn victims ranged from 4 to 85 years (Mean=28.95, SD=±15.65). Pseudomonas spp., were the predominant as 49.33% followed by 22.67% Klebsiella spp., 20% Acinetobacter spp., and 8% Proteus spp., strains. There were 83.33% multidrug resistant isolates and meropenem, imipenem, and amikacin were found to be effective against 28.70%, 25.30%, and 26.00% of cephalosporins resistant strains respectively. Lowest sensitivity of phenotypic tests was observed as 16% ESBLs were detected by double disk synergism test (DDST) and 14% were confirmed by combination disk test (CDT). Molecular detection proved to be effective for the detection of 79.71% bla TEM , 37.68% bla OXA , and 18.84% bla SHV isolates. bla TEM genes were confirmed in 18.18% CDT positive isolates with 62.67% diagnostic accuracy (95% CI = 54.70, 70.00) and 88.42% specificity (95% CI = 80.45, 93.41). All of the bla TEM positive isolates were resistant to cefuroxime and 98.18% were resistant to cephradine, and piperacillin. Conclusion The antimicrobial resistance associated with the ESBLs producing Pseudomonas spp., and Enterobacteriaceae is becoming a challenge for the treatment and survival of burn patients. The higher frequency of MDR isolates and detection of bla TEM , bla OXA , and bla SHV genes confirms that management of burn patients should be improved to prevent the infections.


INTRODUCTION
Burn incidents are frequently reported from low and middle income countries (1).
According to WHO report of 2012, worldwide 195000 are caused by burns (2). WHO also reported 7.1 million fire incidents in 2004 and the incidence rate was 110/10000 cases worldwide. Southeast Asia and Middle-East regions were found to be more affected with 243/10000 and 187/10000 incidence respectively, as compared to lowest incidence of 19/10000 in the United States of America (3). Postburn 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 Streptococcuspyogenes (8).
Pseudomonasaeruginosa is the predominant bacterial pathogen among clinical isolates of burnt patients (9).
Multidrug resistance in Gram-negative isolates is found to be associated with the acquisition of β-lactamase gene variants (10). β-lactamases are either encoded in the plasmids or chromosomal DNA (11). ESBLs are known for hydrolyzing the penicillins, third and fourth generation cephalosporins and monobactams (12).
Particular types of ESBLs are also capable of inactivating the aminoglycosides and sulphonamides (15). Class D oxacillinases (OXA type ESBLs) including bla OXA− 10 and bla OXA− 48 are capable of degrading the cephalosporins and carbapenems respectively (16). Cephamycins and carbapenems are resistant to degradation by ESBLs (13).
To date, 193 variants of bla SHV and 223 variants of bla TEM have been reported worldwide (12). SHV enzymes are commonly found in Enterobacteriaceae including Klebsiella spp., and E. coli but other species also produce them includingP. aeruginosa and Acinetobacterbaumannii (17). bla SHV5 and bla SHV12 from Korea and Japan, bla TEM12 and bla TEM52 from the United Kingdom, bla OXA10 and bla OXA13 have been reported from Iran and France respectively (18). A recent study from Pakistan revealed 40% of ESBLs producing bacteria in burn isolates (19). Horizontal transfer by plasmids and transposons during conjugation is a principal genetic factor for worldwide dissemination of ESBLs encoding genes (20). Self-medication is a contributing factor behind rapidly developing antimicrobial resistance (21). Rapid and accurate diagnosis of infectious agents is necessary for appropriate antibiotic prescription (22). 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 bla OXA , bla SHV , and bla TEM 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 validatetheir use as a routine diagnostic procedure which would reduce the cost and duration of the treatment. The cephalosporins and carbapenems resistant Gram-negative isolates were further processed for the phenotypic identification and genetic profiling of ESBLs by bla OXA , bla SHV , and bla TEM multiplex PCR. Burnt patients suffering from previous infections,those receiving antibiotic therapy, and cephalosporins sensitive Gramnegative and positive isolates were excluded.

Bacteriological profiling and data collection
A total of 358 samples including wounds swabs, blood and tissue biopsy specimens were collected from the patients under treatment in burn unit's OPD, general ward, intensive care unit (ICU), and plastic surgery ward. Gram-positive isolates were excluded from further analysis.Antimicrobial susceptibility testing was performed after identification of bacterial isolates.

Antimicrobial susceptibility testing and phenotypic detection of ESBLs
The antimicrobial resistance and susceptibility patterns were analyzed by performing Kirby Bauer's disk diffusion method according to Clinical Laboratory Standards Institute (CLSI) 2017 guidelines 23 .Bioanalyse®(Turkey) antimicrobial discs were used for AST profiling of Gram-negative bacterial isolates.Preliminary ESBLs detection was performed by double disk synergism test (DDST) and confirmatory combination disk test (CDT) (24).

bla O XA , bla SHV , and bla TEM multiplex PCR
Whole-genomic DNA extraction was performed by the boiling lysis method by preparing the cells suspension of purely isolated bacterial colonies as described previously (25). Previously designed conserved regions specific bla OXA , bla SHV , and bla TEM primers were optimized for multiplex PCR (26,27). PCR amplicons were visualized by agarose-gel electrophoresis with 1% agarose gel and 1X Tris-borate-EDTA (TBE) buffer.

Statistical analysis
Statistical Package for Social Sciences (SPSS) version 23 has been used for the execution of all statistical analyses. The association of ESBLs genotypes with the antimicrobial resistance has been demonstrated by percentages. The p-value of < 0.05 has been taken as significant in the frequency distribution of infections among males and females and validity testing of ESBLs genes detection.

Distribution of clinical isolates
During one year, n=358 specimens were collected from burns patients admitted in cephalosporins resistant MDR isolates with resistance against more than two or three antimicrobial agents. These multidrug resistant strains were also resistant to nalidixic acid and tetracycline ( Pseudomonasspp., was the predominant ESBLs producer as 10(13.51%) strains were confirmed by CDT as ESBLs producers. Klebsiella spp., Acinetobacter spp., and Proteus spp., strains were relatively low in number with 5(14.71%), 4(13.33%), and 2(16.67%) as ESBLs producers respectively. A large number of these cephalosporins resistant isolates were not-determined phenotypically as ESBLs producers.

DISCUSSION
This study includes assessment of cephalosporins and carbapenems resistance in burns 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 some small difference between the community-acquired (50.70%) and nosocomial infections (49.30%). Community acquired infections were less prevalent about a decade ago where only 16.90% 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 ease of access to the commercially available antibiotics and inappropriate prescriptions by physicians may be the contributing factors in the emergence of MDR strains (13,18).Individuals with the younger age work in different factories and industries. Most of the burn victims belonged to the young age of 20-30 years in accordance with earlier research (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 multiple bacterial strains have been isolated from the burn'spatients with Pseudomonas spp., and Acinetobacter spp.,coinfection (32).
There were 83.33% (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 imipenem. Early investigations on burns patients differ where more than 80% isolates were resistant to imipenem and meropenem as all of the isolates were included in the analyses (33). Molecular detection by multiplex PCR was useful as 46% of cephalosporins resistant isolates were positive for ESBLs genes. 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 more advance molecular techniques for the rapid, specific and accurate diagnosis of ESBLs producers (34).
bla TEM was predominant in cephalosporins resistant isolates followed by bla OXA and bla SHV . These findings are in agreement with Bajpai et al., form New Delhi, India where bla TEM was detected in 48.70% isolates followed by bla SHV (35). Shakibaieet al., reported 6.6% bla SHV and of 2.5% bla TEM from burn patients in Iran (30). The

Availability of data and materials
The data sets analyzed during the current study are available from the corresponding author.

Competing interests
Authors declare that they have no competing interests.

Funding
No funding was received for this study.

Declaration
This work is part of Ph.D thesis of Mr. Muhammad Hayat Haider.

Availability of data and materials
The data sets analyzed during the current study are available from the corresponding author.

Competing interests
Authors declare that they have no competing interests.

Funding
No funding was received for this study.

Declaration
This work is part of Ph.D thesis of Mr. Muhammad Hayat Haider.

Muhammad Hayat Haider
Ph.D Scholar Department of Microbiology and Molecular Genetics.
University of the Punjab, Lahore Postal address: