Prevalence of aerobic pathogenic bacteria isolated from patients with burn infection and their antimicrobial susceptibility patterns

Burn infections are one of the most common serious Background: illnesses caused by pathogens, mainly by both gram-negative and gram-positive bacteria. The aim of this study was to detect of the prevalence of multi-drug resistant and extended-spectrum β-lactamase-producing (ESBL) bacteria isolated from inpatients with burn infection and the antimicrobials sensitivity patterns of all bacterial isolates during three years. This cross-sectional study was performed in Al-Najaf Central Methods: Hospital in Al-Najaf City, Iraq from January 2015 to December 2017. A total of 295 burns swabs were collected from hospitalized patients with burn infection. All grown bacterial isolates were identified by standardized microbiological tests. Antimicrobials susceptibility testing was done using the disc diffusion method. Multi-drug, extensive-drug and pan-drug resistant bacteria and extended-spectrum β-lactamase-producing bacteria were determined according to standardized methods and guidelines. Of the 295 burn swabs, 513 different bacteria strains were Results: isolated. was the most common bacteria with Pseudomonas aeruginosa 142 isolates (27.6%) followed by methicillin resistance Staphylococcus 106 isolates (20.6%), while was the least aureus Staphylococcus typhi common bacteria with only 17 isolates (3.3%). 323 (63%) different bacterial strains were isolated from patients who stayed in hospital for 15 days. Most bacterial isolates were resistant to most antimicrobials with high percentages. Out of the 513 bacterial isolates; only 33 isolates (6.4%) were resistant to imipenem 10μg and 464 isolates (90.4%) were multi-drug resistant, 20 isolates (14%) were extensive-drug resistant and 17 isolates (3.3%) were pan-drug resistant. was the most Pseudomonas aeruginosa common ESBL-producing bacteria (51 isolates-35.9%). There was a high prevalence of multi-drug resistant bacteria Conclusions: in burn infection in Al-Najaf hospital. was the Pseudomonas aeruginosa most common multi-drug resistant bacteria, and the most common of ESBL bacteria causing burn infection over the three years. 1


Introduction
Burn infection caused by pathogenic bacteria is one of the most common hospital problems worldwide, particularly in developing countries 1 . Fire leads to skin destruction and simultaneous suppression of both humoral and cellular immune system subsequently resulting burn infection 2 . Complications of burn infection are responsible for more than 70% of death cases among inpatients with burns 3 . These infections mainly caused by multidrug resistant gram-negative and gram-positive bacteria such as Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K.pneumoniae) and Staphylococcus aureus (S.aureus) 4,5 . Nonsterile burns halls and duration of patients stay in hospital in addition to the surface area of burned skin, are the most important factors related to the increase of persistent and multiplication of pathogenic bacteria in the burned areas 6,7 . Multi-drug resistant (MDR) bacteria is one of the most common pathogens causing burn infection in hospitalized patients worldwide 8,9 . These pathogens are resisting to at least three different classes of antimicrobials such as, penicillin's, beta-lactams, cephems, 3rd and 4th generation cephalosporins, aminoglycosides, tetracyclines and quinolones, and is becoming one of the most dangerous health issues in hospitals 10 . In addition, extended-spectrum β-lactamase (ESBL)-producing bacteria are considered as a potent pathogens due to it their resistance to a wide range of antimicrobials like, cefotaxime, ceftriaxone and ceftazidime, that lead to difficulty in the treatment of most infections such as burn infection and urinary tract infection 11,12 . Burn infection is characterized by difficult healing due to administration of unsuitable treatment, long stays in hospital and the contaminates of hospital environments lead to the emergence of new multi-drug resistant bacterial isolates causing dangerous complications such as, bacteremia, septicemia and death 13,14 . Therefore, we must pay attention to all safety standards in hospitals, especially in burns wards through sterilization, performing antimicrobial susceptibility test on all pathogenic bacteria isolated from burn infections, and keeping the burned skin in sterile conditions to prevent the emergence of these pathogens. According to the above, the aim of this work was to investigate of the prevalence of multi-drug resistant bacteria and extended-spectrum β-lactamase-producing bacteria isolated from inpatients with burn infection in Al-Najaf central hospital in Al-Najaf City, Iraq over three years, from January 2015 to December 2017 to increase our understanding of the most prominent bacteria and their resistance to different antimicrobials to prevent the emergence of these isolates in the future.

Ethical considerations
We confirm that we received approval for this study including: patient's swabs and consent from the participants. All swabs were taken by physician and consent by the hospital treatment and care team responsible and then handed the samples over to us. All swabs were provided from the participants physician in Al-Najaf central hospital in Al-Najaf City, (Burns Department). All swabs were immediately transported to the Laboratory of Microbiology in Faculty of Science, University of Kufa to process. Note: Each swab was labeled with the following items: age, sex, duration of stay in the hospital after burning. Al Najaf Central Hospital is part of the University of Kufa and therefore written approval was not sought as there is a pre-existing agreement between the university and hospital regarding clinical sample collection sample collection. Oral consent to take swabs was taken from each patient.
Eligibility criteria for patients Patients will be considered eligible for registration into this study if they fulfill all the inclusion criteria and none of the exclusion criteria as defined below.
1-Patients (Male or female) at least more than 18 years old.
2-Patients should have sufficient capacity for informed consent.

3-Patients should don't have any other infections.
Study design, burns swabs collection and bacterial identification This is a cross-sectional descriptive study performed in Al-Najaf central hospital in Al-Najaf City, Iraq, from January 2015 to December 2017. A total of 295 swabs (emulsion with normal saline) were collected from the burned area of hospitalized patient with burn infections (2nd degree, shown the signs of infection during the change of dressings), ages ranges 18-45 years old (males and females), 3 swabs were taken from each patient at 5, 10 and 15 days of stay. Immediately, all collected swabs were incubated with brain heart infusion broth (Oxoid™, USA, CM1135R) for 24h at 37°C to encourage bacterial growth and then streaked onto blood agar (Oxoid™, USA, CM0055B) using a swab (Himedia, India, PW1210G) and chocolate agar (Oxoid™, USA, R01293) surface and incubated aerobically at 37°C for 24-48 h. All emerged bacterial isolates were identified according to colony morphology and standard microbiological tests such as; colony morphology, blood hemolysis onto blood agar surface (Oxoid™, USA, CM0055B), gram stain, oxidase test, catalase test, imvic test, motility test, coagulase test, growth on MacConkey agar (Oxoid™, USA, R061322) and Mannitol salt agar (Oxoid™, USA, CM0085B) 15 .
Identification of methicillin resistance S.aureus All S.aureus isolates growth was adjusted according to turbidity of standard McFarland tube 0.5 (measured by Vis-Nir spectrophotometer, Biobase, UK, bk-S410). All isolates were streaked onto Mueller Hinton agar (Oxoid™, USA, PO5007A) surface supplemented with 4% NaCl. Five µg of methicillin disc (Oxoid™, USA, CT0159B) was placed at the surface of Mueller Hinton agar and incubated aerobically at 37°C for 24h. All S. aureus isolates that were resistant to methicillin with diameter of inhibition zone < 17 mm were considered as methicillin resistant S.aureus (MRSA), while those isolates with diameters of inhibition zone ≥ 17 mm considered methicillin sensitive S.aureus (MSSA) 18 .

Phenotypic detection of extended spectrum betalactamase-producing bacteria
This test was performed according to modified double disc synergy test (MDDST) 19 as follows: all bacterial isolates (turbidity was adjusted according to McFarland tube 0.5) were streaked by sterile swab (Himedia, India) onto Mueller Hinton agar (OxoidTM, USA) surface, AMC disc 30µg was placed in the center of agar plate, CRO 30µg, CTX 30µg and CAZ 30µg were placed around AMC disc 30µg (15 mm from center to center). All plates were incubated aerobically at 37 °C for 24h. Any increase in the inhibition zone towards AMC disc 30µg was considered as positive for the extended spectrum beta-lactamase.

Statistical analysis
Percentages were used in this study to compare between the prevalence of pathogenic bacteria and their resistant to antimicrobials using Graphpad-prism V.10 computer software.

Discussion
Burn infection is one of the most serious problems in hospitals caused by different pathogens that infect most patients who stay   in hospitals for prolonged periods. In this study, 513 different bacterial strains were isolated from 295 swabs of hospitalized patients with burn infections over the three years. Gram negative bacteria were responsible for more than half of infections while gram positive bacteria accounted for 34.7% of overall bacterial isolates. Pseudomonas aeruginosa was the most common bacteria, accounting for 27.6% of total isolates. These results are in agreement with previous studies 20-23 . Pseudomonas aeruginosa is one of the most important pathogens causing different infections such as bacteremia and burn infections 24 . This pathogen is well adapted to the hospital environments due to biofilm formation that provides long survival advantages for the pathogen, and effectively prevent eradication by the host immune system or antimicrobial drug treatment 25 . Pseudomonas aeruginosa has become responsible for more than 70% of mortality in burn patients 26,27 . The results of this study showed that MRSA was the second most common bacteria isolated from patients with burn infection, 106 isolates (20.6%)     43,44 . In this study, most pathogenic bacteria isolated from burn infection were highly resistant to most antimicrobials, especially against beta-lactams and 3rd generation cephalosporins., All pathogenic bacteria were MDR with high percentages and most of them were XDR,. P. aeruginosa was the most common PDR-bacteria followed by MRSA and A.baumannii. These results are similar with many previous studies 5, [45][46][47] . Biofilm formation by microorganisms is one of the most important mechanisms in antimicrobials resistant, consisting of the irreversible assemblage of bacterial cells associated with a surface and enclosed in matrix of polysaccharides material 48 . Biofilms are regarding as a major factor contribution to many chronic inflammatory diseases such as burn infection due to enabling bacteria to colonize the burned skin, altering growth rate and allowing genes to be transcribed that provide these pathogens to high resistance to antimicrobials and host immune system. The overuse and unsuitability of different antimicrobials to treat burn infections has led to the emergence of new MDR, XDR and PDR-bacterial strains that are able to resistant a wide range of many antimicrobials such as aminoglycosides, beta-lactams, cephalosporins, streptomycin and tetracycline 49,50 . Burn infection in hospitalized patients caused by MDR, XDR and PDR-gram negative and gram positive bacteria such as; P. aeruginosa, K. pneumoniae, MRSA, MSSA and A.baumannii may lead to delays in burn healing, graft lose, as well as development of sepsis and death; therefore, determination of the risk factors for these pathogens infections is essential for infection control 47 . The results of this study showed that P. aeruginosa and K. pneumoniae were the most common ESBLproducing gram negative bacteria followed by A.baumannii and E.coli while there was no any strain of ESBL-S.typhi. These results are similar to previous studies 1,51,52 . Infections caused by ESBL-producing gram negative bacteria are associated with an increase of health care costs, morbidity and mortality 53,54 . Extended spectrum beta-lactamases (ESBLs) have been reported as one of the most important hospital-acquired infections such as burn infection and bacteremia 9,55 . Most bacteria harboring ESBLs are usually resistant to beta-lactam antibiotics and other classes of antimicrobials 56 . These enzymes are carried in and transferred from bacteria to bacteria by plasmids 57,58 . The most important steps to ensure the safety of patients with burn infections are: to control the spread of ESBL-producing bacteria, isolation of colonized patients in sterile wards, and continuously performing antimicrobial sensitivity tests 59 .

Conclusions
There was a high incidence of MDR-bacteria causing burn infections in Al-Najaf hospital in Al-Najaf City, Iraq.
Pseudomonas aeruginosa was the most common MDR, XDR and PDR-bacteria, and the most common of ESBL-producing bacteria causing burn infection over three years followed by MRSA. Imipenem 10µg had good antibacterial activity against more than 93% of bacterial isolates. There was positive correlation between a long stay in hospital and high prevalence of pathogenic bacteria causing burn infection.

Limitation of the study
In this study, some gram negative and gram positive bacterial isolates are excluded because of the small number of isolates (less than seven isolates over the three years) such as proteus spp (5 isolates), citrobacter spp (4 isolates), enterobacter spp (6 isolates) and enterococcus spp (5 isolates). We think this small number of bacterial isolates don't has any significant effect on the results of this study.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work.

Dana M. Walsh
Rebiotix, Roseville, MN, USA This paper provides an observational report of the number of drug resistant bacteria isolated from infected burn wounds in patients in a hospital in Iraq. Increasing numbers of drug resistant organisms are identified with increasing length of hospital stay. The most commonly identified organism was Pseudomonas aeruginosa, a bacteria that commonly identified in burn wound infections.
Overall, the science in this article is sound. Appropriate MDR testing is used and compared to accepted standards.
Methods section: It is unclear if an institutional review board approved this study; this needs to be directly mentioned in the methods section. No statistics were performed on this study; however, a simple Student's T test could be performed to determine statistical significance between two groups or analysis of variance (ANOVA) could be performed to show statistically significant differences between the number of isolates depending on length of hospital stay. This could also be done for number and type of drug resistant organisms to show which antibiotic has the least resistance. Including statistics would strengthen this work.
Discussion section: Why are biofilms mentioned at all? This seems out of place, especially since no testing was done to determine if these microbes are capable of making biofilms. This either needs to be removed or more appropriately addressed in the context of the study.
Conclusions section: This could be strengthened. For example, it seems that a majority of the organisms were not resistant to imipenem. This is an important finding for this hospital and should be mentioned as an antibiotic that could help this facility control MDR infections. It would be helpful if the authors addressed the following questions in this section: What new information does this study provide? Is this important for the hospital's burn ward? Will this increase awareness of the prevalence of MDR organisms and encourage physicians to use imipenem for treatment of infections?
Other comments: Metagenomic sequencing and biofilm testing would both be interesting additions to this study. Metagenomics could be used to determine what other bacteria were present in the infections that were not culturable.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes control committee (HICC), because the our work focused on types of bacteria and antimicrobials.
The name of organism in abstract: Staphylococcus typhi. Yes, this is a print mistake, we are so sorry the right name of organism in abstract must be Salmonella typhi. We can correct this mistake.

○
There was an antibiotic policy in the hospital according to antimicrobial sensitivity test protocol.

○
The role of hospital infection control committee is not in our study, because the only goal of our study is the type of aerobic bacteria and antimicrobials.
○ About your question: what disinfectants were used in hospitals? We don't know, this is hospital work only and we can not know about these security things.
○ About your question: why they did not go for anaerobic culture? This is another different study, our study focused on aerobic bacteria only.
○ About your question: why patients above 60 were not chosen. Because we did not find patients above 60 in our study. The name of organism in abstract: staphylococcus typhi. Yes, this is print mistake, we are so sorry.

○
The write name of organism in abstract must be salmonella typhi. We can correct this mistake.

○
There was an antibiotic policy in the hospital according to antimicrobial sensitivity test protocol.

○
The role of hospital infection control committee is not in our study, because the only goal of our study is the type of aerobic bacteria and antimicrobials.