|Year : 2021 | Volume
| Issue : 1 | Page : 98-104
Hematological analysis of blood cells and isolation of pathogenic microorganisms from second-degree flame burn patient with the prevalence of multidrug resistance traits
Asif Shahriar1, Hossain Ahmed2, Aar Rafi Mahmud3
1 Department of Microbiology, Stamford University Bangladesh, Dhaka, Bangladesh
2 Department of Biotechnology and Genetic Engineering, University of Development Alternative, Dhaka, Bangladesh
3 Department of Biochemistry and Molecular Biology, Mawlana Bhashani Science and Technology University, Tangail, Bangladesh
|Date of Submission||06-Oct-2020|
|Date of Acceptance||20-Dec-2020|
|Date of Web Publication||13-Mar-2021|
Dr. Asif Shahriar
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217
Source of Support: None, Conflict of Interest: None
Burns are traumatic injuries that can occur in the home or workplace. It is a type of injury to flesh or skin caused by heat, electricity, chemicals, or radiation. The aims of this study to identified drug resistance traits of microorganisms, which is lead to wound infection after burn injury and causes hematological imbalanced of blood cells that are lead to the immune response against these pathogenic strains. The patient was burned by flame and seriously injured, with 24% tissue damaged. The whole blood was collected for complete blood count and peripheral blood film to analyze the components and morphological shape of blood cells. In addition, the wound samples were collected from wound infected sites to isolation and identification of microbial contamination as well as the study of antimicrobial susceptibility and resistance traits of microbes which are intensively involved in wound infection along with skin tissue damage. Among the complication period, the patient was seriously infected by pathogenic bacteria with viable bacterial count with log 107 colony-forming unit (CFU)/mL. The predominant pathogens were Escherichia coli, Klebsiella spp., Pseudomonas spp. and Staphylococcus spp. Most of the pathogens were found as drug-resistant, notably against second- and third-line antibiotics. The serious complication of microbial infection during the wound healing period was lead to increase white blood cells range along with terribly lower than the normal range of hemoglobin at 6.8 g/dl. Furthermore, the abnormal morphology of red blood cells was noted as an iron deficiency complication of the patient. Bacterial infection of the postburn patient is alarming for quick treatment. Most of the pathogens are rarely sensitive against most commercial antibiotics. Hence, bacterial colonization is the main threat for the burn-injured patient, which prohibits the immune response against in vivo infection.
Keywords: Anemia, burn wound infection, drug resistance microbes, immune response, white blood cell and hemoglobin
|How to cite this article:|
Shahriar A, Ahmed H, Mahmud AR. Hematological analysis of blood cells and isolation of pathogenic microorganisms from second-degree flame burn patient with the prevalence of multidrug resistance traits. Biomed Biotechnol Res J 2021;5:98-104
|How to cite this URL:|
Shahriar A, Ahmed H, Mahmud AR. Hematological analysis of blood cells and isolation of pathogenic microorganisms from second-degree flame burn patient with the prevalence of multidrug resistance traits. Biomed Biotechnol Res J [serial online] 2021 [cited 2022 Aug 14];5:98-104. Available from: https://www.bmbtrj.org/text.asp?2021/5/1/98/311090
| Introduction|| |
The burn injury produces a state of deregulation of the immune system that predisposes patients to infections. Burns extortion to a significant portion of trauma injuries, and many trauma-associated fatalities occur in burn Patients. Loss of the natural cutaneous barrier is the most apparent effect of wound infection. Beyond this is a more complex interplay of pro and anti-inflammatory signals that result in dysregulation of the innate and adaptive immune responses. As the skin is the body's first and major defense mechanism against microbial invasion in patient body cells, that enduring a burn trauma are particularly susceptible to muscle or tissue infections. The body responds with systemic immunosuppression during immediately following injury. Burn type stimulates wound healing issues consisting of three over-lapping phases: Inflammation, proliferation, and remodeling. Burns can be classified by depth, mechanism of injury, extent, and associated injuries. The first-mentioned classification is based on the depth of injury; first-degree burns involve superficial skin layers; second-degree burns extend deeper in the skin, whereas third-degree burns involve underlying tissues. Leucocytes or white blood cells (WBCs) (neutrophils and monocytes) defend the body against any kind of infections, which in turn provides chemotactic signals that recruit macrophages. The inflammatory cells then phagocytose necrotic tissue, protect against pathogens, and produce growth factors that initiate migratory and proliferative responses. On the other hand, B-lymphocytes have also produced antibodies which induced protection against microbial infection. Injured patients also suffer progressive anemia caused by dilution from resuscitation, blood loss from open wounds, and hemolysis. In addition, other cells in the plasma, such as WBC and platelets (PLT) can either be abnormally high or low following a severe burn injury as a result of systemic inflammation., Besides burn wound infections can be caused by bacteria, fungi, or viruses. The outgrowth of multi-drug-resistant strains of bacteria and fungi have enhanced the incidence of burn wound infections, sepsis, and associated death. The immune suppression in burn patients in addition to the fact that Pseudomonas aeruginosa has a preference for moist and warm wound environments made it a significant challenge for burn patients.,
In advance, in burn care over the past 50 years, infection is now the leading cause of death after extensive burn injuries. Multiple studies over the last decade have shown that 42%–65% of deaths in burn victims are due to different types of infections.,,, Hospital-acquired bacteria infect burn patients by various offensive and inoffensive procedures. Early diagnosis of microbial infections and screening for drug resistance is aimed to institute the appropriate antibacterial therapy and to avoid further Complications. Nowadays, the majority of the bacteria that cause burn infection in hospitals are resistant to at least one of the commonly used drugs. The prevalence of multidrug-resistant (MDR) bacteria in burn centers may result in the empiric selection of antibiotics that target MDR bacteria, thus propagating a vicious cycle of increased antimicrobial resistance. Members of the interleukin (IL)-2 receptor activating family of cytokines, such as IL-7 and IL-15, have an extensive role in treating bacterial infections; IL-15 has been broadly premeditated for its protective anti-tumor efficacy in several cancer preclinical studies. The major immune cells that produce IL-15 include dendritic cells, neutrophils, lymphocytes, macrophages, monocytes, endothelial cells, stromal cells, and renal epithelial cells, which transparent IL-15 in association with the IL-15 receptor alpha chain.,,, IL-15 can be evoked by various excitations, including endotoxin, interferons α/β/γ, double-stranded RNA. Trans presented IL-15 signals its action through a heterodimeric receptor that shares the IL-2R/IL-15Rβ (CD122) beta and common gamma chains. Functionally, IL-15 has been characterized as a T-cell growth factor and stimulates T cell proliferation (memory CD8+ T-cells preferentially), and immunoglobulin synthesis by B cells and is essential for the growth and survival of natural killer (NK) and NKT cells. These are leads to an immune response against microbial infection associated with WBCs equilibrium.
Burn patients are of an infectious etiology, with pneumonia, urinary tract infection (UTI), and Cellulitis. Respiratory tract infections are most frequently reported. Contributing factors include the presence of inhalation injury in some patients and the frequent need for prolonged mechanical ventilation. Furthermore, burn-injured patients are suffering in iron deficiency anemia, imbalanced count in leucocytes components along with incompatibility in PLTs count. Besides, the skin and soft tissue infections occur earlier during hospitalization, generally during the 1st week. In contrast, pneumonia, bloodstream infections, and UTIs attend to occur later in the hospitalization. Several studies have shown that the pathogens of specific concern in the burn population include MDR strains of P. aeruginosa. The mechanism of resistance in MDR P. aeruginosa is possible through the exhibition of several enzymes that modify beta-lactams and carbapenems such as extended-spectrum beta-lactamases (ESBLs), and metallo-β-lactamases (MBLs) are the major problem for treating the burn-injured patient. ESBLs are β-lactamases capable of conferring bacterial resistance to penicillin, first, second, and third-generation cephalosporins, and aztreonam, but not to cephamycin or carbapenems. MBL is a group of carbapenem-hydrolyzing b-lactamase but not aztreonam and resists currently available β-lactamase inhibitors, but are inhibited by chelating agents such as ethylenediamine tetra-acetic acid.,
Though immediate care for burn patients has improved due to treatments such as hemodynamic stabilization, the effort of this study is to investigate the wide range of infection, including anemia during post-burn-injured period by complete blood count (CBC) and peripheral blood film (PBF) assay components and also determine the antimicrobial susceptibility patterns and prevalence of pathogenic microbial infection amongst postburn patients. In addition, the in vitro activity of 13 commonly used antimicrobial agents to treat microbial infections in pediatrics burn patients are analyzed as well as screening for ESBL production along with to evaluate the different risk factors for the development of burn wound infection.
| Methods|| |
Patient selection and sample collection
Wound infected patient with 24% flame burn admitted in Dhaka Medical College Burn unit after 3 days of burn-injured (within March 2019 to May 2019) and also included for skin grafting in the study. Patient (age: 27, sex: female) of second degree (n = 10) was included during this study. Blood samples were collected for investigating a wide range of health disorders, including infection and anemia. In addition, the Burn wound swab was aseptically collected from the depth of the wound using two sterile cotton swabs by following cleaning of 70% ethyl alcohol burn wound infected sites (especially from the Chest, tummy, and hand) to rule out hospital-acquired infections [Figure 1]. The sample was homogenized in 4 mL sterile saline and transported immediately to the laboratory for further processing.
Complete blood count
Whole blood is collected over every week until recovery to determined complete blood cell count, including various blood cell infections. Patient CBC performed twice per week for the 8 weeks after injury. CBC test is performed by an automated hematology analyzer in the clinical pathology department of Dhaka Medical College Hospital, Bangladesh. All of the CBC assays were include WBC, and PLT are reported as 1000 cells/cubic liter (k/L), red blood cell (RBC) count is reported in 1 million cells/cubic liter (m/L), hemoglobin (HGB) is reported in grams/deciliter (gm/dl), neutrophil, lymphocyte, monocyte, eosinophil, and basophil are reported in percentage (%) value. Erythrocyte sedimentation rate (ESR) test also reported detecting disease-causing inflammation with the infectious rate during wound infection.
Peripheral blood film
The PBF exposes the morphology of peripheral blood cells, which ensures its place in the hemato-morphological diagnosis of various primary and secondary blood and blood-related diseases. Red cell morphology, white cell morphology, and PLTs morphology were investigated to detect a number disease include unexplained cytopenia: Anemia, leukopenia or thrombocytopenia, unexplained leukocytosis, lymphocytosis or monocytosis, unexplained jaundice or hemolysis; features of congenital hemolytic anemias such as splenomegaly, jaundice or bone pains; suspected chronic or acute myeloproliferative disease, for example, chronic myeloid leukemia; suspected organ failure such as renal disease, liver failure, features of hyperviscosity syndrome as in paraproteinemia, leukemic hyperleukocytosis, polycythemia, severe bacterial sepsis, and parasitic infections; malignancies with possible bone marrow involvement; suspected cases of nutritional anemia; suspected chronic lymphoproliferative diseases such as chronic lymphocytic leukemia; lymphoma with leukemic spills, evaluation of therapeutic response in hemopathies among others.,
Microbiological and biochemical analysis of pathogenic strains
Wound swabs were cultured on blood agar, pseudomonas agar, and MacConkey agar plates. Pathogenic Microorganisms were isolated and identified following the standard procedures. Blood agar and MacConkey agar media ware used for isolation and identification of Gram-positive and Gram-negative bacteria, respectively; And especially pseudomonas agar was used for isolation and identification of Pseudomonas spp. On the other hand, mannitol salt agar was used for isolation and identification of staphylococcus spp. As the Pseudomonas spp. and Staphylococcus spp. are the common bacterial strain which is randomly colonized and infected in postburn patient and lead to causing wound healing infection. Nutrient agar was used for general isolation and identification of all kinds of microorganisms. After inoculation, agar plates were kept into the incubator at 37°C for 24–48 h. Different biochemical tests were performed following the standard methods to identify the bacteria isolated from the wound samples. Samples were collected four times during the infectious period.
Microscopic identification of pathogenic strains
The Gram staining is used to distinguish between Gram-positive and Gram-negative bacteria, which have been distinct and consistent differences in their cell walls. Under the microscope, stained slides were observed to distinguish between Gram-positive and Gram-negative bacteria and to identify the bacilli, rod, and cocci.,
Determination of antibiogram pattern
Kirby-Bauer method was applied to consider as standard agar disk diffusion method.,, Comparing with McFarland 0.5 solutions, the suspension of experimental organisms were processed by adjusting the turbidity of the broth in phosphate buffer saline. An identical bacterial lawn from the growth medium was prepared on Muller-Hinton agar plates for performing an antibiogram test. Most prescribed and commercially available antimicrobial discs (gentamicin 30 μg, streptomycin 10 μg, erythromycin 15 μg, amikacin 30 μg, neomycin 30 μg, cefepime 30 μg, ampicillin 10 μg, colistin sulfate 30 μg, imipenem 10 μg, cefoxitin 30 μg, chloramphenicol 30 μg, ciprofloxacin 30 μg, aztreonam 30 μg) were applied on the surface of inoculated plates with appropriate spatial technique. The result was interpreted by following the CLSI guidelines, and the susceptibility of specific antibiotics were measured by the presence of clear a zone around the antibiotic discs.
| Results|| |
Imbalanced count of total blood cells and elements
The patient was hospitalized >60 days and her HGB rate was gradually decreased from 2nd week. Especially the rate was very low at (8.5 g/dl and 6.8 g/dl) in 38th and 44th days blood samples severally [Table 1]. There was no significant change in the total count of WBC and PLTs except 44th days samples. WBC range was increased over the normal value besides the PLTs was decreased less than (150-450 × 109/L), and also RBC range was slightly decreased in 23rd, 38th and 44th days blood samples see [Table 1]. On the other hand, the phagocytic cell neutrophils rate was increased >75%, and its reached 96% over the infection period, and lymphocyte rate was decreased from the 1st week to until cured. ESR was remained high from the 1st week to till discharged from hospital [Table 1].
Abnormalities of blood cells by peripheral blood film
Microscopic analysis of blood smear showed abnormal blood cells [Figure 2]. The RBC were hypochromic anisocytosis with microcytes and appeared paler in color, which is lead to Iron deficiency hypochromic anemia [Figure 2]b. WBCs (specially Neutrophils) and PLTs were matured with normal count and distribution [Figure 2]a and [Figure 2]b.
|Figure 2a-d; shows different microscopic examination of peripheral blood film|
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Microbial identification and prevalence in burn wound samples
The patient was infected by pathogenic microorganisms ranging from 105 to 107 CFU/mL. All samples were enormously contaminated with pathogenic bacteria Pseudomonas spp. in range of 104–107 CFU/mL. Others bacteria (Escherichia coli; Klebsiella spp. and Staphylococcus spp.) were also found in days 10, 24 and 38 samples [Table 2]. All these microorganisms were identified based on biochemical confirmation tests [Table 3] and gram staining characterization by microscopic analysis [Figure 3]. Among these microorganisms, the pathogenic bacteria Staphylococcus spp. were observed in the range of 104–105 CFU/mL in particularly days 10 and 24 [Table 2]. The enteric bacteria Klebsiella spp. was found to prevail in (days 10, 24, and 38) samples with a range of 104–105 CFU/mL and very lower frequency of E. coli in range of 104 CFU/mL was monitored in (days 10) samples [Table 2].
|Figure 3: Microscopic analysis of pathogenic strains. (a) Escherichia coli; (b) Klebsiella spp.; (c) Staphylococcus spp.; (d) Pseudomonas spp|
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Microscopic identification of isolates
The Gram staining was used to distinguish between gram-positive and gram-negative bacteria under a microscope, which has been distinct and consistent differences in their cell walls. Stained slides were observed with E. coli is Gram-negative (−ve) rod-shaped bacteria [Figure 3]a, nonmotile, encapsulated rod-shaped Klebsiella spp. [Figure 3]b, Gram-positive (+ve) spherical (cocci), grape-like clusters shaped Staphylococcus spp. [Figure 3]c and Gram-negative (−ve), rod-shaped Pseudomonas spp. [Figure 3]d.
Drug resistance and susceptible traits of identified microorganisms
Among these antibiotics, amikacin (n = 8, 63%); cefepime (n = 2, 70%), neomycin (n = 4, 55%) were found resistant and gentamicin (n = 2, 88%), imipenem (n = 4, 100%) were effectively sensitive against E. coli [Figure 3]a, amikacin (n = 20, 81%), cefepime (n = 16, 78%), chloramphenicol (n = 20, 60%), erythromycin (n = 16, 95%); imipenem (n = 4, 65%), streptomycin (n = 2, 78%) were resistant and only gentamicin (n = 2, 100%) was highly effective against Klebsiella spp. [Figure 3]b; ampicillin (n = 20, 95%), aztreonam (n = 20, 81%); cefoxitin (n = 2, 100%); erythromycin (n = 2, 100%), imipenem (n = 2, 70%) were resistant and colistin sulfate (n = 2, 100%), amikacin (n = 6, 62%); sterptomycin (n = 4, 68%) were sensitive against Pseudomonas spp., gentamicin (n = 2, 50%) was found as both resistant and sensitive equally [Figure 3]c. Amikacin (n = 2, 65%), erythromycin (n = 20, 100%), gentamicin (n = 10, 56%), imipenem (n = 2, 97%) were resistant and cefepime (n = 20, 67.35%), ciprofloxacin (n = 10, 100%), streptomycin (n = 20, 88%) were effectively sensitive against Staphylococcus spp [Figure 3]d. Notably, all pathogens were found to be sensitive against gentamicin 30 μg at least 44% to 100% respectively and most infectious pathogen of wound infection Pseudomonas spp. was extremely sensitive against colistin sulphate 30 μg [Figure 4].
|Figure 4: Resistance and susceptible pattern of (a) Escherichia coli, (b) Klebsiella spp., (c) Pseudomonas spp., (d) Staphylococcus spp. against different types of antibiotics. All experiments were carried out three times with 95% accuracy and presented data ware statistically analyzed by showing the standard errors consider as 5%|
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| Discussion|| |
Infection is the most frequent cause of death in burn patients that survive the initial burn trauma and a common cause of prolonged hospitalization. All types of infections revealed many kinds of health issues with immunological imbalanced against infections. These types of health issues were included blood cells imbalanced, and changes are blood cells morphology, which eventually acts as phagocytosis against pathogenic microorganisms that are responsible for wound infection of the postburn patient. This investigation shows that the components of blood, HGB is decreased over the 2nd week after burn injury and it terribly decreased in 6th and 7th week, respectively. ESR level was also so high from the 1st week that indicates the high level of inflammation. Patient PLTs level slightly decreased during the 7th week of burn injury. The shape of RBC were hypochromic anisocytosis with microcytes and appeared paler in color and lack of development RBCs caused iron deficiency anemia. A similar study also exhibits that abnormal size, the shape of RBCs are responsible for iron deficiency anemia.,, In addition, the WBC count shown that neutrophils parentage were satanically increased from the 1st week to the 6th week and gradually decreased from the 7th week of burn injury. Neutrophils are the primary cellular defense against infections caused by bacteria; neutrophils are phagocytes microbes and release them out of cells., Also, lymphocytes were decreased and below normal range from 1st week of infection that was usually occurring by an excessive cutaneous infection which is previously mentioned in other studies.,
This investigation also shows that cutaneous burn injury and infection was caused by pathogenic microorganisms, including E. coli, Klebsiella spp.; Pseudomonas spp. and Staphylococcus spp. Within a large family of Enterobacteriaceae (E. coli and Klebsiella spp.) and Staphylococcus spp. were found from 1st week to 6th week of infection with supreme bioburden of log 104–105 CFU/mL respectively. The patient was massively propagated by Pseudomonas spp. with extreme bioburden of log 107 CFU/mL in the whole infectious period. High frequency of pathogenic Pseudomonas spp. during wound, infection patient was demonstrated the further possibility of the onward onset of opportunistic complications. Several studies showed that in context to the presence of P. aeruginosa, S. aureus, and E. coli (as have also been identified in the current study), Acinetobacter baumannii was indeed the most prevalent bacterium in the burn wounds.,,
Moreover, the illustration of bacterial resistance appears the MDR isolates of Acinetobacter baumannii and P. aeruginosa were especially anxiety of burn wound infection patients.,, In this study, almost all isolates were exhibited the multidrug-resistance traits against commonly prescribed antibiotics and some antibiotics (imipenem 10 μg, gentamicin 30 μg, colistin sulfate 30 μg, ciprofloxacin 30 μg) were found 100% sensitive against E. coli, Klebsiella spp., Pseudomonas spp. and Staphylococcus spp., respectively. However, an important clinical complication has to be taken on the fact that since E. coli and Klebsiella spp. are well known to be the extended-spectrum β-lactamase(ESBL) producers., The hospital-acquired infections may take place due to insufficient disinfection of hospital surfaces, instruments, and rooms., It increases the wound complication of burn-injured patient.
These types of drug-resistant microorganisms are responsible for wound infection postburn-injured patients. It also causes various health complications along with bacterial infection. Large phagocytic cell (macrophage) usually defended the bacterial colonization during wound infection. WBCs are led to play the phagocytic role against microorganisms and increase the percentage of neutrophils that kill the pathogens intensively. However, the drug resistance traits of pathogens are prohibited the immune response and contains imbalanced blood cells with abnormal morphology and arrangement.
| Conclusions|| |
The early burn-injury is the specific trend in the components of a CBC that is a result of the severity of the secondary burn-injured patient. These trends can be used to determine if a patient's clinical hematologic complication within the expected ranges with severe variation from the 1st-week flame burn injury. The superficial layer of skin ware intensively damaged, and the patient was seriously infected by pathogenic bacteria. Most of them are found as MDR and prevalence of burn wounds and hence increase the possibility of opportunistic infections. The findings of our study sufficiently may address the objective of burn wound management to trim down the drug-resistant bacterial prevalence along with their suggested susceptibility patterns of some drugs to reveal the wound infection. The patient also needed to skin grafting surgery to the developed infected site along with blood transfusion to cure iron deficiency complications. Most possible outcomes of this study are patients should be aware from the pathogenic bacterial infection to increase immune response during wound infection and to avoid the misuse of drugs to be suggestive in the context of overall public health management.
We thank Stamford University Bangladesh for providing us the facilities to carry out the experiments. And also special thanks to Burn and Plastic Surgery Unit, Dhaka Medical College Hospital, Bangladesh for allowing us to collect samples and hematological reports. However, the authors received no specific funding for this work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]